METHODS AND COMPOSITIONS TO ENHANCE ACTIVITY OF CRY ENDOTOXINS

Abstract

Methods and compositions for enhancing the resistance of plants to plant pests are provided. Chimeric pesticidal polypeptides and nucleic acid molecules encoding the chimeric pesticidal polypeptides are provided. The chimeric pesticidal polypeptides comprising a solubility-enhancing polypeptide operably linked to a polypeptide comprising pesticidal activity. The nucleic acid molecules can be used in expression cassettes for making transformed plants with enhanced resistance to plant pests. Further provided are transformed plants, plant tissues, plant cells, other host cells, and seeds as well as pesticidal compositions.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit U.S. Provisional Application No. 61/713,844, filed Oct. 15, 2012, which is incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] An official copy of the Sequence Listing submitted electronically as an ASCII formatted Sequence Listing with a file named "3113PCT_SequenceListing.txt," created on Sep. 17, 2013, having a size of 158 kb and filed concurrently with the Specification is part of the Specification and is incorporated herein by reference as if set forth in its entirety.

FIELD OF THE INVENTION

[0003] The invention relates generally to plant molecular biology and plant pest control, and more particularly to compositions and methods for enhancing the activity of pesticidal polypeptides from Bacillus spp. and for protecting a plant from a plant pest, particularly an insect pest.

BACKGROUND OF THE INVENTION

[0004] Pests, such as insect pests, are a major factor in the loss of the world's agricultural crops. For example, corn rootworm and boll weevil damage can be economically devastating to agricultural producers. Insect pest-related agricultural crop loss from corn rootworm alone has reached one billion dollars a year.

[0005] Traditionally, the primary methods for controlling insect pests, such as corn rootworm, are crop rotation and application of broad-spectrum, synthetic, chemical pesticides. However, consumers and government regulators alike are becoming increasingly concerned with environmental hazards associated with producing and using chemical pesticides. Because of such concerns, regulators have banned or limited the use of some of the more hazardous chemical pesticides. Thus, there is substantial interest in developing alternatives to chemical pesticides that present a lower risk of pollution and environmental hazards and that provide a greater target specificity than is characteristic of chemical pesticides.

[0006] Certain species in the genus Bacillus have polypeptides that possess pesticidal activity against a broad range of insect pests including those in the orders Lepidoptera, Diptera, Coleoptera, Hemiptera and others. For example, Bacillus thuringiensis and Bacillus popilliae are among the most successful species discovered to date having pesticidal activity. Such pesticidal activity also has been attributed to strains of Bacillus larvae, Bacillus lentimorbus, Bacillus sphaericus and Bacillus cereus. See, Biotechnology Handbook 2: Bacillus (Harwood ed., Plenum Press 1989); and Int'l Patent Application Publication No. WO 96/10083.

[0007] Pesticidal polypeptides from Bacillus spp. include the crystal (Cry) endotoxins, cytolytic (Cyt) endotoxins, vegetative proteins (VIPs) and the like. See, e.g., Bravo et al. (2007) Toxicon 49:423-435. The Cry endotoxins (also called .delta.-endotoxins) have been isolated from various strains of B. thuringiensis. A common characteristic of the Cry endotoxins is their expression during the stationary phase of growth, as they generally accumulate in a mother cell compartment to form a crystal inclusion that can account for 23-30% of the dry weight of sporulated cells. The Cry endotoxins initially are produced in an inactive protoxin form, which are proteolytically converted into an active endotoxin through the action of proteases in an insect's gut. Once active, the endotoxins bind to the gut epithelium and form cation-selective channels that cause cell lysis and subsequent death. See, Carroll et al. (1997) J. Invertebr. Pathol. 70:41-49; Oppert (1999) Arch. Insect Biochem. Phys. 42:1-12; and Rukmini et al. (2000) Biochimie 82:109-116.

[0008] Although Cry endotoxins often are highly effective against insect pests, some insect pests are not affected by them or show low susceptibility. Likewise, some insect pests have developed resistance to the Cry endotoxins, which threatens their effectiveness. Methods to address these problems include enhancing or expanding Cry endotoxin activity by site-directed mutagenesis, by introducing cleavage sites in specific regions of the endotoxin or by deleting small fragments from the amino-terminus of the endotoxin. See, e.g., Abdullah & Dean (2004) Appl. Environ. Microbiol. 70:3769-3771; Pardo-Lopez et al. (2009) Peptides 30:589-595; Rajamohan et al. (1996) Proc. Natl. Acad. Sci. USA 93:14338-14343; and Wu et al. (2000) FEBS Lett. 473 227-232. These methods, however, can be time consuming and not certain to produce a desired result.

[0009] For the foregoing reasons, there is a need for compositions and methods to enhance the pesticidal activity of Cry endotoxins.

BRIEF SUMMARY OF THE INVENTION

[0010] Compositions and methods are provided for enhancing pesticidal activity of Cry endotoxins and for protecting a plant from plant pest such as insect pests. The compositions comprise chimeric pesticidal polypeptide-encoding nucleic acid molecules, variants and fragments thereof, as well as chimeric pesticidal polypeptides, active variants and fragments thereof. The chimeric pesticidal polypeptides of the invention comprise a solubility-enhancing polypeptide fused to a Cry endotoxin or biologically active fragment thereof. Also provided are expression cassettes or polynucleotide constructs comprising a nucleotide sequence encoding a chimeric pesticidal polypeptide of the invention, as well as bacteria, plants, plant organs, plants tissues, plant parts, plant cells and seeds comprising the expression cassette or polynucleotide nucleotide construct encoding the chimeric pesticidal polypeptide. Further provided are pesticidal compositions comprising at least one chimeric pesticidal polypeptide of the invention.

[0011] Methods are provided for enhancing the pesticidal activity of Cry endotoxins. The methods involve making a chimeric pesticidal polypeptide comprising the amino acid sequence of a solubility-enhancing polypeptide operably linked to an amino acid sequence of a Cry endotoxin or biologically active fragment thereof. Such chimeric pesticidal polypeptide can be produced by fusing amino acid sequence of the solubility-enhancing polypeptide to the amino acid sequence of a Cry endotoxin or biologically active fragment thereof. Alternatively, the methods involve making a polynucleotide construct comprising a nucleotide sequence encoding the chimeric pesticidal polypeptide and transforming an organism or non-human host cell of interest with the polynucleotide construct for expression of the chimeric pesticidal polypeptide. The nucleotide sequence encoding the chimeric pesticidal polypeptide can be produced by, for example, operably linking a nucleotide sequence encoding a solubility-enhancing polypeptide to a nucleotide sequence encoding a Cry endotoxin or biologically active fragment thereof. Typically, the polynucleotide construct further comprises a promoter that drives expression in the organism or host cell, wherein the promoter is operably linked to the nucleotide sequence encoding the chimeric pesticidal polypeptide.

[0012] Thus, methods are provided for producing a polynucleotide construct comprising a nucleotide sequence encoding a chimeric pesticidal polypeptide, which comprises an amino acid sequence of solubility-enhancing polypeptide fused to an amino acid sequence of a Cry endotoxin or biologically active fragment thereof. The methods involve operably linking a nucleotide sequence encoding solubility-enhancing polypeptide to a nucleotide sequence encoding a Cry endotoxin or biologically active fragment thereof. The polynucleotide construct may additional comprise an operably linked promoter for expression of the chimeric pesticidal polypeptide in a non-human host cell of interest, particularly a plant cell. Such a polynucleotide construct finds use, for example, in methods for expressing the chimeric pesticidal polypeptide in a plant transformed therewith.

[0013] The present invention further provides methods for making plants with enhanced resistance to at least one pest. The methods involved transforming a plant or at least one plant cell with a polynucleotide construct comprising a nucleotide sequence encoding a chimeric pesticidal polypeptide of the invention. Typically, the nucleotide sequence encoding a chimeric pesticidal polypeptide will be operably linked to a promoter that drives expression in a plant cell. The methods can further involve regenerating the plant or the at least one plant cell into a transformed plant, wherein the regenerated plant expresses the chimeric pesticidal polypeptide. Such a transformed plant comprises enhanced resistance to at least one plant pest, particularly an insect pest, when compared to the resistance of a control plant.

[0014] The methods also involve applying a composition such as a pesticidal composition comprising the chimeric pesticidal polypeptide or active variant or fragment thereof, to the environment of an insect pest, particularly on or in the vicinity of a plant by, for example, spraying, dusting, broadcasting or seed coating to protect the plant from the insect pest.

[0015] Further provided are transformed plants, plant cells and other host cells, and seeds comprising a nucleotide sequence encoding the chimeric pesticidal polypeptide of the invention.

[0016] The following embodiments are encompassed by the present invention:

[0017] 1. A method of enhancing pesticidal activity of a Cry endotoxin, the method comprising operably linking a first amino acid sequence of a solubility-enhancing polypeptide to a second amino acid sequence of a Cry endotoxin, whereby a chimeric pesticidal polypeptide is produced, the chimeric pesticidal polypeptide comprising the first amino acid sequence operably linked to second amino acid sequence.

[0018] 2. The method of embodiment 1, wherein the solubility-enhancing polypeptide is selected from the group consisting of a maltose-binding protein (MBP), a thioredoxin, a transcription elongation factor NusA, a glutathione-S-transferase (GST), a mistic, a small ubiquitin-related modifier (SUMO), a protein disulfide isomerase DsbC, and a thiol:disulfide interchange protein DsbD.

[0019] 3. The method of embodiment 1 or 2, wherein the solubility-enhancing polypeptide is a MBP.

[0020] 4. The method of embodiment 3, wherein the MBP is selected from the group consisting of MBPs having an accession number set forth in Table 1.

[0021] 5. The method of embodiment 1 or 2, wherein the solubility-enhancing protein is NusA.

[0022] 6. The method of embodiment 1 or 2, wherein the solubility-enhancing protein is thioredoxin.

[0023] 7. The method of embodiment 1, wherein the solubility-enhancing polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 4, 6, 34, 35, and 36.

[0024] 8. The method of any one of embodiments 1-7, wherein the Cry endotoxin is selected from the group consisting of Cry endotoxins set forth in Table 2.

[0025] 9. The method of any one of embodiments 1-8, wherein the Cry endotoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 10, 12, and 14.

[0026] 10. The method of embodiment 1, wherein the chimeric pesticidal polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 2, 16, 18, 21, 23, 25, 27, and 33.

[0027] 11. The method of embodiment 1, wherein the chimeric pesticidal polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26, and 32.

[0028] 12. The method of any one of embodiments 1-11, wherein the chimeric pesticidal polypeptide comprises a linker amino sequence between the first amino acid sequence and the second amino acid sequence.

[0029] 13. The method of embodiment 1, wherein the linker amino acid sequence is selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 28, 29, 30, and 31.

[0030] 14. The method of any one of embodiments 1-13, further comprising operably linking the linker amino acid sequence between the first amino acid sequence and the amino acid sequence, whereby the chimeric pesticidal polypeptide comprises in linear order the first amino acid sequence, the linker amino acid sequence, and the second amino acid sequence.

[0031] 15. The method of any one of embodiments 1-15, wherein the chimeric pesticidal polypeptide comprises increased pesticidal activity against at least one pest, when compared to the pesticidal activity of the Cry endotoxin against the at least one pest.

[0032] 16. The method of embodiment 15, wherein the at least one pest is an insect pest.

[0033] 17. The method of embodiment 16, wherein the insect pest is an insect pest from the order Coleoptera or Lepidoptera.

[0034] 18. The method of embodiment 16 or 17, wherein the insect pest is from the genus Diabrotica.

[0035] 19. The method of embodiment 16, wherein the insect pest is western corn rootworm.

[0036] 20. The method of embodiment 16, wherein the insect pest is black cutworm.

[0037] 21. A chimeric pesticidal polypeptide comprising a first amino acid sequence of a solubility-enhancing polypeptide operably linked to a second amino acid sequence of a Cry endotoxin.

[0038] 22. The chimeric pesticidal polypeptide of embodiment 21, wherein the operably linked first and second amino sequences comprise an amino acid sequence selected from the group consisting of:

[0039] (a) the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33;

[0040] (b) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32; and

[0041] (c) an amino acid sequence comprising at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33, wherein the nucleotide sequence encodes a polypeptide having pesticidal activity.

[0042] 23. The chimeric pesticidal polypeptide of embodiment 21 or 22, wherein the solubility-enhancing polypeptide is a MBP.

[0043] 24. The chimeric pesticidal polypeptide of embodiment 23, wherein the MBP is selected from the group consisting of MBPs having an accession number set forth in Table 1.

[0044] 25. The chimeric pesticidal polypeptide of embodiment 21 or 22, wherein the solubility-enhancing protein is a NusA.

[0045] 26. The chimeric pesticidal polypeptide of embodiment 21 or 22, wherein the solubility-enhancing protein is a thioredoxin.

[0046] 27. The chimeric pesticidal polypeptide of embodiment 21, wherein the first amino acid sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 4, 6, 34, 35, and 36.

[0047] 28. The chimeric pesticidal polypeptide of any one of embodiments 21-27, wherein the Cry endotoxin is selected from the group consisting of Cry endotoxins set forth in Table 2.

[0048] 29. The chimeric pesticidal polypeptide of embodiment 21, wherein the Cry endotoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 10, 12, and 14.

[0049] 30. The chimeric pesticidal polypeptide of any one of embodiments 21-29, wherein the chimeric pesticidal polypeptide further comprises a linker amino sequence operably linked between the first amino acid sequence and the second amino acid sequence.

[0050] 31. The chimeric pesticidal polypeptide of embodiment 30, wherein the linker amino acid sequence is selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 28, 29, 30, and 31.

[0051] 32. The chimeric pesticidal polypeptide of any one of embodiments 21-31, wherein the chimeric pesticidal polypeptide comprises increased pesticidal activity against at least one pest, when compared to the pesticidal activity of the Cry endotoxin against the at least one pest.

[0052] 33. The chimeric pesticidal polypeptide of embodiment 32, wherein the at least one pest is an insect pest.

[0053] 34. The chimeric pesticidal polypeptide of embodiment 33, wherein the insect pest is an insect pest from the order Coleoptera or Lepidoptera.

[0054] 35. The chimeric pesticidal polypeptide of embodiment 33, wherein the insect pest is from the genus Diabrotica.

[0055] 36. The chimeric pesticidal polypeptide of embodiment 33, wherein the insect pest is western corn rootworm.

[0056] 37. The chimeric pesticidal polypeptide of embodiment 33, wherein the insect pest is black cutworm.

[0057] 38. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric pesticidal polypeptide, the chimeric pesticidal polypeptide comprising a first amino acid sequence of a solubility-enhancing polypeptide operably linked to a second amino acid sequence of a Cry endotoxin.

[0058] 39. The nucleic acid molecule of embodiment 38, wherein the nucleotide sequence comprises a nucleotide sequence selected from the group consisting of:

[0059] (a) the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32;

[0060] (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33;

[0061] (c) a nucleotide sequence comprising at least 80% nucleotide sequence identity to SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32, wherein the nucleotide sequence encodes a polypeptide having pesticidal activity; and

[0062] (e) a nucleotide sequence encoding an amino acid sequence comprising at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33, wherein the nucleotide sequence encodes a polypeptide having pesticidal activity.

[0063] 40. The nucleic acid molecule of embodiment 38 or 39, wherein the solubility-enhancing polypeptide is a MBP.

[0064] 41. The nucleic acid molecule of embodiment 40, wherein the MBP is selected from the group consisting of MBPs having an accession number set forth in Table 1.

[0065] 42. The nucleic acid molecule of embodiment 38 or 39, wherein the solubility-enhancing protein is a NusA.

[0066] 43. The nucleic acid molecule of embodiment 38 or 39, wherein the solubility-enhancing protein is a thioredoxin.

[0067] 44. The nucleic acid molecule of embodiment 38 or 39, wherein the first amino acid sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 4, 6, 34, 35 and 36.

[0068] 45. The nucleic acid molecule of any one of embodiments 38-44, wherein the Cry endotoxin is selected from the group consisting of Cry endotoxins set forth in Table 2.

[0069] 46. The nucleic acid molecule of any one of embodiments 38-44, wherein the second amino acid sequence encodes a Cry endotoxin selected from the group consisting of Cry endotoxins set forth in Table 2.

[0070] 47. The nucleic acid molecule of embodiment 38, wherein the second amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 10, 12 and 14.

[0071] 48. The nucleic acid molecule of any one of embodiments 38-47, wherein the chimeric pesticidal polypeptide further comprises a linker amino sequence operably linked between the first amino acid sequence and the second amino acid sequence.

[0072] 49. The nucleic acid molecule of embodiment 48, wherein the linker amino acid sequence is selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 28, 29, 30, and 31.

[0073] 50. The nucleic acid molecule of any one of embodiments 38-49, wherein the chimeric pesticidal polypeptide comprises increased pesticidal activity against at least one pest, when compared to the pesticidal activity of the Cry endotoxin against the at least one pest.

[0074] 51. The nucleic acid molecule of any one of embodiments 38-50, wherein the at least one pest is an insect pest.

[0075] 52. The nucleic acid molecule of embodiment 51, wherein the insect pest is an insect pest from the order Coleoptera or Lepidoptera.

[0076] 53. The nucleic acid molecule of embodiment 51, wherein the insect pest is from the genus Diabrotica.

[0077] 54. The nucleic acid molecule of embodiment 51, wherein the insect pest is western corn rootworm.

[0078] 55. The nucleic acid molecule of embodiment 51, wherein the insect pest is black cutworm.

[0079] 56. An expression cassette comprising a promoter that drives expression in a host cell operably linked to a nucleic acid molecule of any one of embodiments 38-55.

[0080] 57. The expression cassette of embodiment 56, wherein the host cell is a plant cell.

[0081] 58. The expression cassette of embodiment 57, wherein the promoter is selected from the group consisting of a chemical-inducible promoter, constitutive promoter, pest-inducible promoter, tissue-specific promoter and wound-inducible promoter.

[0082] 59. The expression cassette of embodiment 58, wherein the tissue-specific promoter is selected from the group consisting of a leaf-preferred promoter, root-preferred promoter, seed-preferred promoter, stalk-preferred promoter and vascular tissue-preferred promoter.

[0083] 60. A vector comprising the expression cassette of any one of embodiments 56-59.

[0084] 61. A transformed plant, plant part, plant cell or seed comprising in its genome the expression cassette of any one of embodiments 56-60.

[0085] 62. The transformed plant, plant part or plant host cell of embodiment 61, wherein the nucleotide sequence is stably incorporated into the genome of the transformed plant, plant part, plant cell or seed.

[0086] 63. A pesticidal composition comprising an effective amount of a chimeric pesticidal polypeptide of any one of embodiments 21-37 or an active variant or fragment thereof having pesticidal activity.

[0087] 64. The pesticidal composition of embodiment 63, further comprising bacteria expressing a nucleotide sequence encoding the chimeric pesticidal polypeptide or a biologically active fragment or variant thereof.

[0088] 65. The pesticidal composition of embodiment 63, further comprising at least one agricultural protectant selected from the group consisting of an acaricide, bactericide, fertilizer or micronutrient donor, fungicide, insecticide, nematocide and semiochemical.

[0089] 66. The pesticidal composition of embodiment 65, wherein the semiochemical is selected from the group consisting of an allomone, attractant, feeding pheromone, kairomone, repellent and stimulant.

[0090] 67. A method of protecting a plant from an insect pest, the method comprising providing an effective amount of a pesticidal composition of any one of embodiments 63-66 to reduce insect pest-related damage to the plant.

[0091] 68. The method of embodiment 67, wherein the pesticidal composition is applied by a procedure selected from the group consisting of spraying, dusting, broadcasting and seed coating.

[0092] 69. The method of embodiment 67 or 68, wherein the chimeric pesticidal polypeptide has pesticidal activity against an insect pest in the order Coleoptera, an insect pest in the order Lepiedoptera or both.

[0093] 70. The method of any one of embodiments 67-69, wherein the insect pest is selected from the group consisting of species in the genus Diabrotica.

[0094] 71. A plant comprising a polynucleotide construct stably incorporated in its genome, the polynucleotide construct comprising a nucleotide sequence operably linked to a promoter that drives expression in the plant, wherein the nucleotide sequence encodes a chimeric pesticidal polypeptide comprising a first amino acid sequence of a solubility-enhancing polypeptide operably linked to a second amino acid sequence of a Cry endotoxin.

[0095] 72. The plant of embodiment 71, wherein the nucleotide sequence is selected from the group consisting of:

[0096] (a) the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32;

[0097] (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33;

[0098] (c) a nucleotide sequence comprising at least 80% nucleotide sequence identity to SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32, wherein the nucleotide sequence encodes a polypeptide having pesticidal activity; and

[0099] (e) a nucleotide sequence encoding an amino acid sequence comprising at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33, wherein the nucleotide sequence encodes a polypeptide having pesticidal activity.

[0100] 73. The plant of embodiment 71 or 72, wherein the solubility-enhancing polypeptide is MBP.

[0101] 74. The plant of embodiment 73, wherein the MBP is selected from the group consisting of MBPs having an accession number set forth in Table 1.

[0102] 75. The plant of embodiment 71 or 72, wherein the solubility-enhancing protein is NusA.

[0103] 76. The plant of embodiment 71 or 72, wherein the solubility-enhancing protein is thioredoxin.

[0104] 77. The plant of embodiment 71, wherein the first amino acid sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 4, 6, 34, 35 and 36.

[0105] 78. The plant of any one of embodiments 71-77, wherein the Cry endotoxin is selected from the group consisting of Cry endotoxins set forth in Table 2.

[0106] 79. The plant of any one of embodiments 71-78, wherein the second amino acid sequence encodes a Cry endotoxin selected from the group consisting of Cry endotoxins set forth in Table 2.

[0107] 80. The plant of embodiment 71 or 72, wherein the second amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 10, 12 and 14.

[0108] 81. The plant of any one of embodiments 71-80, wherein the chimeric pesticidal polypeptide further comprises a linker amino sequence operably linked between the first amino acid sequence and the second amino acid sequence.

[0109] 82. The nucleic acid molecule of embodiment 81, wherein the linker amino acid sequence is selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 28, 29, 30 and 31.

[0110] 83. The plant of any one of embodiments 71-82, wherein the chimeric pesticidal polypeptide comprises increased pesticidal activity against at least one pest, when compared to the pesticidal activity of the Cry endotoxin against the at least one pest.

[0111] 84. The plant of embodiment 83, wherein the at least one pest is an insect pest.

[0112] 85. The plant of embodiment 84, wherein the insect pest is an insect pest from the order Coleoptera or Lepidoptera.

[0113] 86. The plant of embodiment 84, wherein the insect pest is from the genus Diabrotica.

[0114] 87. The plant of embodiment 84, wherein the insect pest is western corn rootworm.

[0115] 88. The plant of embodiment 84, wherein the insect pest is black cutworm.

[0116] 89. The plant of any one of embodiments 71-88, wherein the plant is a monocot.

[0117] 90. The plant of embodiment 89, wherein the monocot is barley, maize, rice, rye, sorghum, sugarcane or wheat.

[0118] 91. The plant of any one of embodiments 71-88, wherein the plant is a dicot.

[0119] 92. The plant of embodiment 91, wherein the dicot is alfalfa, Brassica, cotton, soybean or sunflower.

[0120] 93. The plant of any one of embodiment 71-92, wherein the plant is a seed.

[0121] 94. A method of protecting a plant, plant part or plant host cell from an insect pest, the method comprising the steps of:

[0122] (a) introducing into the plant, plant part or plant host cell an expression cassette of any one of embodiments 56-59; and

[0123] (b) regenerating the plant, plant part or plant host cell into a morphologically normal fertile plant, wherein the plant or part thereof comprises a chimeric pesticidal polypeptide.

[0124] 95. The method of embodiment 94, wherein the chimeric pesticidal polypeptide has pesticidal activity against an insect pest in the order Coleoptera, an insect pest in the order Lepidoptera or both.

[0125] 96. The method of embodiment 94, wherein the insect pest is selected from the group consisting of species in the genus Diabrotica.

[0126] 97. A method of enhancing the resistance of a plant to at least one pest, the method comprising introducing into a plant or at least one plant cell a polynucleotide construct comprising a nucleotide sequence operably linked to a promoter that drives expression in the plant, wherein the nucleotide sequence encodes a chimeric pesticidal polypeptide comprising a first amino acid sequence of a solubility-enhancing polypeptide operably linked to a second amino acid sequence of a Cry endotoxin.

[0127] 98. The method of embodiment 97, wherein the nucleotide sequence comprises a nucleotide sequence selected from the group consisting of:

[0128] (a) the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32;

[0129] (b) a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33;

[0130] (c) a nucleotide sequence comprising at least 80% nucleotide sequence identity to SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32, wherein the nucleotide sequence encodes a polypeptide having pesticidal activity; and

[0131] (e) a nucleotide sequence encoding an amino acid sequence comprising at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33, wherein the nucleotide sequence encodes a polypeptide having pesticidal activity.

[0132] 99. The method of embodiment 97 or 98, wherein solubility-enhancing polypeptide is MBP.

[0133] 100. The method of embodiment 99, wherein the MBP is selected from the group consisting of MBPs having an accession number set forth in Table 1.

[0134] 101. The method of embodiment 97 or 98, wherein the solubility-enhancing protein is NusA.

[0135] 102. The method of embodiment 97 or 98, wherein the solubility-enhancing protein is thioredoxin.

[0136] 103. The method of embodiment 97, wherein the first amino acid sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 4, 6, 34, 35 and 36.

[0137] 104. The method of any one of embodiments 97-103, wherein the Cry endotoxin is selected from the group consisting of Cry endotoxins set forth in Table 2.

[0138] 105. The method of any one of embodiments 97-104, wherein the second amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 10, 12 and 14.

[0139] 106. The method of any one of embodiments 97-105, wherein the chimeric pesticidal polypeptide further comprises a linker amino sequence operably linked between the first amino acid sequence and the second amino acid sequence.

[0140] 107. The method of embodiment 106, wherein the linker amino acid sequence is selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 28, 29, 30 and 31.

[0141] 108. The method of any one of embodiments 97-107, wherein the chimeric pesticidal polypeptide comprises increased pesticidal activity against at least one pest, when compared to the pesticidal activity of the Cry endotoxin against the at least one pest.

[0142] 109. The method of any one of embodiments 97-108, further comprising regenerating a plant comprising the polynucleotide construct.

[0143] 110. The method of any one of embodiments 97-109, wherein the plant has enhanced resistance to at least one pest, when compared to a control plant lacking the polynucleotide construct.

[0144] 111. The method of embodiment 110, wherein the at least one pest is an insect pest.

[0145] 112. The method of embodiment 111, wherein the insect pest is an insect pest from the order Coleoptera or Lepidoptera.

[0146] 113. The method of embodiment 111, wherein the insect pest is from the genus Diabrotica.

[0147] 114. The method of embodiment 111, wherein the insect pest is western corn rootworm.

[0148] 115. The method of embodiment 111, wherein the insect pest is black cutworm.

[0149] 116. The method of any one of embodiments 97-115, wherein the plant is a monocot.

[0150] 117. The method of embodiment 116, wherein the monocot is barley, maize, rice, rye, sorghum, sugarcane or wheat.

[0151] 118. The method of any one of embodiments 97-115, wherein the plant is a dicot.

[0152] 119. The method of embodiment 118, wherein the dicot is alfalfa, Brassica, cotton, soybean or sunflower.

BRIEF DESCRIPTION OF THE DRAWINGS

[0153] The present invention will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0154] FIG. 1 depicts the structure of E. coli maltose-binding protein (MBP)-Bt Cry protein fusion of the present invention. =maltose binding protein =Bt Cry protein.

DETAILED DESCRIPTION OF THE INVENTION

[0155] The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

[0156] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0157] The present invention is based on the discovery that pesticidal activity of Cry endotoxins can be enhanced by operably linking a solubility-enhancing polypeptide, such as, for example, a maltose-binding protein (MBP), to a Cry endotoxin or insecticidally active fragment thereof. Moreover, insect pest resistance can be overcome by operably linking an MBP to a Cry endotoxin by the methods disclosed herein. The present invention therefore provides compositions and methods for enhancing Cry endotoxin activity, for overcoming plant pest resistance, and for protecting plants from pests, especially insect pests.

[0158] While the invention does not depend on a particular biological mechanism, the fusion of a solubility-enhancing polypeptide to a Cry endotoxin or insecticidally active fragment thereof may act to raise the solubility of the Cry protein, particularly in the insect gut environment, more particularly in the gut environment of a Coleopteran insect. That is the solubility of the fusion protein has a higher solubility at a slightly acidic pH than the Cry protein has at the same pH. In a preferred embodiment of the invention, with activated Cyr toxin that is used in making the fusion protein of the invention has an isoelectric point (pI) from about pH 7 to about pH 8. It is recognized that in the digestive system in Lepidopteran insects, pH is relatively high. In such alkaline conditions, an activated Bt Cry toxin typically is soluble but in the slightly acidic pH of the Coleopteran gut environment in which pH is slightly acidic, solubility of the activated Bt Cry toxin can be much lower.

[0159] Compositions and methods are provided for enhancing the pesticidal activity of Cry endotoxins by operably linking a solubility-enhancing protein to a Cry endotoxin and for protecting plants from insect pests. The compositions and methods disclosed herein include recombinant nucleic acid molecules that encode chimeric pesticidal polypeptides, expression cassettes and other nucleotide constructs including the nucleic acid molecules described herein organisms transformed with the nucleic acid molecules described herein, isolated chimeric pesticidal polypeptides, and pesticidal compositions having the chimeric pesticidal polypeptides, as well as methods of using the same. The compositions and methods therefore find use in enhancing pesticidal activity of pesticidal proteins and in protecting plants from insect pests.

[0160] As used herein, a "solubility-enhancing polypeptide" is any polypeptide that when operably linked to a Cry endotoxin or insecticidally active fragment thereof enhances the pesticidal activity of the Cry endotoxin or insecticidally active fragment against at least one insect pest, preferably a Coleopteran insect pest, when compared to the pesticidal activity of the same Cry endotoxin or insecticidally active fragment thereof in the absence of an operably linked solubility-enhancing polypeptide. In certain embodiments, the solubility enhancing polypeptides of the present invention are capable of increasing the solubility of an operable linked Cry endotoxin or insecticidally active fragment thereof at a slightly acidic pH that is found, for example, in the gut environment of a Coleopteran insect. Preferably, a chimeric pesticidal polypeptide of the present invention, which comprises a solubility-enhancing polypeptide of the present invention operably linked to a Cry endotoxin or insecticidally active fragment thereof, comprises an increased solubility in an environment that has a slightly acidic pH such as, for example, the gut environment of a Coleopteran insect, than the same Cry endotoxin or insecticidally active fragment thereof lacking the operably linked solubility-enhancing polypeptide.

[0161] As used herein, "pest" or "plant pest" means an organism that interferes with or is harmful to plant development and/or growth. Such plant pests include, but are not limited to, nematodes, insect, viruses, viroids, mites, fungal pathogens, bacteria and any other plant pests disclosed herein. Accordingly, the polynucleotides and polypeptides of the invention can be used to enhance resistance of plants to plant pests. One of skill in the art, however, understands that not all polypeptides are equally effective against all plant pests. The chimeric pesticidal polypeptides described herein display activity against plant pests such as insect pests, which may include economically important agronomic, forest, greenhouse, nursery ornamentals, food and fiber, public and animal health, domestic and commercial structure, household and stored product pests. Therefore, of interest herein are chimeric pesticidal polypeptides for use in protecting plants from insect pests.

[0162] As used herein, "pesticidal polypeptide" means a peptide, polypeptide or protein that has biological activity against insect pests (i.e., is pesticidal).

[0163] As used herein, "pesticidal" or "pesticidal activity" means capable of killing insect pests. Likewise, "pesticidal" or "pesticidal activity" means capable of inhibiting insect growth. As such, the chimeric pesticidal polypeptides described herein are capable of inhibiting growth or reproduction of or of killing, at least one plant pest, particularly at least one insect pest.

[0164] By "insecticidal activity" is intended the ability of a polypeptide of the invention or a composition comprising the polypeptide (or other agent, chemical or composition) to inhibit the growth of or damage to a plant caused by, at least one insect pest.

[0165] As used herein, "enhance" and the like means increasing an activity/effectiveness of a pesticidal polypeptide such as a Cry endotoxin against a particular pest by operably linking the Cry endotoxin to a solubility-enhancing polypeptide, where the activity/effectiveness of the resulting chimeric pesticidal polypeptide is increased by about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90% or about 90% to about 100% when compared to a wild-type Cry endotoxin. Alternatively, the activity/effectiveness of the chimeric pesticidal polypeptide is increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more when compared to the wild-type Cry endotoxin. In addition, "enhance" can mean that the host pest range of the pesticidal polypeptide is expanded to include additional pests.

[0166] As used herein, "about" means within a statistically meaningful range of a value such as a stated concentration, length, molecular weight, percentage, pH, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.

[0167] As used herein, "chimeric polypeptide" or "chimeric pesticidal polypeptide" means a polypeptide having a first amino acid sequence derived from a first source operably linked, covalently or non-covalently, to a second amino acid sequence derived from a second source, where the first and second source are not the same. A first source and a second source that are not the same can include two different organisms or two different proteins from the same organism or a biological source and a synthetic source or even two different synthetic sources. A biological source can include any non-synthetically produced nucleotide or amino acid sequence (e.g., a genomic or cDNA sequence, a plasmid or viral vector, a native virion or a mutant or analog). A synthetic source can include a nucleotide or amino acid sequence produced chemically and not by a biological source (e.g., solid phase synthesis of amino acid sequences). The chimeric pesticidal polypeptide can be produced by expressing a recombinant nucleic add molecule encoding a polypeptide having at least two parts or can be produced synthetically.

[0168] A chimeric pesticidal polypeptide of the present can further comprise, for example, a linker molecule (also referred to here as a "linker) between the first and second amino acid sequences. Examples of linkers that can be used in the methods of the present invention are the NEB pMAL, SA, NusA, and TrxA linkers having the amino acid sequences set forth in SEQ ID NOS: 28, 29, 30, and 31, respectively.

[0169] Solubility-enhancing polypeptides of the present include, but are not limited to, maltose-binding protein (MBP), thioredoxin (e.g., TrxA), transcription elongation factor NusA, glutathione-S-transferase (GST), mistic, small ubiquitin-related modifier (SUMO), protein disulfide isomerase DsbC, and thiol:disulfide interchange protein DsbD, and variants and fragments thereof that when operably linked to a Cry endotoxin or insecticidally active fragment thereof enhance the pesticidal activity of an operably linked Cry endotoxin or insecticidally active fragment against at least one insect pest, preferably a Coleopteran insect pest, when compared to the pesticidal activity of the same Cry endotoxin or insecticidally active fragment thereof in the absence of an operably linked solubility-enhancing polypeptide.

[0170] As used herein, "maltose-binding protein" or "MBP" means a polypeptide that is a member of the maltodextrin transport system that binds maltodextrins (e.g., maltose, maltotriose and trehalose) with micromolar affinity and that is essential for an energy-dependent translocation of maltodextrins through a cytoplasmic membrane of some prokaryotes. See, Boos & Shuman (1998) Microbiol. Mol. Biol. Rev. 62:204-229. For example, in Escherichia coli, the maIE gene encodes a 396 amino acid residue pre-MBP, which subsequently is processed into MBP upon cleavage of a 26 amino acid N-terminal signal peptide. See, e.g., Duplay et al. (1984) J. Biol. Chem. 259:10606-10613.

[0171] In one embodiment of the invention, the chimeric pesticidal polypeptide comprises an amino acid sequence of an MBP operably linked to an amino acid sequence of a Cry endotoxin including, for example, the chimeric pesticidal polypeptides comprising the amino acid sequences set forth in SEQ ID NOS: 2, 21, 23, 25, 27, and 33 and encoded by the nucleotide sequences set forth in SEQ ID NOS: 1, 20, 22, 24, 26, and 32, respectively.

[0172] In another embodiment, the chimeric pesticidal polypeptide comprises an amino acid sequence of NusA operably linked to an amino acid sequence of a Cry endotoxin including, for example, the chimeric pesticidal polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16 and encoded by the nucleotide sequence set forth in SEQ ID NO: 15.

[0173] In yet another embodiment, the chimeric pesticidal polypeptide comprises an amino acid sequence of thioredoxin operably linked to an amino acid sequence of a Cry endotoxin including, for example, the chimeric pesticidal polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18 and encoded by the nucleotide sequence set forth in SEQ ID NO: 17.

[0174] The present invention comprises the use of an solubility-enhancing polypeptide that when fused to a Cry endotoxin polypeptide increases the pesticidial activity of against at least one pest, preferably an insect pest, more preferably an insect pest from the order Coleoptera or Lepidoptera, even more preferably an insect pest from the genus Diabrotica, most preferably the western corn rootworm (Diabrotica virgifera virgifera). In certain embodiments, the solubility-enhancing polypeptide selected from the group consisting of MBP, NusA, and TrxA.

[0175] The present invention does not depend on the use of a particular solubility-enhancing polypeptide. Any solubility-enhancing polypeptide that, when fused to a Cry endotoxin polypeptide, is capable increasing the pesticidial activity of the Cry endotoxin polypeptide against at least one pest can be used in the methods and compositions disclosed herein. Such solubility enhancing polypeptides include, for example, pre-proteins and mature proteins, and variants and fragments thereof. In certain embodiments, the solubility-enhancing polypeptide is MBP. MBPs of the present invention include, but are not limited, full-length MBP (also referred to as "pre-MBP", a mature form of an MBP, a fragment of a full-length or mature MBP or a variant. Unless expressed stated herein or otherwise apparent form the context, the terms "maltose-binding protein" and "MBP" encompass such full-length and mature forms of MBPs as well as fragments and variants thereof.

[0176] The amino acid and/or nucleotide sequences for a number of MBPs have been disclosed and can be used in the compositions and methods of the present invention including, but not limited to, the following those MBPs having the following accession numbers in Table 1.

TABLE-US-00001 TABLE 1 GenBank Accession Number* NP_290668 ZP_03064501 CBG37227 NP_709885 ACI74011 YP_002415175 YP_001726921 EFK20320 ZP_02999303 YP_312946 ZP_06651607 ACC91724 NP_756856 ACF57854 ACF57853 ABO28850 AAB86559 AAC83813 BAI57431 AAQ93661 ACI46135 ACI46133 AAK55118 AAB87675 ZP_02904048 YP_002385139 ZP_06356417 YP_003367151 YP_002639794 NP_458527 YP_001338045 ACY91424 ZP_03381315 YP_219095 AAX68014 AAL23053 NP_463094 ZP_04558600 YP_002228802 ZP_02700756 YP_002149143 CAA38189 CBK84491 ZP_06390929 YP_001455393 YP_003610806 YP_001174979 YP_001572421 YP_002241024 P18815 YP_003212142 YP_001436222 YP_001480694 ZP_06712852 ZP_04616043 ZP_04638283 ZP_06191619 ZP_04626936 ZP_04633703 ZP_04613648 NP_667372 ZP_04641958 YP_001161578 YP_001008013 ZP_04625082 YP_003294268 YP_002931694 ZP_06638363 BAE44434 YP_003743846 YP_003039205 NP_927811 ZP_05729913 YP_003714385 YP_003469920 YP_003729949 YP_003520206 YP_002891253 ZP_03366279 ZP_05920010 YP_003007381 ZP_06636620 YP_001343631 YP_003255618 ZP_04976824 YP_089260 ZP_06157313 YP_002474876 YP_001447347 ZP_01984309 ZP_04754507 ZP_06177188 NP_800911 ZP_06180148 ZP_02477508 ZP_01258612 ZP_05878943 ZP_05629984 YP_001053928 YP_132094 ZP_05882865 ZP_02194153 ZP_01221443 ZP_05927269 NP_763457 ZP_05943387 ZP_01865449 ZP_05717553 ZP_06032225 ZP_06079586 ZP_05239461 YP_002812521 NP_233329 ZP_01970977 ZP_04918366 ZP_05719582 ZP_05886194 ZP_05117778 ZP_06941932 ZP_04415908 ZP_01950896 ZP_01676462 ZP_01897017 YP_001343632 YP_132089 ZP_06938154 YP_003007380 ZP_03352864 ZP_01221449 ZP_00988394 ZP_01811558 YP_856203 YP_001142458 NP_936112 ZP_01062901 NP_763137 YP_206757 ZP_00988462 ZP_04403894 ZP_05120494 ZP_01947956 ZP_01062959 ZP_01865441 YP_431775 ZP_01895948 YP_001173956 ZP_03375554 YP_001188617 ZP_00135549 ZP_03699982 YP_003269532 YP_003548264 ZP_01893753 ZP_01112736 YP_431868 ZP_01157584 ZP_01115171 ZP_03369842 ZP_01955972 ZP_01955970 ZP_06938864 YP_002352611 YP_002250432 YP_002534909 NP_229009 YP_001245135 YP_002534307 YP_001739017 YP_001244560 YP_003346096 YP_002250571 CAA72193 NP_229635 YP_002352749 YP_001470540 NP_623418 ZP_05493657 YP_001662811 YP_002941091 YP_003672376 YP_146557 ZP_04392773 YP_003677351 ZP_05336993 YP_003477456 AAY89718 YP_001568299 YP_001410291 ZP_03385039 YP_001568464 YP_001124738 YP_604964 YP_003514612 ZP_03146818 ZP_04152680 ZP_04158384 YP_001305848 YP_001142178 ZP_04307633 YP_002509363 ZP_06220504 ZP_04121933 ZP_04204735 ZP_03232052 YP_003148220 ZP_04086064 ZP_04258265 ZP_04296475 ZP_02397670 ZP_00236932 ZP_04187645 ZP_03105903 ZP_03238270 YP_896372 YP_038073 YP_085351 NP_846464 ZP_04128078 ZP_04285672 YP_003186266 ZP_02948002 YP_002447583 CAB65651 NP_980358 ZP_04302212 ZP_03493519 ZP_02878398 ZP_04176050 ZP_05183404 ZP_04290906 ZP_04199019 ZP_04263630 YP_001646638 ZP_04229423 ZP_04170363 YP_002334500 BAB40635 ZP_04218748 ZP_02866107 YP_697032 YP_003258733 ZP_02953587 YP_177014 YP_051264 ZP_02638879 YP_003018517 ZP_04607307 ZP_03828721 YP_003116555 ZP_03832162 YP_002316535 NP_294284 NP_563259 YP_002785582 YP_002886868 ZP_06681977 ZP_05667047 ZP_05664221 ZP_03981553 ZP_06699376 YP_001488817 YP_001375943 ZP_00603967 ZP_05132228 YP_699603 YP_002574412 YP_001814130 YP_003445304 ZP_04878976 ZP_03055079 ZP_01861955 YP_002918149 YP_002240053 YP_001334109 YP_001813248 ZP_06549508 ZP_06329195 ZP_04864724 YP_001179261 YP_144918 YP_039671 NP_645005 NP_370738 YP_003465347 ZP_05877552 ZP_04016117 YP_001439475 BAI87082 ADI96692 CBI48099 YP_850342 YP_005257 ZP_00233442 YP_003613139 ZP_06315382 YP_946709 NP_391341 AAD42742 EFK15520 ZP_05300115 ZP_05298949 YP_001695468 ZP_02185856 YP_830242 YP_288893 NP_471563 YP_003208908 YP_002958966 YP_001836789 ZP_01827852 YP_415662 NP_359509 BAB18102 YP_002739191 YP_003154662 ZP_03497002 YP_003696753 YP_003684577 ZP_07053052 ZP_05231671 ZP_02711433 ZP_01821820 ZP_01821214 NP_346527 ZP_01408156 ZP_02186034 BAC10980 YP_003336687 NP_670540 YP_071605 ZP_06640983 YP_003723548 ZP_04853514 YP_002743432 YP_001399898 YP_077886 YP_002038696 NP_391296 ZP_06611384 ZP_06198009 YP_003339007 YP_003729851 ZP_03958786 ZP_01170933 YP_081353 YP_001719722 ZP_06059512 YP_001449426 ZP_06872846 ZP_04851795 YP_003241054 YP_002473273 YP_001396549 YP_920853 ZP_06190079 BAI87023 YP_001664668 YP_003704868 ZP_06899649 NP_693481 YP_003314160 YP_003334475 ZP_05647136 YP_003203952 YP_056254 ZP_06014674 ZP_05687198 YP_003506902 ZP_04449975 YP_002958266 YP_002948859 YP_003677653 ZP_06428097 YP_003781558 ZP_03925962 YP_001199499 NP_579667 YP_003003618 YP_003678645 ZP_03225712 ZP_01460386 YP_003326738 YP_002744243 YP_002746719 YP_002123617 AAA26922 YP_003477807 ZP_02329884 ZP_04431587 ZP_01461944 NP_242885 YP_003161478 ZP_06533915 YP_001137791 YP_003009537 NP_125870 YP_002308182 ZP_06885100 ZP_04876799 YP_001559408 YP_003699585 ZP_01130620 YP_184184 ZP_05621839 NP_243792 YP_002883083 YP_003637265 YP_003425427 YP_003061750 YP_002997014 ZP_07076764 ZP_05749331 YP_602654 NP_269430 NP_737480 ZP_06808450 YP_003009727 YP_002565304 YP_598732 YP_280516 YP_002881503 NP_784007 YP_002246441 ZP_02045060 YP_002509787 ZP_06608272 YP_002136599 ZP_06363693 EFK02214 ZP_05431814 YP_136875 ZP_06662656 YP_001880661 YP_177522 ZP_06048003 YP_002387348 ZP_01983428 YP_003229678 ZP_04402673 YP_467337 ZP_06365288 CAL69747 YP_002881799 ZP_04412511 ZP_01951248 *The amino acid and corresponding nucleotide sequences of the accession numbers in Table 1 are herein incorporated by reference.

[0177] In certain embodiments of the invention, the MBP comprises the amino acid sequence set forth in SEQ ID NO: 4 or 6 or fragment or variant thereof. Any nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 4 or 6 or fragment or variant thereof, can be used in the methods and compositions of the present invention. In some embodiments of the invention, the nucleotide sequences of the invention will be optimized for expression in a host organism or cell of interest, particularly a plant, more particularly a crop plant, most particularly a maize plant.

[0178] As used herein, "Cry endotoxin" means a .delta.-endotoxin encoded by cry (crystal protein) genes that are located mainly on large plasmids in members of the genus Bacillus, although chromosomally encoded Cry endotoxins have been reported. See, Ben-Dov et al. (1996) Appl. Environ. Microbiol. 62:3140-3145; Berry et al. (2002) Appl. Environ. Microbiol. 68:5082-5095; Gonzales et al. (1981) Plasmid 5:351-365; Lereclus et al. (1982) Mol. Gen. Genet. 186:391-398; and Trisrisook et al. (1990) Appl. Environ. Microbiol. 56:1710-1716. Cry endotoxins do not have a broad spectrum of activity, so they typically do not kill beneficial insects. Furthermore, Cry endotoxins are non-toxic to mammals, including humans, domesticated animals and wildlife.

[0179] Cry endotoxins generally have five conserved sequence domains, and three conserved structural domains. See, e.g., de Maagd et al. (2001) Trends Genetics 17:193-199. The first conserved structural domain (Domain I) consists of seven alpha helices and is involved in membrane insertion and pore formation. The second conserved structural domain (Domain II) consists of three beta-sheets arranged in a Greek key configuration, and the third conserved structural domain (Domain III) consists of two antiparallel beta-sheets in "jelly-roll" formation. Domains II and III are involved in receptor recognition and binding, and are therefore considered determinants of toxin specificity.

[0180] As used herein, "insect pest" means an organism in the phylum Arthropoda that interferes with or is harmful to plant development and/or growth, and more specifically means an organism in the class Insecta. The class Insecta can be divided into two groups historically treated as subclasses: (1) wingless insects, known as Apterygota; and (2) winged insects, known as Pterygota. The insect pests can be adults, larvae or even ova. A preferred developmental stage for testing for pesticidal activity is larvae or other immature form of the insect pest. Methods of rearing insect larvae and performing bioassays are well known in the art. See, e.g., Czapla & Lang (1990) J. Econ. Entomol. 83:2480-2485; Griffith & Smith (1977) J. Aust. Ent. Soc. 16:366; and Keiper & Foote (1996) Hydrobiologia 339:137-139; as well as U.S. Pat. No. 5,351,643. For example, insect pests can be reared in total darkness at about 20.degree. C. to about 30.degree. C. and from about 30% to about 70% relative humidity.

[0181] Compositions comprising chimeric pesticidal polypeptide-encoding nucleic acid molecules and chimeric pesticidal polypeptides also are provided. The compositions include chimeric pesticidal polypeptides that comprise a solubility-enhancing polypeptide fused to a Cry endotoxin or biologically active fragment thereof. Nucleotide sequences that encode the chimeric pesticidal polypeptides can be derived from the chimeric pesticidal polypeptide sequence and produced using any method known in the art. The compositions also include variants and fragments of the chimeric pesticidal-encoding nucleic acid molecules and chimeric pesticidal polypeptides. The isolated, chimeric pesticidal-encoding nucleic acid molecules can be used to create transgenic organisms, such as plants, that are resistant to an insect pest susceptible to the pesticidal polypeptide.

[0182] The presently disclosed methods and compositions provides for chimeric pesticidal polypeptides with improved efficacy and nucleic acid molecules encoding the chimeric pesticidal polypeptides. The chimeric pesticidal polypeptides comprise solubility-enhancing polypeptide fused (i.e., operably linked) to a Cry endotoxin or biologically active fragment thereof and have enhanced pesticidal activity against at least one plant pest, when compared to the pesticidal activity of the Cry endotoxin or biologically active fragment thereof that has not been fused to a solubility-enhancing polypeptide.

[0183] As used herein, "pesticide," "pesticidal polypeptide," or "pesticidal protein" mean a polypeptide that is capable of killing the pest or inhibiting its growth, feeding or reproduction. One of skill in the art understands that not all substances or mixtures thereof are equally effective against all pests. "Chimeric pesticidal polypeptides" or "chimeric pesticidal proteins" are "pesticidal polypeptides" or "pesticidal proteins", to which a solubility-enhancing polypeptide has been fused as disclosed herein. Of particular interest herein are pesticidal polypeptides and chimeric pesticidal polypeptides that act as insecticides and thus have biological activity against insect pests.

[0184] As used herein, "pest" means an organism that interferes with or is harmful to plant development and/or growth. Examples of pests include, but are not limited to, algae, arachnids (e.g., acarids including mites and ticks), bacteria (e.g., plant pathogens including Xanthomonas spp. and Pseudomonas spp.), crustaceans (e.g., pillbugs and sowbugs); fungi (e.g., members in the phylum Ascomycetes or Basidiomycetes, and fungal-like organisms including Oomycetes such as Pythium spp. and Phytophthora spp.), insects, mollusks (e.g., snails and slugs), nematodes (e.g., soil-transmitted nematodes including Clonorchis spp., Fasciola spp., Heterodera spp., Globodera spp., Opisthorchis spp. and Paragonimus spp.), protozoans (e.g., Phytomonas spp.), viruses (e.g., Comovirus spp., Cucumovirus spp., Cytorhabdovirus spp., Luteovirus spp., Nepovirus spp., Potyvirus spp., Tobamovirus spp., Tombusvirus spp. and Tospovirus spp.), viroids, parasitic plants, and weeds.

[0185] Of particular interest herein are insect pests. As used herein, "insect pest" means an organism in the phylum Arthropoda that interferes with or is harmful to plant development and/or growth, and more specifically means an organism in the class Insecta. The class Insecta can be divided into two groups historically treated as subclasses: (1) wingless insects, known as Apterygota; and (2) winged insects, known as Pterygota. Examples of insect pests include, but are not limited to, insects in the orders Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Isoptera, Lepidoptera, Mallophaga orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera and Thysanura, particularly Coleoptera and Lepidoptera. While technically not insects, arthropods such as arachnids, especially in the order Acari, are included in "insect pest." Insect pests include economically important agronomic, forest, greenhouse, nursery ornamentals, food and fiber, public and animal health, domestic and commercial structure, household, and stored product pests.

[0186] Insects of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the family Noctuidae Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. segetum Denis & Schiffermuller (turnip moth); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hubner (cotton leaf worm); Anticarsia gemmatalis Hubner (velvetbean caterpillar); Athetis mindara Barnes and McDunnough (rough skinned cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Egira (Xylomyges) curialis Grote (citrus cutworm); Euxoa messoria Harris (darksided cutworm); Helicoverpa armigera Hubner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Heliothis virescens Fabricius (tobacco budworm); Hypena scabra Fabricius (green cloverworm); Hyponeuma taltula Schaus; (Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Melanchra picta Harris (zebra caterpillar); Mocis latipes Guenee (small mocis moth); Pseudaletia unipuncta Haworth (armyworm); Pseudoplusia includens Walker (soybean looper); Richia albicosta Smith (Western bean cutworm); Spodoptera frugiperda J E Smith (fall armyworm); S. exigua Hubner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Trichoplusia ni Hubner (cabbage looper); borers, casebearers, webworms, coneworms, and skeletonizers from the families Pyralidae and Crambidae such as Achroia grisella Fabricius (lesser wax moth); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo partellus Swinhoe (spotted stalk borer); C. suppressalis Walker (striped stem/rice borer); C. terrenellus Pagenstecher (sugarcane stemp borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia funeralis Hubner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea flavipennella Box; D. grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (sugarcane borer); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hubner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Hedylepta accepta Butler (sugarcane leafroller); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Loxostege sticticalis Linnaeus (beet webworm); Maruca testulalis Geyer (bean pod borer); Orthaga thyrisalis Walker (tea tree web moth); Ostrinia nubilalis Hubner (European corn borer); Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Adoxophyes orana Fischer von Rosslerstamm (summer fruit tortrix moth); Archips spp. including A. argyrospila Walker (fruit tree leaf roller) and A. rosana Linnaeus (European leaf roller); Argyrotaenia spp.; Bonagota salubricola Meyrick (Brazilian apple leafroller); Choristoneura spp.; Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (codling moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hubner (vine moth); Grapholita molesta Busck (oriental fruit moth); Lobesia botrana Denis & Schiffermuller (European grape vine moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Spilonota ocellana Denis & Schiffermuller (eyespotted bud moth); and Suleima helianthana Riley (sunflower bud moth).

[0187] Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese Oak Silkmoth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Collas eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden looper); Erechthias flavistriata Walsingham (sugarcane bud moth); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Heliothis subflexa Guenee; Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Malacosoma spp.; Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Orgyia spp.; Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper); Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Telchin licus Drury (giant sugarcane borer); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer) and Yponomeuta padella Linnaeus (ermine moth).

[0188] Of interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae, and Curculionidae including, but not limited to: Anthonomus grandis Boheman (boll weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (Diaprepes root weevil); Hypera punctata Fabricius (clover leaf weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian cane weevil); M. hemipterus sericeus Olivier (silky cane weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug); S. livis Vaurie (sugarcane weevil); Rhabdoscelus obscurus Boisduval (New Guinea sugarcane weevil); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae including, but not limited to: Chaetocnema ectypa Horn (desert corn flea beetle); C. pulicaria Melsheimer (corn flea beetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberi Smith & Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn rootworm); D. virgifera virgifera LeConte (western corn rootworm); Leptinotarsa decemlineata Say (Colorado potato beetle); Oulema melanopus Linnaeus (cereal leaf beetle); Phyllotreta cruciferae Goeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family Coccinellidae including, but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle); chafers and other beetles from the family Scarabaeidae including, but not limited to: Antitrogus parvulus Britton (Childers cane grub); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Dermolepida albohirtum Waterhouse (Greyback cane beetle); Euetheola humilis rugiceps LeConte (sugarcane beetle); Lepidiota frenchi Blackburn (French's cane grub); Tomarus gibbosus De Geer (carrot beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga crinita Burmeister (white grub); P. latifrons LeConte (June beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European chafer); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp. including M. communis Gyllenhal (wireworm); Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae such as, but not limited to, Migdolus fryanus Westwood (longhorn beetle); and beetles from the Buprestidae family including, but not limited to, Aphanisticus cochinchinae seminulum Obenberger (leaf-mining buprestid beetle).

[0189] Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicomis Loew (corn blotch leafminer); midges including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Neolasioptera murtfeldtiana Felt, (sunflower seed midge); Sitodiplosis mosellana Gehin (wheat midge); fruit flies (Tephritidae), Oscinella frit Linnaeus (frit flies); maggots including, but not limited to: Delia spp. including Delia platura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly); Fannia canicularis Linnaeus, F. femoralis Stein (lesser house flies); Meromyza americana Fitch (wheat stem maggot); Musca domestica Linnaeus (house flies); Stomoxys calcitrans Linnaeus (stable flies)); face flies, horn flies, blow flies, Chrysomya spp.; Phormia spp.; and other muscoid fly pests, horse flies Tabanus spp.; bot flies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer flies Chrysops spp.; Melophagus ovinus Linnaeus (keds); and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; biting midges, sand flies, sciarids, and other Nematocera.

[0190] Included as insects of interest are those of the order Hemiptera such as, but not limited to, the following families: Adelgidae, Aleyrodidae, Aphididae, Asterolecaniidae, Cercopidae, Cicadellidae, Cicadidae, Cixiidae, Coccidae, Coreidae, Dactylopiidae, Delphacidae, Diaspididae, Eriococcidae, Flatidae, Fulgoridae, Issidae, Lygaeidae, Margarodidae, Membracidae, Miridae ortheziidae, Pentatomidae, Phoenicococcidae, Phylloxeridae, Pseudococcidae, Psyllidae, Pyrrhocoridae and Tingidae.

[0191] Agronomically important members from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Acyrthisiphon pisum Harris (pea aphid); Adelges spp. (adelgids); Adelphocoris rapidus Say (rapid plant bug); Anasa tristis De Geer (squash bug); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacaspis tegalensis Zehntner (sugarcane scale); Aulacorthum solani Kaltenbach (foxglove aphid); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Blissus leucopterus leucopterus Say (chinch bug); Blostomatidae spp.; Brevicoryne brassicae Linnaeus (cabbage aphid); Cacopsylla pyricola Foerster (pear psylla); Calocoris norvegicus Gmelin (potato capsid bug); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Cimicidae spp.; Coreidae spp.; Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); C. notatus Distant (suckfly); Deois flavopicta Stl (spittlebug); Dialeurodes citri Ashmead (citrus whitefly); Diaphnocoris chlorionis Say (honeylocust plant bug); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid); Duplachionaspis divergens Green (armored scale); Dysaphis plantaginea Paaserini (rosy apple aphid); Dysdercus suturellus Herrich-Schaffer (cotton stainer); Dysmicoccus boninsis Kuwana (gray sugarcane mealybug); Empoasca fabae Harris (potato leafhopper); Eriosoma lanigerum Hausmann (woolly apple aphid); Erythroneoura spp. (grape leafhoppers); Eumetopina flavipes Muir (Island sugarcane planthopper); Eurygaster spp.; Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); and Hyalopterus pruni Geoffroy (mealy plum aphid); Icerya purchasi Maskell (cottony cushion scale); Labopidicola allii Knight (onion plant bug); Laodelphax striatellus Fallen (smaller brown planthopper); Leptoglossus corculus Say (leaf-footed pine seed bug); Leptodictya tabida Herrich-Schaeffer (sugarcane lace bug); Lipaphis erysimi Kaltenbach (turnip aphid); Lygocoris pabulinus Linnaeus (common green capsid); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Macrosiphum euphorbiae Thomas (potato aphid); Macrosteles quadrilineatus Forbes (aster leafhopper); Magicicada septendecim Linnaeus (periodical cicada); Mahanarva fimbriolata Stl (sugarcane spittlebug); M. posticata Stl (little cicada of sugarcane); Melanaphis sacchari Zehntner (sugarcane aphid); Melanaspis glomerata Green (black scale); Metopolophium dirhodum Walker (rose grain aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stl (rice leafhopper); Nezara viridula Linnaeus (southern green stink bug); Nilaparvata lugens Stl (brown planthopper); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Orthops campestris Linnaeus; Pemphigus spp. (root aphids and gall aphids); Peregrinus maidis Ashmead (corn planthopper); Perkinsiella saccharicida Kirkaldy (sugarcane delphacid); Phylloxera devastatrix Pergande (pecan phylloxera); Planococcus citri Risso (citrus mealybug); Plesiocoris rugicollis Fallen (apple capsid); Poecilocapsus lineatus Fabricius (four-lined plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Pseudococcus spp. (other mealybug complex); Pulvinaria elongata Newstead (cottony grass scale); Pyrilla perpusilla Walker (sugarcane leafhopper); Pyrrhocoridae spp.; Quadraspidiotus perniciosus Comstock (San Jose scale); Reduviidae spp.; Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Saccharicoccus sacchari Cockerell (pink sugarcane mealybug); Scaptacoris castanea Perty (brown root stink bug); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Sogatella furcifera Horvath (white-backed planthopper); Sogatodes oryzicola Muir (rice delphacid); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Therioaphis maculata Buckton (spotted alfalfa aphid); Tinidae spp.; Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Trioza diospyri Ashmead (persimmon psylla); and Typhlocyba pomaria McAtee (white apple leafhopper).

[0192] Also included are adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (wheat curl mite); Panonychus ulmi Koch (European red mite); Petrobia latens Muller (brown wheat mite); Steneotarsonemus bancrofti Michael (sugarcane stalk mite); spider mites and red mites in the family Tetranychidae, Oligonychus grypus Baker & Pritchard, O. indicus Hirst (sugarcane leaf mite), O. pratensis Banks (Banks grass mite), O. stickneyi McGregor (sugarcane spider mite); Tetranychus urticae Koch (two spotted spider mite); T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite), flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Eriophyidae and other foliar feeding mites and mites important in human and animal health, i.e. dust mites in the family Epidermoptidae, follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deer tick); I. holocyclus Neumann (Australian paralysis tick); Dermacentor variabilis Say (American dog tick); Amblyomma americanum Linnaeus (lone star tick); and scab and itch mites in the families Psoroptidae, Pyemotidae, and Sarcoptidae.

[0193] Insect pests of the order Thysanura are of interest, such as Lepisma saccharina Linnaeus (silverfish); Thermobia domestica Packard (firebrat).

[0194] Additional arthropod pests covered include: spiders in the order Araneae such as Loxosceles reclusa Gertsch & Mulaik (brown recluse spider); and the Latrodectus mactans Fabricius (black widow spider); and centipedes in the order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (house centipede). In addition, insect pests of the order Isoptera are of interest, including those of the termitidae family, such as, but not limited to, Cornitermes cumulans Kollar, Cylindrotermes nordenskioeldi Holmgren and Pseudacanthotermes militaris Hagen (sugarcane termite); as well as those in the Rhinotermitidae family including, but not limited to Heterotermes tenuis Hagen. Insects of the order Thysanoptera are also of interest, including but not limited to thrips, such as Stenchaetothrips minutus van Deventer (sugarcane thrips).

[0195] The insect pests can be adults, larvae or even ova. A preferred developmental stage for testing for pesticidal activity is larvae or other immature form of the insect pest. Methods of rearing insect larvae and performing bioassays are well known in the art. See, e.g., Czapla & Lang (1990) J. Econ. Entomol. 83:2480-2485; Griffith & Smith (1977) J. Aust. Ent. Soc. 16:366; Keiper & Foote (1996) Hydrobiologia 339:137-139; and U.S. Pat. No. 5,351,643. For example, insect pests can be reared in total darkness at about 20.degree. C. to about 30.degree. C. and from about 30% to about 70% relative humidity.

[0196] The novel chimeric pesticidal polypeptides can exhibit improved pesticidal activity when compared to a pesticidal polypeptide lacking a solubility-enhancing polypeptide. As used herein, the term "improved pesticidal activity" refers to a polypeptide that has enhanced pesticidal activity following the presently disclosed methods relative to the activity of the corresponding pesticidal polypeptide lacking a solubility-enhancing polypeptide as made by the methods disclosed herein and/or to a polypeptide that is effective against a broader range of pests, and/or a polypeptide having specificity for a pest that is not susceptible to the toxicity of the polypeptide prior to modification of its sequence using the presently disclosed methods. A finding of improved or enhanced pesticidal activity requires a demonstration of an increase of pesticidal activity of at least 10%, against the pest target or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200% or 300% or greater increase of pesticidal activity relative to the pesticidal activity of the polypeptide prior to the addition of a solubility-enhancing polypeptide, as determined against the same pest.

[0197] For example, an improved pesticidal activity is provided where a wider or narrower range of pests is impacted by the polypeptide relative to the range of pests that is affected by the polypeptide prior to sequence modification. A wider range of impact may be desirable where versatility is desired, while a narrower range of impact may be desirable where, for example, beneficial insects might otherwise be impacted by use or presence of the pesticidal polypeptide. While the invention is not bound by any particular mechanism of action, an improved pesticidal activity may also be provided by changes in one or more characteristics of a polypeptide; for example, the stability or longevity of a polypeptide in an insect gut may be increased relative to the stability or longevity of a corresponding wild-type protein.

[0198] A "subject plant or plant cell" is one in which genetic alteration, such as transformation, has been affected as to a gene of interest or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in phenotype of the subject plant or plant cell.

[0199] A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e. with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

[0200] As noted above, the methods involve generating novel chimeric pesticidal polypeptide sequences and the nucleotide sequences that encode such polypeptides. The novel chimeric pesticidal polypeptide sequences are produced by making a fusion protein comprising an amino acid sequence of a solubility-enhancing polypeptide operably linked to an amino acid sequence of a pesticidal protein, particularly a cry protein.

[0201] Any pesticidal protein can be used in the presently disclosed methods. In some embodiments, the pesticidal protein is a .delta.-endotoxin of Bacillus spp. The specific activity of .delta.-endotoxins is considered highly beneficial. Unlike most insecticides, the .delta.-endotoxins do not have a broad spectrum of activity, so they typically do not kill beneficial insects. Furthermore, the .delta.-endotoxins are non-toxic to mammals, including humans, domesticated animals, and wildlife. In particular embodiments, the .delta.-endotoxin is a Cry protein.

[0202] It is well known that naturally occurring .delta.-endotoxins are synthesized by B. thuringiensis sporulating cells as a proteinaceous crystalline inclusion protoxin. Upon being ingested by susceptible insect larvae, the microcrystals dissolve in the midgut, and the protoxin is transformed into a biologically active moiety by proteases characteristic of digestive enzymes located in the insect gut. The activated .delta.-endotoxin binds with high affinity to protein receptors on brush-border membrane vesicles. The epithelial cells lining the midgut are the primary target of the endotoxin and are rapidly destroyed as a consequence of membrane perforation resulting from the formation of gated, cation-selective channels by the toxin. Both Cry and Cyt toxins are pore-forming toxins. The .alpha.-helix regions of the Cry toxins form the trans-membrane pore, whereas Cyt toxins insert into the membrane by forming a .beta.-barrel comprised of .beta.-sheets from each monomer.

[0203] Bt Cry proteins have five conserved sequence domains, and three conserved structural domains (see, e.g., de Maagd et al. (2001) Trends Genetics 17:193-199). The most amino-terminal conserved structural domain (Domain I) consists of seven alpha helices, with a central hydrophobic helix-.alpha.5 encircled by six other amphipathic helices, and is involved in membrane insertion and pore formation. The second conserved structural domain (Domain II) consists of three antiparallel beta-sheets implicated in cell binding, and the most carboxy-terminal conserved structural domain (Domain III) consists of a beta-sandwich. Exposed regions in domains II and III are involved in receptor recognition and binding, and are therefore considered determinants of toxin specificity. The location and properties of these domains are known to those of skill in the art. See, for example, Grochulski et al. (1995) J Mol Biol 254:447-464; Morse, Yamamoto, and Stroud (2001) Structure 9:409-417; Li et al. (1991) Nature 353:815-821; Galitsky et al. (2001) Acta Cryst D57:1101-1109; Boonserm et al. (2006) J Bacteriol 188:3391-3401; Boonserm et al. (2005) J Mol Biol 348:363-382; and Guo et al. (2009) J Struct Biol 168:259-266.

[0204] Bt Cyt proteins have a single .alpha.-.beta. domain comprising two outer layers of .alpha.-helix hairpins wrapped around a .beta.-sheet (Li, Koni, and Ellar (1996) J Mol Biol 257:129-152; and Cohen et al. (2008) J Mol Biol 380:820-827). The .beta.-sheet is involved in membrane insertion.

[0205] A list of some known .delta.-endotoxins (Cry and Cyt endotoxins) and their GenBank Accession Nos. are listed in Table 2, which can be used as a source for nucleic and amino acid sequences for use in the methods disclosed herein.

TABLE-US-00002 TABLE 2 Endotoxin Accession # Endotoxin Accession # Cry1Aa1 AAA22353 Cry1Aa2 AAA22552 Cry1Aa3 BAA00257 Cry1Aa4 CAA31886 Cry1Aa5 BAA04468 Cry1Aa6 AAA86265 Cry1Aa7 AAD46139 Cry1Aa8 I26149 Cry1Aa9 BAA77213 Cry1Aa10 AAD55382 Cry1Aa11 CAA70856 Cry1Aa12 AAP80146 Cry1Aa13 AAM44305 Cry1Aa14 AAP40639 Cry1Aa15 AAY66993 Cry1Aa16 HQ439776 Cry1Aa17 HQ439788 Cry1Aa18 HQ439790 Cry1Aa19 HQ685121 Cry1Aa20 JF340156 Cry1Aa21 JN651496 Cry1Aa22 KC158223 Cry1Ab1 AAA22330 Cry1Ab2 AAA22613 Cry1Ab3 AAA22561 Cry1Ab4 BAA00071 Cry1Ab5 CAA28405 Cry1Ab6 AAA22420 Cry1Ab7 CAA31620 Cry1Ab8 AAA22551 Cry1Ab9 CAA38701 Cry1Ab10 A29125 Cry1Ab11 I12419 Cry1Ab12 AAC64003 Cry1Ab13 AAN76494 Cry1Ab14 AAG16877 Cry1Ab15 AAO13302 Cry1Ab16 AAK55546 Cry1Ab17 AAT46415 Cry1Ab18 AAQ88259 Cry1Ab19 AAW31761 Cry1Ab20 ABB72460 Cry1Ab21 ABS18384 Cry1Ab22 ABW87320 Cry1Ab23 HQ439777 Cry1Ab24 HQ439778 Cry1Ab25 HQ685122 Cry1Ab26 HQ847729 Cry1Ab27 JN135249 Cry1Ab28 JN135250 Cry1Ab29 JN135251 Cry1Ab30 JN135252 Cry1Ab31 JN135253 Cry1Ab32 JN135254 Cry1Ab33 AAS93798 Cry1Ab34 KC156668 Cry1Ab-like AAK14336 Cry1Ab-like AAK14337 Cry1Ab-like AAK14338 Cry1Ab-like ABG88858 Cry1Ac1 AAA22331 Cry1Ac2 AAA22338 Cry1Ac3 CAA38098 Cry1Ac4 Cry1Ac4 Cry1Ac5 AAA22339 Cry1Ac6 AAA86266 Cry1Ac7 AAB46989 Cry1Ac8 AAC44841 Cry1Ac9 AAB49768 Cry1Ac10 CAA05505 Cry1Ac11 CAA10270 Cry1Ac12 I12418 Cry1Ac13 AAD38701 Cry1Ac14 AAQ06607 Cry1Ac15 AAN07788 Cry1Ac16 AAU87037 Cry1Ac17 AAX18704 Cry1Ac18 AAY88347 Cry1Ac19 ABD37053 Cry1Ac20 ABB89046 Cry1Ac21 AAY66992 Cry1Ac22 ABZ01836 Cry1Ac23 CAQ30431 Cry1Ac24 ABL01535 Cry1Ac25 FJ513324 Cry1Ac26 FJ617446 Cry1Ac27 FJ617447 Cry1Ac28 ACM90319 Cry1Ac29 DQ438941 Cry1Ac30 GQ227507 Cry1Ac31 GU446674 Cry1Ac32 HM061081 Cry1Ac33 GQ866913 Cry1Ac34 HQ230364 Cry1Ac35 JF340157 Cry1Ac36 JN387137 Cry1Ac37 JQ317685 Cry1Ad1 AAA22340 Cry1Ad2 CAA01880 Cry1Ae1 AAA22410 Cry1Af1 AAB82749 Cry1Ag1 AAD46137 Cry1Ah1 AAQ14326 Cry1Ah2 ABB76664 Cry1Ah3 HQ439779 Cry1Ai1 AAO39719 Cry1Ai2 HQ439780 Cry1A-like AAK14339 Cry1Ba1 CAA29898 Cry1Ba2 CAA65003 Cry1Ba3 AAK63251 Cry1Ba4 AAK51084 Cry1Ba5 AB020894 Cry1Ba6 ABL60921 Cry1Ba7 HQ439781 Cry1Bb1 AAA22344 Cry1Bb2 HQ439782 Cry1Bc1 CAA86568 Cry1Bd1 AAD10292 Cry1Bd2 AAM93496 Cry1Be1 AAC32850 Cry1Be2 AAQ52387 Cry1Be3 ACV96720 Cry1Be4 HM070026 Cry1Bf1 CAC50778 Cry1Bf2 AAQ52380 Cry1Bg1 AAO39720 Cry1Bh1 HQ589331 Cry1Bi1 KC156700 Cry1Ca1 CAA30396 Cry1Ca2 CAA31951 Cry1Ca3 AAA22343 Cry1Ca4 CAA01886 Cry1Ca5 CAA65457 Cry1Ca6 AAF37224 Cry1Ca7 AAG50438 Cry1Ca8 AAM00264 Cry1Ca9 AAL79362 Cry1Ca10 AAN16462 Cry1Ca11 AAX53094 Cry1Ca12 HM070027 Cry1Ca13 HQ412621 Cry1Ca14 JN651493 Cry1Cb1 M97880 Cry1Cb2 AAG35409 Cry1Cb3 ACD50894 Cry1Cb-like AAX63901 Cry1Da1 CAA38099 Cry1Da2 I76415 Cry1Da3 HQ439784 Cry1Db1 CAA80234 Cry1Db2 AAK48937 Cry1Dc1 ABK35074 Cry1Ea1 CAA37933 Cry1Ea2 CAA39609 Cry1Ea3 AAA22345 Cry1Ea4 AAD04732 Cry1Ea5 A15535 Cry1Ea6 AAL50330 Cry1Ea7 AAW72936 Cry1Ea8 ABX11258 Cry1Ea9 HQ439785 Cry1Ea10 ADR00398 Cry1Ea11 JQ652456 Cry1Eb1 AAA22346 Cry1Fa1 AAA22348 Cry1Fa2 AAA22347 Cry1Fa3 HM070028 Cry1Fa4 HM439638 Cry1Fb1 CAA80235 Cry1Fb2 BAA25298 Cry1Fb3 AAF21767 Cry1Fb4 AAC10641 Cry1Fb5 AAO13295 Cry1Fb6 ACD50892 Cry1Fb7 ACD50893 Cry1Ga1 CAA80233 Cry1Ga2 CAA70506 Cry1Gb1 AAD10291 Cry1Gb2 AAO13756 Cry1Gc1 AAQ52381 Cry1Ha1 CAA80236 Cry1Hb1 AAA79694 Cry1Hb2 HQ439786 Cry1H-like AAF01213 Cry1Ia1 CAA44633 Cry1Ia2 AAA22354 Cry1Ia3 AAC36999 Cry1Ia4 AAB00958 Cry1Ia5 CAA70124 Cry1Ia6 AAC26910 Cry1Ia7 AAM73516 Cry1Ia8 AAK66742 Cry1Ia9 AAQ08616 Cry1Ia10 AAP86782 Cry1Ia11 CAC85964 Cry1Ia12 AAV53390 Cry1Ia13 ABF83202 Cry1Ia14 ACG63871 Cry1Ia15 FJ617445 Cry1Ia16 FJ617448 Cry1Ia17 GU989199 Cry1Ia18 ADK23801 Cry1Ia19 HQ439787 Cry1Ia20 JQ228426 Cry1Ia21 JQ228424 Cry1Ia22 JQ228427 Cry1Ia23 JQ228428 Cry1Ia24 JQ228429 Cry1Ia25 JQ228430 Cry1Ia26 JQ228431 Cry1Ia27 JQ228432 Cry1Ia28 JQ228433 Cry1Ia29 JQ228434 Cry1Ia30 JQ317686 Cry1Ia31 JX944038 Cry1Ia32 JX944039 Cry1Ia33 JX944040 Cry1Ib1 AAA82114 Cry1Ib2 ABW88019 Cry1Ib3 ACD75515 Cry1Ib4 HM051227 Cry1Ib5 HM070028 Cry1Ib6 ADK38579 Cry1Ib7 JN571740 Cry1Ib8 JN675714 Cry1Ib9 JN675715 Cry1Ib10 JN675716 Cry1Ib11 JQ228423 Cry1Ic1 AAC62933 Cry1Ic2 AAE71691 Cry1Id1 AAD44366 Cry1Id2 JQ228422 Cry1Ie1 AAG43526 Cry1Ie2 HM439636 Cry1Ie3 KC156647 Cry1Ie4 KC156681 Cry1If1 AAQ52382 Cry1Ig1 KC156701 Cry1I-like AAC31094 Cry1I-like ABG88859 Cry1Ja1 AAA22341 Cry1Ja2 HM070030 Cry1Ja3 JQ228425 Cry1Jb1 AAA98959 Cry1Jc1 AAC31092 Cry1Jc2 AAQ52372 Cry1Jd1 CAC50779 Cry1Ka1 AAB00376 Cry1Ka2 HQ439783 Cry1La1 AAS60191 Cry1La2 HM070031 Cry1Ma1 FJ884067 Cry1Ma2 KC156659 Cry1Na1 KC156648 Cry1Nb1 KC156678 Cry1-like AAC31091 Cry2Aa1 AAA22335 Cry2Aa2 AAA83516 Cry2Aa3 D86064 Cry2Aa4 AAC04867 Cry2Aa5 CAA10671 Cry2Aa6 CAA10672 Cry2Aa7 CAA10670 Cry2Aa8 AA013734 Cry2Aa9 AAO13750 Cry2Aa10 AAQ04263 Cry2Aa11 AAQ52384 Cry2Aa12 ABI83671 Cry2Aa13 ABL01536 Cry2Aa14 ACF04939 Cry2Aa15 JN426947 Cry2Ab1 AAA22342 Cry2Ab2 CAA39075 Cry2Ab3 AAG36762 Cry2Ab4 AAO13296 Cry2Ab5 AAQ04609 Cry2Ab6 AAP59457 Cry2Ab7 AAZ66347 Cry2Ab8 ABC95996 Cry2Ab9 ABC74968 Cry2Ab10 EF157306 Cry2Ab11 CAM84575 Cry2Ab12 ABM21764 Cry2Ab13 ACG76120 Cry2Ab14 ACG76121 Cry2Ab15 HM037126 Cry2Ab16 GQ866914 Cry2Ab17 HQ439789 Cry2Ab18 JN135255 Cry2Ab19 JN135256 Cry2Ab20 JN135257 Cry2Ab21 JN135258 Cry2Ab22 JN135259 Cry2Ab23 JN135260 Cry2Ab24 JN135261 Cry2Ab25 JN415485 Cry2Ab26 JN426946 Cry2Ab27 JN415764 Cry2Ab28 JN651494 Cry2Ac1 CAA40536 Cry2Ac2 AAG35410 Cry2Ac3 AAQ52385 Cry2Ac4 ABC95997 Cry2Ac5 ABC74969 Cry2Ac6 ABC74793 Cry2Ac7 CAL18690 Cry2Ac8 CAM09325 Cry2Ac9 CAM09326 Cry2Ac10 ABN15104 Cry2Ac11 CAM83895 Cry2Ac12 CAM83896 Cry2Ad1 AAF09583 Cry2Ad2 ABC86927 Cry2Ad3 CAK29504 Cry2Ad4 CAM32331 Cry2Ad5 CAO78739 Cry2Ae1 AAQ52362 Cry2Af1 ABO30519 Cry2Af2 GQ866915 Cry2Ag1 ACH91610 Cry2Ah1 EU939453 Cry2Ah2 ACL80665 Cry2Ah3 GU073380 Cry2Ah4 KC156702 Cry2Ai1 FJ788388 Cry2Aj Cry2Ak1 KC156660 Cry2Ba1 KC156658 Cry3Aa1 AAA22336 Cry3Aa2 AAA22541 Cry3Aa3 CAA68482 Cry3Aa4 AAA22542 Cry3Aa5 AAA50255 Cry3Aa6 AAC43266 Cry3Aa7 CAB41411 Cry3Aa8 AAS79487 Cry3Aa9 AAW05659 Cry3Aa10 AAU29411 Cry3Aa11 AAW82872 Cry3Aa12 ABY49136 Cry3Ba1 CAA34983 Cry3Ba2 CAA00645 Cry3Ba3 JQ397327 Cry3Bb1 AAA22334 Cry3Bb2 AAA74198 Cry3Bb3 I15475 Cry3Ca1 CAA42469 Cry4Aa1 CAA68485 Cry4Aa2 BAA00179 Cry4Aa3 CAD30148 Cry4Aa4 AFB18317 Cry4A-like AAY96321 Cry4Ba1 CAA30312 Cry4Ba2 CAA30114 Cry4Ba3 AAA22337 Cry4Ba4 BAA00178 Cry4Ba5 CAD30095 Cry4Ba-like ABC47686 Cry4Ca1 EU646202 Cry4Cb1 FJ403208 Cry4Cb2 FJ597622 Cry4Cc1 FJ403207 Cry5Aa1 AAA67694 Cry5Ab1 AAA67693 Cry5Ac1 I34543 Cry5Ad1 ABQ82087 Cry5Ba1 AAA68598 Cry5Ba2 ABW88931 Cry5Ba3 AFJ04417 Cry5Ca1 HM461869 Cry5Ca2 ZP_04123426 Cry5Da1 HM461870 Cry5Da2 ZP_04123980 Cry5Ea1 HM485580 Cry5Ea2 ZP_04124038 Cry6Aa1 AAA22357 Cry6Aa2 AAM46849 Cry6Aa3 ABH03377 Cry6Ba1 AAA22358 Cry7Aa1 AAA22351 Cry7Ab1 AAA21120 Cry7Ab2 AAA21121 Cry7Ab3 ABX24522 Cry7Ab4 EU380678 Cry7Ab5 ABX79555 Cry7Ab6 AC144005 Cry7Ab7 ADB89216 Cry7Ab8 GU145299 Cry7Ab9 ADD92572 Cry7Ba1 ABB70817 Cry7Bb1 KC156653 Cry7Ca1 ABR67863 Cry7Cb1 KC156698 Cry7Da1 ACQ99547 Cry7Da2 HM572236 Cry7Da3 KC156679 Cry7Ea1 HM035086 Cry7Ea2 HM132124 Cry7Ea3 EEM19403 Cry7Fa1 HM035088 Cry7Fa2 EEM19090 Cry7Fb1 HM572235 Cry7Fb2 KC156682 Cry7Ga1 HM572237 Cry7Ga2 KC156669 Cry7Gb1 KC156650 Cry7Gc1 KC156654 Cry7Gd1 KC156697 Cry7Ha1 KC156651 Cry71a1 KC156665 Cry7Ja1 KC156671 Cry7Ka1 KC156680 Cry7Kb1 BAM99306 Cry7La1 BAM99307 Cry8Aa1 AAA21117 Cry8Ab1 EU044830 Cry8Ac1 KC156662 Cry8Ad1 KC156684 Cry8Ba1 AAA21118 Cry8Bb1 CAD57542 Cry8Bc1 CAD57543 Cry8Ca1 AAA21119 Cry8Ca2 AAR98783 Cry8Ca3 EU625349 Cry8Ca4 ADB54826 Cry8Da1 BAC07226 Cry8Da2 BD133574 Cry8Da3 BD133575 Cry8Db1 BAF93483 Cry8Ea1 AAQ73470 Cry8Ea2 EU047597 Cry8Ea3 KC855216 Cry8Fa1 AAT48690 Cry8Fa2 HQ174208 Cry8Fa3 AFH78109 Cry8Ga1 AAT46073 Cry8Ga2 ABC42043 Cry8Ga3 FJ198072 Cry8Ha1 AAW81032 Cry81a1 EU381044 Cry81a2 GU073381 Cry81a3 HM044664 Cry81a4 KC156674 Cry81b1 GU325772 Cry81b2 KC156677 Cry8Ja1 EU625348 Cry8Ka1 FJ422558 Cry8Ka2 ACN87262 Cry8Kb1 HM123758 Cry8Kb2 KC156675 Cry8La1 GU325771 Cry8Ma1 HM044665 Cry8Ma2 EEM86551 Cry8Ma3 HM210574 Cry8Na1 HM640939 Cry8Pa1 HQ388415 Cry8Qa1 HQ441166 Cry8Qa2 KC152468 Cry8Ra1 AFP87548 Cry8Sa1 JQ740599 Cry8Ta1 KC156673 Cry8-like FJ770571 Cry8-like ABS53003 Cry9Aa1 CAA41122 Cry9Aa2 CAA41425 Cry9Aa3 GQ249293 Cry9Aa4 GQ249294 Cry9Aa5 JX174110 Cry9Aa like AAQ52376 Cry9Ba1 CAA52927 Cry9Ba2 GU299522 Cry9Bb1 AAV28716 Cry9Ca1 CAA85764 Cry9Ca2 AAQ52375 Cry9Da1 BAA19948 Cry9Da2 AAB97923 Cry9Da3 GQ249293 Cry9Da4 GQ249297 Cry9Db1 AAX78439 Cry9Dc1 KC156683 Cry9Ea1 BAA34908 Cry9Ea2 AAO12908 Cry9Ea3 ABM21765 Cry9Ea4 ACE88267 Cry9Ea5 ACF04743 Cry9Ea6 ACG63872 Cry9Ea7 FJ380927 Cry9Ea8 GQ249292 Cry9Ea9 JN651495 Cry9Eb1 CAC50780 Cry9Eb2 GQ249298

Cry9Eb3 KC156646 Cry9Ec1 AAC63366 Cry9Ed1 AAX78440 Cry9Ee1 GQ249296 Cry9Ee2 KC156664 Cry9Fa1 KC156692 Cry9Ga1 KC156699 Cry9-like AAC63366 Cry10Aa1 AAA22614 Cry10Aa2 E00614 Cry10Aa3 CAD30098 Cry10Aa4 AFB18318 Cry10A-like DQ167578 Cry11Aa1 AAA22352 Cry11Aa2 AAA22611 Cry11Aa3 CAD30081 Cry11Aa4 AFB18319 Cry11Aa-like DQ166531 Cry11Ba1 CAA60504 Cry11Bb1 AAC97162 Cry11Bb2 HM068615 Cry12Aa1 AAA22355 Cry13Aa1 AAA22356 Cry14Aa1 AAA21516 Cry14Ab1 KC156652 Cry15Aa1 AAA22333 Cry16Aa1 CAA63860 Cry17Aa1 CAA67841 Cry18Aa1 CAA67506 Cry18Ba1 AAF89667 Cry18Ca1 AAF89668 Cry19Aa1 CAA68875 Cry19Ba1 BAA32397 Cry19Ca1 AFM37572 Cry20Aa1 AAB93476 Cry20Ba1 ACS93601 Cry20Ba2 KC156694 Cry20-like GQ144333 Cry21Aa1 I32932 Cry21Aa2 I66477 Cry21Ba1 BAC06484 Cry21Ca1 JF521577 Cry21Ca2 KC156687 Cry21Da1 JF521578 Cry22Aa1 I34547 Cry22Aa2 CAD43579 Cry22Aa3 ACD93211 Cry22Ab1 AAK50456 Cry22Ab2 CAD43577 Cry22Ba1 CAD43578 Cry22Bb1 KC156672 Cry23Aa1 AAF76375 Cry24Aa1 AAC61891 Cry24Ba1 BAD32657 Cry24Ca1 CAJ43600 Cry25Aa1 AAC61892 Cry26Aa1 AAD25075 Cry27Aa1 BAA82796 Cry28Aa1 AAD24189 Cry28Aa2 AAG00235 Cry29Aa1 CAC80985 Cry30Aa1 CAC80986 Cry30Ba1 BAD00052 Cry30Ca1 BAD67157 Cry30Ca2 ACU24781 Cry30Da1 EF095955 Cry30Db1 BAE80088 Cry30Ea1 ACC95445 Cry30Ea2 FJ499389 Cry30Fa1 ACI22625 Cry30Ga1 ACG60020 Cry30Ga2 HQ638217 Cry31Aa1 BAB11757 Cry31Aa2 AAL87458 Cry31Aa3 BAE79808 Cry31Aa4 BAF32571 Cry31Aa5 BAF32572 Cry31Aa6 BAI44026 Cry31Ab1 BAE79809 Cry31Ab2 BAF32570 Cry31Ac1 BAF34368 Cry31Ac2 AB731600 Cry31Ad1 BAI44022 Cry32Aa1 AAG36711 Cry32Aa2 GU063849 Cry32Ab1 GU063850 Cry32Ba1 BAB78601 Cry32Ca1 BAB78602 Cry32Cb1 KC156708 Cry32Da1 BAB78603 Cry32Ea1 GU324274 Cry32Ea2 KC156686 Cry32Eb1 KC156663 Cry32Fa1 KC156656 Cry32Ga1 KC156657 Cry32Ha1 KC156661 Cry32Hb1 KC156666 Cry32Ia1 KC156667 Cry32Ja1 KC156685 Cry32Ka1 KC156688 Cry32La1 KC156689 Cry32Ma1 KC156690 Cry32Mb1 KC156704 Cry32Na1 KC156691 Cry32Oa1 KC156703 Cry32Pa1 KC156705 Cry32Qa1 KC156706 Cry32Ra1 KC156707 Cry32Sa1 KC156709 Cry32Ta1 KC156710 Cry32Ua1 KC156655 Cry33Aa1 AAL26871 Cry34Aa1 AAG50341 Cry34Aa2 AAK64560 Cry34Aa3 AAT29032 Cry34Aa4 AAT29030 Cry34Ab1 AAG41671 Cry34Ac1 AAG50118 Cry34Ac2 AAK64562 Cry34Ac3 AAT29029 Cry34Ba1 AAK64565 Cry34Ba2 AAT29033 Cry34Ba3 AAT29031 Cry35Aa1 AAG50342 Cry35Aa2 AAK64561 Cry35Aa3 AAT29028 Cry35Aa4 AAT29025 Cry35Ab1 AAG41672 Cry35Ab2 AAK64563 Cry35Ab3 AY536891 Cry35Ac1 AAG50117 Cry35Ba1 AAK64566 Cry35Ba2 AAT29027 Cry35Ba3 AAT29026 Cry36Aa1 AAK64558 Cry37Aa1 AAF76376 Cry38Aa1 AAK64559 Cry39Aa1 BAB72016 Cry40Aa1 BAB72018 Cry40Ba1 BAC77648 Cry40Ca1 EU381045 Cry40Da1 ACF15199 Cry41Aa1 BAD35157 Cry41Ab1 BAD35163 Cry41Ba1 HM461871 Cry41Ba2 ZP_04099652 Cry42Aa1 BAD35166 Cry43Aa1 BAD15301 Cry43Aa2 BAD95474 Cry43Ba1 BAD15303 Cry43Ca1 KC156676 Cry43Cb1 KC156695 Cry43Cc1 KC156696 Cry43-like BAD15305 Cry44Aa Cry44Aa Cry45Aa BAD22577 Cry46Aa BAC79010 Cry46Aa2 BAG68906 Cry46Ab BAD35170 Cry47Aa AAY24695 Cry48Aa CAJ18351 Cry48Aa2 CAJ86545 Cry48Aa3 CAJ86546 Cry48Ab CAJ86548 Cry48Ab2 CAJ86549 Cry49Aa CAH56541 Cry49Aa2 CAJ86541 Cry49Aa3 CAJ86543 Cry49Aa4 CAJ86544 Cry49Ab1 CAJ86542 Cry50Aa1 BAE86999 Cry50Ba1 GU446675 Cry50Ba2 GU446676 Cry51Aa1 ABI14444 Cry51Aa2 GU570697 Cry52Aa1 EF613489 Cry52Ba1 FJ361760 Cry53Aa1 EF633476 Cry53Ab1 FJ361759 Cry54Aa1 ACA52194 Cry54Aa2 GQ140349 Cry54Ba1 GU446677 Cry55Aa1 ABW88932 Cry54Ab1 JQ916908 Cry55Aa2 AAE33526 Cry56Aa1 ACU57499 Cry56Aa2 GQ483512 Cry56Aa3 JX025567 Cry57Aa1 ANC87261 Cry58Aa1 ANC87260 Cry59Ba1 JN790647 Cry59Aa1 ACR43758 Cry60Aa1 ACU24782 Cry60Aa2 EAO57254 Cry60Aa3 EEM99278 Cry60Ba1 GU810818 Cry60Ba2 EAO57253 Cry60Ba3 EEM99279 Cry61Aa1 HM035087 Cry61Aa2 HM132125 Cry61Aa3 EEM19308 Cry62Aa1 HM054509 Cry63Aa1 BAI44028 Cry64Aa1 BAJ05397 Cry65Aa1 HM461868 Cry65Aa2 ZP_04123838 Cry66Aa1 HM485581 Cry66Aa2 ZP_04099945 Cry67Aa1 HM485582 Cry67Aa2 ZP_04148882 Cry68Aa1 HQ113114 Cry69Aa1 HQ401006 Cry69Aa2 JQ821388 Cry69Ab1 JN209957 Cry70Aa1 JN646781 Cry70Ba1 ADO51070 Cry70Bb1 EEL67276 Cry71Aa1 JX025568 Cry72Aa1 JX025569 Cyt1Aa X03182 Cyt1Ab X98793 Cyt1B U37196 Cyt2A Z14147 Cyt2B U52043 * The amino acid and corresponding nucleotide sequences of the accession numbers in Table 2 are herein incorporated by reference.

[0206] Examples of .delta.-endotoxins also include but are not limited to Cry1A proteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of .alpha.-helix 1 and/or .alpha.-helix 2 variants of cry proteins such as Cry1A, Cry3A) of U.S. Pat. Nos. 8,304,604, 8,304,605 and 8,476,226; Cry1B of U.S. patent application Ser. No. 10/525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9F families; a Cry15 protein of Naimov, et al., (2008) Applied and Environmental Microbiology, 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 and cryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006/0191034, 2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US Patent Application Publication Number 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006/033867; TIC853 toxins of U.S. Pat. No. 8,513,494, AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US Patent Application Publication Number 2004/0250311; AXMI-006 of US Patent Application Publication Number 2004/0216186; AXMI-007 of US Patent Application Publication Number 2004/0210965; AXMI-009 of US Patent Application Number 2004/0210964; AXMI-014 of US Patent Application Publication Number 2004/0197917; AXMI-004 of US Patent Application Publication Number 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US Patent Application Publication Number 2011/0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of US Patent Application Publication Number 2011/0263488; AXMI-R1 and related proteins of US Patent Application Publication Number 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO 2011/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent Application Publication Number 2010/0298211; AXMI-066 and AXMI-076 of US Patent Application Publication Number 2009/0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXM1188, AXM1189 of U.S. Pat. No. 8,318,900; AXM1079, AXM1080, AXM1081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXM1102, AXM1103, AXM1104, AXM1107, AXM1108, AXM1109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US Patent Application Publication Number 2010/0005543, cry proteins such as Cry1A and Cry3A having modified proteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011/0064710. Other Cry proteins are well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/which can be accessed on the world-wide web using the "www" prefix). The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database, which can be accessed on the world-wide web using the "www" prefix).

[0207] The primary structure of a pesticidal polypeptide can be used in its native form or modified in order to introduce amino acid residues (either through addition or substitution) that contribute to or are suspected of contributing to the pesticidal activity of at least one pesticidal polypeptide. Functional data and results from structure-function analyses can be consulted to identify sequences and/or amino acid residues that contribute to pesticidal activity that should be retained or introduced into the candidate polypeptide sequence. Those residues that contribute to or are suspected of contributing to pesticidal activity include those residues that enhance efficacy or those that dictate pesticidal specificity, including those residues that narrow or broaden the range of pests of pesticidal proteins.

[0208] For example, the aromaticity of the tyrosine and phenylalanine residues at position 249 and 264, respectively, in helix 7 of the Bt Cry4Ba toxin has been found to be important for toxicity (Tiewsiri and Angsuthanasombat (2007) J Biochem Mol Biol 40:163-171). Thus, in those embodiments wherein the candidate polypeptide is homologous with the Bt Cry4Ba toxin, the residues at the positions corresponding to positions 249 and 264 of Cry4Ba should be maintained if they are aromatic or modified if they are non-aromatic.

[0209] Further, additional mutations can be introduced into pesticidal polypeptide sequence to improve the pesticidal activity. For example, as described in U.S. Pat. Nos. 7,462,760 and 7,105,332 (each of which are herein incorporated by reference in its entirety), mutations can be introduced into the candidate polypeptide sequence to destroy proteolytic sites to protect the polypeptide from degradative digestion, for example, by plant proteases. As a further example, the toxicity of Bt Cry proteins can be improved by introducing at least one more protease-sensitive site (e.g., trypsin cleavage site) into the region located between alpha helices 3 and 4 of domain 1 of the endotoxin protein (see U.S. Patent Application Publication No. US2004/0091505, which is herein incorporated by reference in its entirety). As another non-limiting example, a protease-sensitive site that is readily cleaved by insect chymotrypsin, e.g., a chymotrypsin found in the bertha armyworm or the corn earworm (Hegedus et al. (2003) Arch. Insect Biochem. Physiol. 53: 30-47; and Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212), may be added to the candidate polypeptide sequence to provide improved toxicity to the polypeptide.

[0210] In certain embodiments of the invention, the Cry protein comprises the amino acid sequence set forth in SEQ ID NO: 8, 10, 12 or 14 or fragment or variant thereof. Any nucleotide sequence encoding the amino acid sequence set forth in 8, 10, 12 or 14 or fragment or variant thereof, can be used in the methods and compositions of the present invention including, but not limited to the nucleotides sequences encoding SEQ ID NOS: 8, 10, 12, and 14 which are set forth in SEQ ID NOS: 7, 9, 11, and 13, respectively. In some embodiments of the invention, the nucleotide sequences of the invention will be optimized for expression in a host organism or cell of interest, particularly a plant, more particularly a crop plant, most particularly a maize plant.

[0211] Methods of measuring pesticidal activity by insect bioassays are well known in the art. See, e.g., Brooke et al. (2001) Bull. Entomol. Res. 91:265-272; Chen et al. (2007) Proc. Natl. Acad. Sci. USA 104:13901-13906; Crespo et al. (2008) Appl. Environ. Microb. 74:130-135; Kham bay et al. (2003) Pest Manag. Sci. 59:174-182; Liu & Dean (2006) Protein Eng. Des. Sel. 19:107-111; Marrone et al. (1985) J. Econ. Entomol. 78:290-293; Robertson et al., Pesticide Bioassays with Arthropods (2.sup.nd ed., CRC Press 2007); Scott & McKibben (1976) J. Econ. Entomol. 71:343-344; Strickman (1985) Bull. Environ. Contam. Toxicol. 35:133-142; and Verma et al. (1982) Water Res. 16 525-529; as well as U.S. Pat. No. 6,268,181. Examples of insect bioassays include, but are not limited to, pest mortality, pest weight loss, pest repellency, pest attraction, and other behavioral and physical changes of the pest after feeding and exposure to a pesticide or pesticidal polypeptide for an appropriate length of time. General methods include addition of the pesticide, pesticidal polypeptide or an organism having the pesticidal polypeptide to the diet source in an enclosed container. See, e.g., U.S. Pat. Nos. 6,339,144 and 6,570,005.

[0212] Further, a nucleic acid encoding the novel chimeric pesticidal polypeptide can be derived from the amino acid sequence and can be generated using any method known in the art. Therefore, novel isolated pesticidal polypeptides and isolated nucleic acid molecules encoding the same are provided. An "isolated" or "purified" polynucleotide or protein or biologically active fragment thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active fragment thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0213] Further provided are novel chimeric pesticidal polypeptides comprising fragments and variants of a solubility-enhancing polypeptide and/or pesticidal protein as well as nucleic acid molecules encoding such novel chimeric pesticidal polypeptides. Fragments and variants of the novel chimeric pesticidal polypeptides and chimeric pesticidal polypeptide-encoding nucleic acid molecules are also provided. By "fragment" is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain solubility-enhancing activity and/or pesticidal activity. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode fragment proteins retaining a solubility-enhancing and/or pesticidal activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide encoding the novel chimeric pesticidal polypeptides.

[0214] A fragment of a pesticide-encoding polynucleotide that encodes a biologically active fragment of a solubility-enhancing polypeptide or pesticidal protein will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 or 1,100 contiguous amino acids or up to the total number of amino acids present in a chimeric pesticidal polypeptide, a solubility-enhancing polypeptide or a pesticidal protein of the invention. Fragments of a chimeric pesticidal polypeptide, solubility-enhancing polypeptide or a pesticidal protein that are useful as hybridization probes or PCR primers generally need not encode a biologically active fragment of a chimeric pesticidal polypeptide, a solubility-enhancing polypeptide or a pesticidal protein.

[0215] Thus, a fragment of a chimeric pesticidal polypeptide, a solubility-enhancing polypeptide or a pesticidal protein may encode a biologically active fragment of a pesticidal protein or it may be a fragment that can be used as a hybridization probe or PCR primer using methods well known in the art and disclosed elsewhere herein. A biologically active fragment of a pesticidal protein can be prepared by isolating a portion of one of the pesticide-encoding polynucleotides, expressing the encoded portion of the pesticidal protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the pesticidal protein. Polynucleotides that are fragments of a chimeric pesticidal polypeptide, a solubility-enhancing polypeptide or a pesticidal protein sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500 or 3000 contiguous nucleotides or up to the number of nucleotides present in a full-length pesticide-encoding polynucleotide discovered using the methods disclosed herein.

[0216] "Variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the chimeric pesticidal polypeptide, solubility-enhancing polypeptides or pesticidal proteins of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a chimeric pesticidal polypeptide, a solubility-enhancing polypeptide or a pesticidal protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.

[0217] Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, an isolated polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 2 is disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0218] "Variant" protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, pesticidal activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a chimeric pesticidal polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or even 1 amino acid residue.

[0219] The novel chimeric pesticidal polypeptide made using the presently disclosed methods may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions to the solubility-enhancing polypeptide and/or the pesticidal polypeptide portion of the chimeric pesticidal polypeptide. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the chimeric pesticidal polypeptides, pesticidal polypeptides, and solubility-enhancing polypeptide s can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.

[0220] The deletions, insertions, and substitutions of the novel chimeric pesticidal polypeptide sequences, pesticidal polypeptide sequences and solubility-enhancing polypeptide sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the pesticidal activity can be evaluated using an insect feeding bioassays as described elsewhere herein. Preferably, the biologically activity of a solubility-enhancing polypeptide or fragment or variant thereof is evaluated by operably linking the solubility-enhancing polypeptide, fragment or variant to a pesticidal polypeptide and assaying the pesticidal activity of the resulting chimeric pesticidal polypeptide using the methods disclosed herein or otherwise known in the art. The pesticidal activity of the chimeric pesticidal polypeptide can be compared to the pesticidal activity of the pesticidal peptide (i.e., without the solubility-enhancing polypeptide, fragment or variant) to determine if the solubility-enhancing polypeptide, fragment or variant increases the insecticidal activity the chimeric pesticidal polypeptide. Preferably, in any insect feeding assays in which the chimeric pesticidal polypeptide its corresponding pesticidal polypeptide, the amount of the respective polypeptides that are fed to the insects will be adjusted to take into the account the large molecular weight of the chimeric pesticidal polypeptide such that pesticidal activity comparisons will effectively be made on a per molecule or per mole basis. Furthermore, it is recognized that insecticidal activity of chimeric pesticidal polypeptides with different solubility-enhancing polypeptides or fragments or variants of a solubility-enhancing polypeptide can also evaluated and compared to one another in a like manner and preferably with adjustments for any differences in molecular weights between the chimeric pesticidal polypeptides that are being compared.

[0221] Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different pesticide-coding sequences can be manipulated to create a new pesticidal polypeptide possessing the desired properties and these new pesticidal proteins can be used in the methods disclosed herein to make chimeric pesticidal polypepties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0222] The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", and, (d) "percentage of sequence identity."

[0223] (a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence or the complete cDNA or gene sequence.

[0224] (b) As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

[0225] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0226] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.

[0227] Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0228] GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

[0229] GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0230] (c) As used herein, "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

[0231] (d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0232] In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides, and may be labeled with a detectable group such as .sup.32P or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the babyboom polynucleotide. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0233] For example, the entire pesticide-encoding polynucleotide or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding pesticide-encoding polynucleotides and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among pesticide-encoding polynucleotide sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding pesticide-encoding polynucleotides from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0234] Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optimally less than 500 nucleotides in length.

[0235] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times. SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

[0236] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with .gtoreq.90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T.sub.m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0237] The use of the term "polynucleotide" is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0238] Nucleic acids encoding the novel chimeric pesticidal polypeptides can be used in DNA constructs for expression in various hosts, including plants. Thus, expression cassettes comprising the novel chimeric pesticidal polypeptide-encoding nucleic acids for expression in the organism of interest are provided. The cassette will include 5' and 3' regulatory sequences operably linked to a novel chimeric pesticidal polypeptide-encoding polynucleotide. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. In the case of fusion proteins or fusion polypeptides, an operable linkage between, for example, a first amino amino sequence of interest and a second amino acid of interest results in a single amino acid sequence, which comprises both the first and second amino acid sequences, and wherein the C-terminal amino acid the first amino amino sequence is covalently attached to the N-terminal amino acid of the second amino acid sequence by a peptide bond. Operably linked elements may be contiguous or non-contiguous. The cassette may additionally contain at least one additional polynucleotide of interest to be cotransformed into the organism. Alternatively, the additional polynucleotide(s) of interest can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the pesticide-encoding polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0239] The chimeric pesticidal polypeptides of the present invention comprise an amino acid sequence of a solubility-enhancing polypeptide operably linked to an amino acid sequence of a Cry endotoxin. It is recognized that in such an operable linkage, the amino acid sequence of the solubility-enhancing polypeptide can be contiguous to the amino acid sequence of the Cry endotoxin or can be separated by an intervening linker or other amino acid sequence. Likewise, the chimeric pesticidal polypeptide-encoding nucleic acid molecules of the present invention comprise a nucleotide sequence encoding a solubility-enhancing polypeptide operably linked to a nucleotide sequence encoding a Cry endotoxin. It is further recognized that in such an operable linkage, the nucleotide sequence encoding the solubility-enhancing polypeptide can be contiguous to the nucleotide sequence encoding the Cry endotoxin or can be separated by an intervening linker-encoding nucleotide sequence or other nucleotide sequence.

[0240] The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a novel pesticide-encoding polynucleotide, and a transcriptional and translational termination region (i.e., termination region) functional in the organism to which the expression cassette is introduced. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the pesticide-encoding polynucleotide may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the pesticide-encoding polynucleotide may be heterologous to the host cell or to each other. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus or the promoter is not the native promoter for the operably linked polynucleotide.

[0241] The termination region may be native with the transcriptional initiation region, may be native with the operably linked pesticidal polynucleotide of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous) to the promoter, the pesticidal polynucleotide of interest, the plant host or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0242] Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0243] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0244] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0245] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0246] The chimeric pesticidal polypeptide-encoding nucleic acid molecules, expression cassettes comprising the same or the chimeric pesticidal polypeptides can be introduced into an organism or host cell, particularly a non-human host cell. Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. In some examples, host cells are monocotyledonous or dicotyledonous plant cells.

[0247] The nucleic acid encoding the novel chimeric pesticidal polypeptides can be introduced into microorganisms that multiply on plants (epiphytes) to deliver chimeric pesticidal polypeptides to potential target pests. Alternatively, the chimeric pesticidal polypeptides can be directly introduced into such microorganisms. Epiphytes, for example, can be gram-positive or gram-negative bacteria.

[0248] Root-colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a Bacillus cereus strain that colonizes roots can be isolated from roots of a plant (see, for example, Handelsman et al. (1991) Appl. Environ. Microbiol. 56:713-718). Nucleic acids encoding the pesticidal proteins of the invention or the chimeric pesticidal polypeptides can be introduced into a root-colonizing Bacillus cereus by standard methods known in the art.

[0249] Nucleic acids encoding chimeric pesticidal polypeptides can be introduced, for example, into the root-colonizing Bacillus by means of electrotransformation. Specifically, nucleic acids encoding the pesticidal proteins can be cloned into a shuttle vector, for example, pHT3101 (Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218. The shuttle vector pHT3101 containing the coding sequence for the particular chimeric pesticidal polypeptide-encoding nucleic acid can, for example, be transformed into the root-colonizing Bacillus by means of electroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218).

[0250] Expression systems can be designed so that chimeric pesticidal polypeptides are secreted outside the cytoplasm of gram-negative bacteria, such as E. coli, for example. Advantages of having chimeric pesticidal polypeptides secreted are: (1) avoidance of potential cytotoxic effects of the chimeric pesticidal polypeptides expressed; and (2) improvement in the efficiency of purification of the chimeric pesticidal polypeptides, including, but not limited to, increased efficiency in the recovery and purification of the protein per volume cell broth and decreased time and/or costs of recovery and purification per unit protein.

[0251] Chimeric pesticidal polypeptides can be made to be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino-terminal end of the pesticidal protein. Signal peptides recognized by E. coli can be found in proteins already known to be secreted in E. coli, for example the OmpA protein (Ghrayeb et al. (1984) EMBO J, 3:2437-2442). OmpA is a major protein of the E. coli outer membrane, and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified before processing as may be the case for other signal peptides, for example lipoprotein signal peptide (Duffaud et al. (1987) Meth. Enzymol. 153: 492).

[0252] Chimeric pesticidal polypeptides of the invention can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bacillus thuringiensis strains have been used as insecticidal sprays. In the case of a chimeric pesticidal polypeptide(s) that is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the chimeric pesticidal polypeptide(s) into the growth medium during the fermentation process. The chimeric pesticidal polypeptides are retained within the cell, and the cells are then processed to yield the encapsulated chimeric pesticidal polypeptides. Any suitable microorganism can be used for this purpose. Pseudomonas has been used to express Bacillus thuringiensis endotoxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide (Gaertner et al. (1993), in: Advanced Engineered Pesticides, ed. Kim).

[0253] Alternatively, the chimeric pesticidal polypeptides are produced by introducing a heterologous nucleic acid encoding the chimeric pesticidal polypeptide into a cellular host. Expression of the heterologous nucleic acid results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated chimeric pesticidal polypeptides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.

[0254] The novel chimeric pesticidal polypeptide-encoding polynucleotides and chimeric pesticidal polypeptides can be introduced into plant cells or plants to produce a transgenic plant that is resistant to pests that are susceptible to the chimeric pesticidal polypeptide.

[0255] The novel chimeric pesticidal polypeptide-encoding polynucleotides can be combined with constitutive, tissue-preferred or other promoters for expression in plants. The promoters can be selected based on the desired outcome.

[0256] Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0257] Generally, it will be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, herein incorporated by reference.

[0258] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

[0259] Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter may be used in the constructions of the invention. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like, herein incorporated by reference.

[0260] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

[0261] Tissue-preferred promoters can be utilized to target enhanced chimeric pesticidal polypeptide expression within a particular plant tissue. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

[0262] Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0263] Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a .beta.-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed roIC and roID root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1' gene, fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and roIB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

[0264] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seed-germinating" promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; herein incorporated by reference). Gamma-zein is an endosperm-specific promoter. Globulin 1 (Glb-1) is a representative embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean .beta.-phaseolin, napin, .beta.-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference.

[0265] Where low level expression is desired, weak promoters will be used. Generally, by "weak promoter" is intended a promoter that drives expression of a coding sequence at a low level. By low level is intended at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Alternatively, it is recognized that weak promoters also encompasses promoters that are expressed in only a few cells and not in others to give a total low level of expression. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.

[0266] Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, U.S. Pat. No. 6,177,611, herein incorporated by reference.

[0267] The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as .beta.-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFP.TM. from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Ad. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairn et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.

[0268] The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.

[0269] In one embodiment, the polynucleotide of interest is targeted to the chloroplast for expression. In this manner, where the polynucleotide of interest is not directly inserted into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

[0270] Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem. 270(11):6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem. 272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

[0271] Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0272] The polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotide of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.

[0273] The methods of the invention involve introducing a polypeptide or polynucleotide into a plant or other organism. "Introducing" is intended to mean presenting to the organism the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the organism. The methods of the invention do not depend on a particular method for introducing a sequence into an organism, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the organism. Methods for introducing polynucleotides or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0274] "Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.

[0275] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

[0276] In specific embodiments, the chimeric pesticidal polypeptide-encoding sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the pesticidal protein or variants and fragments thereof directly into the plant or the introduction of the a pesticide-encoding transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the pesticide-encoding polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include a viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine.

[0277] In other embodiments, the novel chimeric pesticidal polypeptide-encoding polynucleotide may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct within a viral DNA or RNA molecule. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.

[0278] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

[0279] As used herein, the term plant also includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.

[0280] The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables ornamentals, and conifers.

[0281] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.

[0282] Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean and sugarcane plants are optimal, and in yet other embodiments corn plants are optimal.

[0283] Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

[0284] The present invention also provides a pesticidal composition comprising an effective amount of at least one chimeric pesticidal polypeptide of the invention to reduce pest damage to the plant and methods of protecting a plant from a pest involving providing to the plant or to the vicinity of the plant an effective amount of a pesticidal composition. By "effective amount" and "effective concentration" is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a plant pest. Methods for determining an "effective amount" and "effective concentration" are known to those of ordinary skill in art and include, for example, assays involving placing varying amounts of concentrations of the active ingredient (e.g., a chimeric pesticidal polypeptide) in contact with and/or in the vicinity of a pest and monitoring the survival and/or growth of the pest over time.

[0285] As described above, the effective amount of a chimeric pesticidal polypeptide can vary depending on the formulation and method in which the formulation is applied to the plant or plant environment. As such, bacteria can be transformed with a nucleotide sequence encoding a chimeric pesticidal polypeptide and can be used in the pesticidal compositions as described herein. Thus, the pesticidal composition can be an organism that is transformed to express a chimeric pesticidal polypeptide. Alternatively, a chimeric pesticidal polypeptide can be purified from the bacteria as described above. In some embodiments, the pesticidal composition or pesticidal formulation can include other agricultural protectants, as described above. As used herein, the term "pesticidal composition" and "pesticidial formulation" are equivalent terms that have the same meaning unless indicated otherwise or apparent from the context as used.

[0286] As described above, the effective amount of a chimeric pesticidal polypeptide can vary depending on the formulation and method in which the formulation is applied to the plant or plant environment. As such, bacteria can be transformed with a nucleotide sequence encoding a chimeric pesticidal polypeptide and can be used in the pesticidal compositions as described herein. Thus, the pesticidal composition can be an organism that is transformed to express a chimeric pesticidal polypeptide. Alternatively, the chimeric pesticidal polypeptide can be purified from the bacteria as described above. In some embodiments, the pesticidal compositions or pesticidal formulation can include other agricultural protectants, as described above.

[0287] As described above, the pesticidal composition can be, for example, a dust, emulsion, solid (e.g., particle or pellets) or liquid.

[0288] The pesticidal composition can be provided to the plant by applying the pesticidal composition directly to the environment of the plant or to the vicinity of the plant such as, for example, in the soil or other growth medium surrounding the plant to protect the plant from pest attacks. For example, the pesticidal composition can be applied directly to the plant by atomizing, broadcasting, coating or pouring, dusting spraying, scattering, soil drenching, sprinkling or seed coating at the time when the pest such as, for example, an insect pest has begun to appear on the plant or before the appearance of insect pests as a protective measure.

[0289] Alternatively, the pesticidal composition can be introduced into irrigation water and then applied to the plant during watering. It is preferred to obtain good control of pest in the early stages of plant growth as this is the time when the plant can be most severely damaged. To maintain protection as plants grow and to obtain the greatest protection of large plants, repeated applications of the pesticidal composition can be beneficial.

[0290] The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest. When the pesticidal composition not only includes the chimeric pesticidal polypeptide and/or a chimeric pesticidal polypeptide-expressing bacteria, but also includes another agricultural protectant, the formulation can be applied to the crop area or plant to be treated simultaneously or in succession (i.e., sequentially).

[0291] The pesticidal composition will reduce pest-related damage by at least about 5% to about 50%, at least about 10% to about 60%, at least about 30% to about 70%, at least about 40% to about 80% or at least about 50% to about 90% or greater. Hence, the methods can be utilized to protect plants from pests. Protection may vary from a slight decrease in plant damage caused by the pest (e.g., partial inhibition) to total decrease such that the plant is unaffected by the presence of the pest.

[0292] The present invention therefore provides methods of protecting plants, plant parts and plant host cells by providing a pesticidal composition comprising a chimeric pesticidal polypeptide.

[0293] In certain embodiments the polynucleotides of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired trait. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. For example, the polynucleotides of the present invention may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as other Bacillus thuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825, pentin (described in U.S. Pat. No. 5,981,722), and the like. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122) and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12:123)); increased digestibility (e.g., modified storage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)); the disclosures of which are herein incorporated by reference.

[0294] The polynucleotides of the present invention can also be stacked with traits desirable for disease or herbicide resistance (e.g., fumonisin detoxification genes, U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene); and traits desirable for processing or process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5.602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the present invention with polynucleotides providing agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5.583,210), stalk strength, flowering time or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); the disclosures of which are herein incorporated by reference.

[0295] These stacked combinations can be created by any method including, but not limited to, cross-breeding plants by any conventional or TopCross methodology or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference.

[0296] Various changes in phenotype are of interest including modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the plant. These changes result in a change in phenotype of the transformed plant.

[0297] Genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for transformation will change accordingly. General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest include, generally, those involved in oil, starch, carbohydrate or nutrient metabolism as well as those affecting kernel size, sucrose loading, and the like.

[0298] Agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson et al. (1987) Eur. J. Biochem. 165:99-106, the disclosures of which are herein incorporated by reference.

[0299] Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO 98/20133, the disclosures of which are herein incorporated by reference. Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp. 497-502; herein incorporated by reference); corn (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; both of which are herein incorporated by reference); and rice (Musumura et al. (1989) Plant Mol. Biol. 12:123, herein incorporated by reference). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.

[0300] Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); and the like.

[0301] Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); and the like.

[0302] Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene); glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example, U.S. Publication No. 20040082770 and WO 03/092360); or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron.

[0303] Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.

[0304] The quality of grain is reflected in traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. In corn, modified hordothionin proteins are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0305] Commercial traits can also be encoded on a gene or genes that could increase for example, starch for ethanol production or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321. Genes such as .beta.-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhyroxyalkanoates (PHAs).

[0306] Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. The level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.

[0307] Throughout this specification and the claims, the words "comprise," "comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

[0308] As used herein, the term "about," when referring to a value is meant to encompass variations of, in some embodiments .+-.50%, in some embodiments .+-.20%, in some embodiments .+-.10%, in some embodiments .+-.5%, in some embodiments .+-.1%, in some embodiments .+-.0.5%, and in some embodiments .+-.0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0309] Further, when an amount, concentration or other value or parameter is given as either a range, preferred range or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the presently disclosed subject matter be limited to the specific values recited when defining a range.

[0310] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Enhancement of the Insecticidal Activity of Cry Proteins Against Western Corn Rootworms and Black Cutworms When Fused to MBP

[0311] A computer-designed and artificially synthesized Cry3Aa-type protein called IP3-1 (SEQ ID NO: 8), a truncated RX002 protein (SEQ ID NO: 12) cloned from a Bt strain, a shuffled Cry8Bb-type protein called 2A-12 (SEQ ID NO: 10), and a truncated native Cry1Bd protein called 4c6 (SEQ ID NO: 14) were expressed in E. coli BL21 as fusion proteins comprising an operably linked MBP. The amino acid sequences of the MBP-IP3-1, MBP-RX002, MBP-2A-12, and MBP-4c6 fusion proteins are set forth in SEQ ID NOS: 21, 23, 25, and 27, respectively. Nucleotide sequences encoding the MBP-IP3-1, MBP-RX002, MBP-2A-12, and MBP-4c6 fusion proteins are set forth in SEQ ID NOS: 20, 22, 24, and 26, respectively.

[0312] To express MBP with these Bt Cry proteins, corresponding genes were cloned in an NEB pMAL vector and the proteins were purified using amylose resin following the manufacturer's recommended method (New England Biolabs, Inc., Ipswich, Mass., USA; Catalog No. E8021L). The target protein eluted from amylase resin was concentrated in Amicon Centricon concentrator (10 kDa cutoff). The final fusion proteins were dissolved in 25 mM HEPES-NaOH buffer pH8 at a concentration around 2 mg/ml. The protein concentration was determined by SDS-PAGE using bovine serum albumin as the reference. The pMAL vector has a protease site specific to Factor Xa that cleaves MBP and the linker off the Cry protein. All Cry proteins included in this application were resistant to Factor Xa. The protease digestion was carried out in the HEPES buffer at 1:25 protease and substrate ration at 20.degree. C. for 16 hr. The digestion leading to the complete MBP removal was confirmed by SDS-PAGE.

[0313] MBP fusion and MBP free Cry proteins were diluted in 25 mM HEPES-NaOH buffer pH 8 and 10 .mu.L of diluted samples were mixed with 40 .mu.L of molten artificial insect diet made with low temperature melting agarose. The diet mixture was then placed in each well of a 96-well micro-titer plate and allowed to feed with neonate insect larvae. After 4 days at 27.degree. C., the responses of insects towards the Cry proteins were scored using a 0-3 numerical scoring system based on the size and mortality. If no response or normal growth was seen, Score 0 was given. When the growth was somewhat retarded without any mortality, it was Score 1. Score 2 meant partial death (multiple insects were used in each well) and strong growth inhibition. Score 3 indicated the complete mortality. Each treatment was repeated 6 times for possible highest score of 18 (3.times.6). In this scoring system, Score 9 with 6 repeats of one treatment means the 50% response (9 out of 18) of the treatment and called ILC50 (growth Inhibition and Lethal Concentration for 50% response). The results of these assays are shown in Tables 3 and 4.

[0314] The results shown in Tables 3 and 4 with four different Cry proteins demonstrate that MBP-Cry fusion proteins have significantly increased insecticidal activity against both Coleopteran (Western Corn Rootworm) and Lepidopteran (Black Cutworm) insect species, when compared to the insecticidial activity of their respective free Cry proteins. These results further demonstrate the general applicability of the methods of the present invention to enhancing the insecticidal activity of Cry proteins. Table 3 shows ILC50 (based on the size or weight of the Cry protein portion) values for insecticidal activity against Western Corn Rootworm of MBP-Cry Fusion Proteins and Free Cry Proteins.

TABLE-US-00003 TABLE 3 IP3-1 RX002 2A-12 MBP- free MBP- free MBP- free fusion Cry fusion Cry fusion Cry 7 ppm 103 ppm 89 ppm No activity* 32 ppm 152 ppm *Only an 11% response was observed at 2480 ppm.

[0315] Table 4 shows the ILC50 values for insecticidal activity against Black Cutworm of a MBP-4c6 Cry Fusion Protein and Free 4c6 Cry Protein.

TABLE-US-00004 TABLE 4 MBP-4c6 fusion free 4c6 75 ppm 148 ppm

EXAMPLE 2

MBP-Cry Fusion Protein With Increased Activity Against WCRW

[0316] A new vector, pMAL-SA (SEQ ID NO: 19), was constructed for making MBP-Cry fusion proteins. This vector is based on NEB pMAL vector. It has the cry gene cloning site delineated with SphI and BamHI recognition sequences at the end of MBP and a specially designed linker called "SA" linker between MBP and the Cry cloning site.

[0317] In order to clone a Bt cry gene, the cry coding region is amplified by PCR using appropriate forward and reverse primers. In the forward primer, there is an SphI site over the ATG translation initiation site. In the reverse primer, there is a stop codon at the end of the cry gene coding region and a BamHI site.

[0318] A computer-designed cry3Aa sequence, IP3-1, was synthesized and used as the PCR template. The IP3-1 nucleotide sequence is set forth in SEQ ID NO: 7. This IP3-1 gene was cloned in pMAL-SA as described above to produce a plasmid that contains MBP, the SA linker and IP3-1 coding region (SEQ ID NO: 1). The second amino acid residue of IP3-1 was mutated back to Asn from His to produce the MBP-SA-IP3-1 nucleotide sequence set forth in SEQ ID NO: 2.

[0319] The MBP-SA-IP3-1 fusion protein was expressed in E. coli BL21 and purified by amylose resin affinity chromatography according to the method described in Example 1. The column eluate was concentrated in Amicon Centricon and its buffer was exchanged to 25 mM HEPES-NaOH buffer pH 8. The fusion protein was digested with trypsin at 1:25 trypsin-substrate ratio at 37.degree. C. for 1 hr. Trypsin cleaves the protein at the end of the linker to liberate the Cry protein from MBP and the SA linker. IP3-1 was resistant to trypsin under this digestion condition. The digestion was confirmed with SDS-PAGE. Both the fusion and MBP-free IP3-1 proteins were assayed against WCRW. Table 5 shows ILC50 values for insecticidal activity against Western Corn Rootworm of a MBP-SA-IP3-1 and MBP-free IP3-1. The assay results in Table 5 demonstrate that there is a significant enhancement of WCRW activity when IP3-1 is operable linked to MBP-SA (i.e., MBP-SA-IP3-1), when compared to the insecticidal activity of MBP-free IP3-1 against WCRW.

TABLE-US-00005 TABLE 5 Protein ICL50 MBP-SA-IP3-1 6 ppm MBP-free IP3-1 269 ppm

EXAMPLE 3

Enhancement of Cry3Aa Protein Activity When Fused to NusA and Trx

[0320] A computer-designed and artificially synthesized cry3Aa-type gene called IP3-1 was cloned in pET-43.1 EK/LIC for NusA fusion and pET-32 for TrxA fusion by following the manufacturer's directions (EMD Biosciences, Madison, Wis., USA). These vectors were used to express NusA-IP3-1 and TrxA-IP3-1 fusion proteins. The amino acid sequences of the NusA-IP3-1 and TrxA-IP3-1 fusion proteins are set forth in SEQ ID NOS: 16 and 18, respectively. Nucleotide sequences encoding the NusA-IP3-1 and TrxA-IP3-1 fusion proteins are set forth in SEQ ID NOS: 15 and 17, respectively. The amino acid sequence of IP3-1 is set forth in SEQ ID NO: 8. A nucleotide sequence encoding IP3-1 is set forth in SEQ ID NO: 7.

[0321] The fusion proteins were purified by affinity chromatography using Ni-NTA agarose (Qiagen Inc., Valencia, Calif., USA) according to the manufacturer's directions. The Cry3Aa protein was then digested away from the tags including NusA, TrxA, 6XHis etc. with enterokinase and the insecticidal activity of the free Cry3A protein was compared with its NusA and TrxA fusion proteins. The insect assay was conducted using WCRW as described above. Table 6 shows the ILC50 values for insecticidal activity against Western Corn Rootworm of a MBP-SA-IP3-1 and MBP-free IP3-1 protein. The IP3-1 protein sample was produced from the NusA-Cry3Aa fusion by enterokinase digestion. As demonstrated in the results in Table 6, both the NusA-IP3-1-and TrxA-IP3-1-fusion proteins, which comprise a solubility-enhancing polypeptide and a Cry3A protein (IP3-1), have an increased insecticidal activity against WCRW as evidenced by the significantly lower ICL50 values, when compared to insecticidal activity of free IP3-1.

TABLE-US-00006 TABLE 6 Protein ICL50 NusA-IP3-1-fusion 26 ppm TrxA-IP3-1-fusion 31 ppm Tag free IP3-1 554 ppm

EXAMPLE 4

Generation of a MBP-SA-RX002 Transformation Construct

[0322] The maltose-binding protein (MBP-SA) (SEQ ID NO: 4) was fused to the N-terminus of RX002 (SEQ ID NO:11) in a synthetic gene designed for this invention (SEQ ID NO:32 encoding SEQ ID NO:33). The gene was cloned as a BamHI--Stul fragment into a Gateway entry vector containing a plant expression cassette with the BSV(AY) TR PROMOTER--ADH1 INTRON1 sequence and the potato PIN II terminator sequence. The resulting plant expression cassette contains the following components operatively linked together in this order; BSV (AY) TR PRO-ADH1-INTRON1, the MBP-SA-RX002 gene, and the PIN II terminator. The expression cassette is flanked by Gateway attL3 and attL4 recombination sites and this entry vector was used to transfer the expression cassette into an attR3 and attR4 containing binary destination transformation vector. The final transformation vector contains the MBP-SA-RX002 expression cassette upstream of a cassette containing the maize Ubiquitin1 promoter-5'UTR-Ubiquitin intron1 controlling expression of a PAT selectable marker gene with the 35S terminator sequence.

EXAMPLE 5

Agrobacterium-Mediated Transformation of Maizeand Regeneration of Transgenic Plants

[0323] For Agrobacterium-mediated transformation of maize with a promoter sequence of the invention, the method of Zhao was employed (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos were isolated from maize and the embryos contacted with a suspension of Agrobacterium under conditions whereby the bacteria were capable of transferring the promoter sequence of the invention to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos were immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos were co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos were cultured on solid medium following the infection step. Following the co-cultivation period and an optional "resting" step was performed. In this resting step, the embryos were incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos were cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos were cultured on medium containing a selective agent and growing transformed callus was recovered (step 4: the selection step). The immature embryos were cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus was then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium were cultured on solid medium to regenerate the plants.

EXAMPLE 6

Expression of MBP-RX002 Fusion in Transgenic Maize Tissue

[0324] Transgenic events derived from the testing vector were evaluated for expression of MBP-RX002 by Western analysis. Leaf and root material for transgenic maize expressing MBP-RX002 were lyophilized then powdered with 5/32 inch BBs (i.e., birdshot) using a Geno/Grinder 2000 homogenizer at 1700 beats per minute for 30 seconds. 80 .mu.L of grinding buffer (1.times.PBS+0.1% Tween-20+1% 2-mercaptoethanol containing Roche cOmplete protease inhibitor (Roche Applied Science, Indianapolis, Ind., USA; Catalog No. 04693124001; at one tablet per 7 mL) was added to each sample. Pulverization was repeated for an additional 30 seconds, then the samples were sonicated for 5 minutes at room temperature in a VWR 75D sonicator. After centrifugation at 21,000 g/4.degree. C. for 15 minutes, supernatants were collected. Protein concentrations were determined from the supernatants using Thermo Scientific Coomassie Plus Kit (23236) and a SpectraMAX 190 spectrophotometer. Samples were normalized for total protein in 21 .mu.L using grinding buffer as diluent. Seven microliters of 4.times. LDS dye containing 1% 2-mercaptoethanol was added to each sample prior to heating at 80.degree. C. for 10 minutes. Twenty-five microliters of sample was loaded per lane on a NuPAGE Novex 4-12% Bis-Tris midi gel (Invitrogen WB1402BOX) and electrophoresed at 200 V for 1 hour. Protein was transferred to a nitrocellulose membrane using an Invitrogen iBlot with a transfer stack (IB301001). The membrane was blocked for 30 minutes in 1.times.PBS+0.1% Tween-20+5% powdered milk w/v (blocking buffer), then incubated overnight at 4.degree. C. with rabbit polyclonal antibody against RX002, diluted in blocking buffer at 1:4000. Membrane was washed 4.times.5 minutes in PBST (1.times.PBS+0.1% Tween-20), then incubated for 2 hours with goat anti-rabbit HRP conjugated secondary antibody (Pierce 31460; 10 .mu.g/mL working stock) at a 1:5000 dilution in blocking buffer. The membrane was washed 4.times.5 minutes in PBST and developed using Thermo Super Signal West Dura Extended Duration Substrate (34076). Visualization of hybridization signal was accomplished using a Fujifilm LAS-4000 imaging system.

[0325] The results of this analysis are shown in Table 7. Accumulation of MBP-RX002 was detected in 8 of the 10 events sampled for Western analysis in either root or leaf and root tissue. An 118 kD protein band corresponding to the expected size of the MBP-RX002 fusion was observed in both leaf and root or root tissue demonstrating that the fusion can be expressed in planta. In addition to the full length protein, a 75 kD immunoreactive protein band corresponding to the expected size of RX002 was also observed in some events indicating that some proportion of the full-length fusion protein was processed in planta to release RX002.

TABLE-US-00007 TABLE 7 Event No. Expression in leaf Expression in root Control - - 123434603 - - 123434607 +++ ++++ 123434610 - - 123434613 ++++ ++++ 123434614 - + 123434615 - ++ 123434616 ++++ ++++ 123434617 ++++ ++++ 123434618 ++++ ++++ 123434619 - +++

[0326] The article "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more element.

[0327] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0328] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Sequence CWU 1

1

3613093DNAArtificial SequenceMBP-SA-IP3-1misc_feature(1)...(1104)MBPmisc_feature(1105)...(1161- )Linkermisc_feature(1162)...(3093)IP3-1 1atggacaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080gaagccctga aagacgcgca gactcgtatc accaagagca tgtctgcatc tgcatctgca 1140tctgcatctg catctgcacg catgaaccct aacaacaggt cagagcacga cacgatcaag 1200acaaccgaga acaacgaggt gccgacgaac cacgtccagt acccactcgc cgaaacaccg 1260aaccctaccc tggaggacct caactacaag gagttcctga ggatgacagc ggacaacaac 1320accgaagccc tcgactctag cacgaccaag gacgtgattc agaagggcat ctctgtggtc 1380ggcgacctgc ttggcgttgt cgggttcccg ttcggcggcg ctctcgtcag cttctacacg 1440aacttcttga acaccatctg gccgtccgaa gaccccctca aggcgttcat ggagcaggtg 1500gaggccctca tcgaccagaa gatcgctgac tatgccaaga acaaggctct ggcggagctg 1560caaggcctgc agaacaactt cgaggactat gtgagcgccc tcagctcttg gcagaagaac 1620ccagtcagct cccgcaaccc gcacagccag ggccgcatca gggagctgtt cagccaggcc 1680gagagccact tccgcaactc catgccgagc ttcgccgtga gcggctacga ggtcctcttc 1740ctgactacct atgcccaggc tgccaacacc cacttgcttc tcctgaagga cgcccagatc 1800tacggcgaag agtggggcta cgagaaggag gacatcgccg agttctacca caggcagctc 1860aagctgacgc aggagtacac cgaccactgc gtgaagtggt acaacgtcgg ccttgacaag 1920ctgagagggt ctacatacga gagctgggtc aacttcaacc gctataggcg cgagatgaca 1980cttaccgtcc tcgacctgat cgcgctcttc cctctgtacg acgtcagact ttactcgaag 2040ggtgtcaaga ccgagttgac cagggacgtg cttaccgacc caatcgtcgg cgtcaacaac 2100ctgcgcggct acgggaccac cttcagcaac atcgagaact acatcaggaa gccgcacctg 2160ttcgactacc tgcaccgcat tcagttccac accaggctgc agcccggcta ctacggcaac 2220gacagcttca actactggag cggcaactac gtgtctacca gacccagcat cggctccaac 2280gacatcatca cgtccccgtt ctacggcaac aagagcagcg agccggtcca gaacctggag 2340ttcaacggcg agaaggtcta tcgcgctgtc gccaacacca acctcgcggt ctggccgagc 2400gcggtgtaca gcggcgtcac taaggtcgag ttcagccagt acaacgacca gaccgacgag 2460gcgtccacgc agacctacga cagcaagagg aacgttggcg ccgtctcctg ggacagcatc 2520gaccagctcc cgccagagac cactgacgag ccacttgaga aggcttatag ccaccagctg 2580aactacgtca tgtgcttcct catgcaaggc tctcgcggca ccattccggt gttcacctgg 2640acacacaaga gcgttgactt cttcaacacc atcgacagca agaagatcac ccagctcccg 2700cttgtgaagg cctacaagct ccagagcggc gcgagcgtgg tcgccgggcc tggtttcaca 2760ggcggcgaca tcattcagtg tacggagaac ggcagcgcgg ccaccatcta cgtcacgccg 2820gacgtgagct actcccagaa gtacagagcc cgcatccact acgcctccac gagccagatc 2880accttcaccc tgagcctcga cggcgcgccc ttcaaccagt actacttcga caagaccatg 2940aacaagggcg acacactgac ctacaacagc ttcaacctgg ctagcttctc taccccattc 3000gagctgagcg gcaacaacct gcagatcggc gtcacaggcc tcagcgccgg cgacaaggtg 3060tacatcgaca agattgagtt catcccggtc tga 309321030PRTArtificial SequenceMBP-SA-IP3-1 2Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250 255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 Arg Ile Thr Lys Ser Met Ser Ala Ser Ala Ser Ala Ser Ala Ser Ala 370 375 380 Ser Ala Arg Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys385 390 395 400 Thr Thr Glu Asn Asn Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu 405 410 415 Ala Glu Thr Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe 420 425 430 Leu Arg Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr 435 440 445 Thr Lys Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu 450 455 460 Gly Val Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr465 470 475 480 Asn Phe Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Leu Lys Ala Phe 485 490 495 Met Glu Gln Val Glu Ala Leu Ile Asp Gln Lys Ile Ala Asp Tyr Ala 500 505 510 Lys Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu 515 520 525 Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser 530 535 540 Arg Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala545 550 555 560 Glu Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Gly Tyr 565 570 575 Glu Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu 580 585 590 Leu Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu 595 600 605 Lys Glu Asp Ile Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln 610 615 620 Glu Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys625 630 635 640 Leu Arg Gly Ser Thr Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg 645 650 655 Arg Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu 660 665 670 Tyr Asp Val Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg 675 680 685 Asp Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr 690 695 700 Gly Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu705 710 715 720 Phe Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Leu Gln Pro Gly 725 730 735 Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser 740 745 750 Thr Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr 755 760 765 Gly Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu 770 775 780 Lys Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser785 790 795 800 Ala Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp 805 810 815 Gln Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val 820 825 830 Gly Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 835 840 845 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Val Met 850 855 860 Cys Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Phe Thr Trp865 870 875 880 Thr His Lys Ser Val Asp Phe Phe Asn Thr Ile Asp Ser Lys Lys Ile 885 890 895 Thr Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser 900 905 910 Val Val Ala Gly Pro Gly Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr 915 920 925 Glu Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr 930 935 940 Ser Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile945 950 955 960 Thr Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe 965 970 975 Asp Lys Thr Met Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn 980 985 990 Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln 995 1000 1005 Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys 1010 1015 1020 Ile Glu Phe Ile Pro Val1025 103031179DNAArtificial SequenceNEB- pMAL-MBPmisc_feature(1)...(1104)MBPmisc_feature(1105)...(1179)Linker 3atggacaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac 1140aacaacctcg ggatcgaggg aaggatttca gaattaggc 11794393PRTArtificial SequenceNEB-pMAL-MBP 4Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250 255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly 370 375 380 Ile Glu Gly Arg Ile Ser Glu Leu Gly385 390 51161DNAArtificial SequenceMBP-SAmisc_feature(1)...(1104)MBPmisc_feature(1105)...(1161)Linke- r 5atggacaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct

1020ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080gaagccctga aagacgcgca gactcgtatc accaagagca tgtctgcatc tgcatctgca 1140tctgcatctg catctgcacg c 11616387PRTArtificial SequenceMBP-SA 6Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250 255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 Arg Ile Thr Lys Ser Met Ser Ala Ser Ala Ser Ala Ser Ala Ser Ala 370 375 380 Ser Ala Arg385 71932DNAArtificial SequenceIP3-1 7atgaacccta acaacaggtc agagcacgac acgatcaaga caaccgagaa caacgaggtg 60ccgacgaacc acgtccagta cccactcgcc gaaacaccga accctaccct ggaggacctc 120aactacaagg agttcctgag gatgacagcg gacaacaaca ccgaagccct cgactctagc 180acgaccaagg acgtgattca gaagggcatc tctgtggtcg gcgacctgct tggcgttgtc 240gggttcccgt tcggcggcgc tctcgtcagc ttctacacga acttcttgaa caccatctgg 300ccgtccgaag accccctcaa ggcgttcatg gagcaggtgg aggccctcat cgaccagaag 360atcgctgact atgccaagaa caaggctctg gcggagctgc aaggcctgca gaacaacttc 420gaggactatg tgagcgccct cagctcttgg cagaagaacc cagtcagctc ccgcaacccg 480cacagccagg gccgcatcag ggagctgttc agccaggccg agagccactt ccgcaactcc 540atgccgagct tcgccgtgag cggctacgag gtcctcttcc tgactaccta tgcccaggct 600gccaacaccc acttgcttct cctgaaggac gcccagatct acggcgaaga gtggggctac 660gagaaggagg acatcgccga gttctaccac aggcagctca agctgacgca ggagtacacc 720gaccactgcg tgaagtggta caacgtcggc cttgacaagc tgagagggtc tacatacgag 780agctgggtca acttcaaccg ctataggcgc gagatgacac ttaccgtcct cgacctgatc 840gcgctcttcc ctctgtacga cgtcagactt tactcgaagg gtgtcaagac cgagttgacc 900agggacgtgc ttaccgaccc aatcgtcggc gtcaacaacc tgcgcggcta cgggaccacc 960ttcagcaaca tcgagaacta catcaggaag ccgcacctgt tcgactacct gcaccgcatt 1020cagttccaca ccaggctgca gcccggctac tacggcaacg acagcttcaa ctactggagc 1080ggcaactacg tgtctaccag acccagcatc ggctccaacg acatcatcac gtccccgttc 1140tacggcaaca agagcagcga gccggtccag aacctggagt tcaacggcga gaaggtctat 1200cgcgctgtcg ccaacaccaa cctcgcggtc tggccgagcg cggtgtacag cggcgtcact 1260aaggtcgagt tcagccagta caacgaccag accgacgagg cgtccacgca gacctacgac 1320agcaagagga acgttggcgc cgtctcctgg gacagcatcg accagctccc gccagagacc 1380actgacgagc cacttgagaa ggcttatagc caccagctga actacgtcat gtgcttcctc 1440atgcaaggct ctcgcggcac cattccggtg ttcacctgga cacacaagag cgttgacttc 1500ttcaacacca tcgacagcaa gaagatcacc cagctcccgc ttgtgaaggc ctacaagctc 1560cagagcggcg cgagcgtggt cgccgggcct ggtttcacag gcggcgacat cattcagtgt 1620acggagaacg gcagcgcggc caccatctac gtcacgccgg acgtgagcta ctcccagaag 1680tacagagccc gcatccacta cgcctccacg agccagatca ccttcaccct gagcctcgac 1740ggcgcgccct tcaaccagta ctacttcgac aagaccatga acaagggcga cacactgacc 1800tacaacagct tcaacctggc tagcttctct accccattcg agctgagcgg caacaacctg 1860cagatcggcg tcacaggcct cagcgccggc gacaaggtgt acatcgacaa gattgagttc 1920atcccggtct ga 19328643PRTArtificial SequenceIP3-1 8Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Thr Thr Glu1 5 10 15 Asn Asn Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu Ala Glu Thr 20 25 30 Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys Asp 50 55 60 Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val65 70 75 80 Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Glu Asp Pro Leu Lys Ala Phe Met Glu Gln 100 105 110 Val Glu Ala Leu Ile Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys 115 120 125 Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val 130 135 140 Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro145 150 155 160 His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His 165 170 175 Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Gly Tyr Glu Val Leu 180 185 190 Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu Leu 195 200 205 Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp 210 215 220 Ile Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr225 230 235 240 Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly 245 250 255 Ser Thr Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met 260 265 270 Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val 275 280 285 Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Val Leu 290 295 300 Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr305 310 315 320 Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr 325 330 335 Leu His Arg Ile Gln Phe His Thr Arg Leu Gln Pro Gly Tyr Tyr Gly 340 345 350 Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro 355 360 365 Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys 370 375 380 Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr385 390 395 400 Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr 405 410 415 Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp 420 425 430 Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val 435 440 445 Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro 450 455 460 Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu465 470 475 480 Met Gln Gly Ser Arg Gly Thr Ile Pro Val Phe Thr Trp Thr His Lys 485 490 495 Ser Val Asp Phe Phe Asn Thr Ile Asp Ser Lys Lys Ile Thr Gln Leu 500 505 510 Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala 515 520 525 Gly Pro Gly Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly 530 535 540 Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys545 550 555 560 Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr 565 570 575 Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr 580 585 590 Met Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser 595 600 605 Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val 610 615 620 Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe625 630 635 640 Ile Pro Val92049DNAArtificial Sequence2A-12 9atgcgaatga gtccaaataa tcaaaatgaa tatgaaatta tagatgcgac accttctact 60tctgtatcca atgattctaa cagataccct tttgcgaatg agccaacaaa tgcgctacaa 120aatatggatt ataaagatta tttaaaaatg tctgcgggaa atgctagtga ataccctggt 180tcacctgaag tacttgtcag cggacaagat gcagctaagg ccgcaattga tatagtaggt 240aaattactat caggtttagg ggtcccattt gttgggccga tagtgagtct ttatactcaa 300cttattgata ttctgtggcc ttcagggcaa aagagtcaat gggagatttt tatggaacaa 360gtagaagaac tcataaatca aaaaatagca gaatatgcaa ggaataaagc gctttcggaa 420ttagaaggat taggtaataa ttaccaatta tatctaaccg cgcttgaaga atggaaagaa 480aatccatttc gtagaggatt tagaagaggt gccttacgag atgcgcgaaa tcgatttgaa 540atcctggata gtttatttac gcaatatatg ccatctttta gagtgacaaa ttttgaagta 600ccattcctta ctgtatatgc aatggcagcc aaccttcatt tactgttatt aaaggacgcg 660tcaatttttg gagaagaatg gggatggtca acaactacta ttaataacta ttatgatcgt 720caaatgaaac ttactgcaga atattctgat cactgtgtaa agtggtatga aactggttta 780gcaaaattaa aaggcacgag cgctaaacaa tgggtcgact acaaccaatt ccgtagagaa 840atgacactga cggttttaga tgttgttgca ttattcccaa attatgacac acgcacgtac 900ccaatggaaa cgaaagcaca actaacaagg gaagtatata cagatccact gggcgcggta 960aacgtgtctt caattggttc ctggtatgac aaagcacctt ctttcggagt gatagaatca 1020tccgttattc gaccacccca tgtatttgat tatataacgg gactcacagt gtatacacaa 1080tcaagaagca tttcttccgc tcgctatata agacattggg ctggtcatca aataagctac 1140catcgtgtca gtaggggtag taatcttcaa caaatgtatg gaactaatca aaatctacac 1200agcaccagta cctttgattt tacgaattat gatatttaca agacgttatc aaaagatgcg 1260gtgctccttg atattgtttt tcctggttat acgtatatat tttttggaat gccagaagtc 1320gagtttttca tggtaaacca attgaataat accagaaaga cgttaaagta taatccggtt 1380tccaaagata ttatagcggg gacaagagat tcggaattag aattgcctcc agaaacttca 1440gatcaaccaa attatgagtc aaatagccat agattatgtc atatcacaag tattcccgcg 1500acgggtaaca ctaccggatt agtgcctgta ttttcttgga cacatcgaag tgcagattta 1560aacaatacaa tatattcaga taaaatcact caaattccgg ccgttaaatg ttgggataat 1620ttaccgtttg ttccagtggt aaaaggacca ggacatacag gaggggattt attacagtat 1680aatagaagta ctggttctgt aggaacctta tttctagctc gatatggcct agcattagaa 1740aaagcaggaa aatatcgtgt aagactgaga tatgctactg atgcagatat tgtattgcat 1800gtaaacgatg ctcagattca gatgccaaaa acaatgaacc caggtgagga tctgacatct 1860aaaactttta aagttgcaga tgctatcaca acagttaatt tagcaacaga tagttcggtt 1920gcagttaaac ataatgttgg tgaagaccct aattcaacag tttctggtat agtttacgtt 1980gaccgaatcg aattcatccc agtagatgag acatatgaag cggaacaaga tttagaagcg 2040gccaaatga 204910682PRTArtificial Sequence2A-12 10Met Arg Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala1 5 10 15 Thr Pro Ser Thr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala 20 25 30 Asn Glu Pro Thr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu 35 40 45 Lys Met Ser Ala Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val 50 55 60 Leu Val Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly65 70 75 80 Lys Leu Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser 85 90 95 Leu Tyr Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Gln Lys Ser 100 105 110 Gln Trp Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile Asn Gln Lys 115 120 125 Ile Ala Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu 130 135 140 Gly Asn Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu Trp Lys Glu145 150 155 160 Asn Pro Phe Arg Arg Gly Phe Arg Arg Gly Ala Leu Arg Asp Ala Arg 165 170 175 Asn Arg Phe Glu Ile Leu Asp Ser Leu Phe Thr Gln Tyr Met Pro Ser 180 185 190 Phe Arg Val Thr Asn Phe Glu Val Pro Phe Leu Thr Val Tyr Ala Met 195 200 205 Ala Ala Asn Leu His Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly 210 215 220 Glu Glu Trp Gly Trp Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg225 230 235 240 Gln Met Lys Leu Thr Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr 245 250 255 Glu Thr Gly Leu Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val 260 265 270 Asp Tyr Asn Gln Phe Arg Arg Glu Met Thr Leu Thr Val Leu Asp Val 275 280 285 Val Ala Leu Phe Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr 290 295 300 Lys Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val305 310 315 320 Asn Val Ser Ser Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly 325 330 335 Val Ile Glu Ser Ser Val Ile Arg Pro Pro His Val Phe Asp Tyr Ile 340 345 350 Thr Gly Leu Thr Val Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg 355 360 365 Tyr Ile Arg His Trp Ala Gly His Gln Ile Ser Tyr His Arg Val Ser 370 375 380 Arg Gly Ser Asn Leu Gln Gln Met Tyr Gly Thr Asn Gln Asn Leu His385 390 395 400 Ser Thr Ser Thr Phe Asp Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu 405 410 415 Ser Lys Asp Ala Val Leu Leu Asp Ile Val Phe Pro Gly Tyr Thr Tyr 420 425 430 Ile Phe Phe Gly Met Pro Glu Val Glu Phe Phe Met Val Asn Gln Leu 435 440 445 Asn Asn Thr Arg Lys Thr Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile 450 455 460 Ile Ala Gly Thr Arg Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser465 470 475 480 Asp Gln Pro Asn Tyr Glu Ser Asn Ser His Arg Leu Cys His Ile Thr 485 490 495 Ser Ile Pro Ala Thr Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser 500 505 510 Trp Thr His Arg Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys 515 520 525 Ile Thr Gln Ile Pro Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val 530 535 540 Pro Val Val Lys Gly Pro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr545 550 555 560 Asn Arg Ser Thr Gly Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly 565 570 575 Leu Ala Leu Glu Lys Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala 580 585 590 Thr Asp Ala Asp Ile Val Leu His Val Asn Asp Ala Gln Ile Gln Met 595 600

605 Pro Lys Thr Met Asn Pro Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys 610 615 620 Val Ala Asp Ala Ile Thr Thr Val Asn Leu Ala Thr Asp Ser Ser Val625 630 635 640 Ala Val Lys His Asn Val Gly Glu Asp Pro Asn Ser Thr Val Ser Gly 645 650 655 Ile Val Tyr Val Asp Arg Ile Glu Phe Ile Pro Val Asp Glu Thr Tyr 660 665 670 Glu Ala Glu Gln Asp Leu Glu Ala Ala Lys 675 680 112010DNAArtificial SequenceRX002-truncated 11atgcgtccaa ataatcaaaa tgaatatgaa attatagata caccatctcg tacgtctgta 60tctaatgatt ctgtcagata tccttttgcg aatgaaccaa caactgattt aaacaatatg 120aattataaag atttccttaa aacagtaaat ggttacaata ctggagatct ttctggatct 180gaagcattta tcagtcaaac agcaattagt actgcaggta aagctgtggg tacagtacta 240gggttattgg gtgttccatt agccggagca gttggcccct taataacctt ctatggtacc 300atcgcactat tattctgggg gccgggagat ccatggcaag cttttatgac ccaagtagag 360gcattagtta accaaaaaat agcagattat gcaagaagta aagcaatctc agaattacaa 420ggattaagga atattctcga tttatatcgt tcagcactta tagattggca agagaaccca 480acaagaacaa gatcagtaac aaatatccgt tctcaatttg aaactgtaaa taattttttc 540gaatatcaaa tgccatcttt tgcagtggca ggttatgagg ttccattgtt agcagtatat 600gcacaggcgg caaatcttca tttatcaata ttaagagatg ccgcgacatt cggagcacaa 660tggggaatgt ctcaaactgc tattaataac atatatgacc ttcagcagag aagaactgct 720gagtatacta atcattgtgt gaaatggtat aataatggtt tagataaatt aagaggttcg 780aatgctgggc aatgggttaa ttttaatcgc taccgtagag agatgacact aatggtattg 840gatattgtag cgatatttcc aaactatgat acacgtacgt atccaagtgg aattggaact 900agtgtccaac ttacaagaga agtatatacg gatcctattg gttcgacagc aacacaaggt 960ggcgtttctt ggtatgacga agcaccttct tttacagcta ttgaaagttc cgtggttcga 1020ccacttcact tatttgattt actaacaggc gttacagtct atgccgctag tagttcttgg 1080gattcaagtc attattttag attttggaat gggcataaag tagacacaaa gggaattaat 1140agttctattc aatatagtaa tgtatatggt tctactagta atgcggttag tacaactact 1200atatcatttt cgggttttga agtttttaaa accatttcaa tagctggtgt actatttgct 1260tggacaacaa ggtattttgg agtccctaaa gttctttttt ctaaaataga ccctatttct 1320ggtattggaa gggattcaga atttagcgaa aggtatgcag ggattggaga acaaataaag 1380aattcacttg aggaattacc tttacaaaca gaagatgagc cggattataa atcttatagt 1440cataaattga atcatatttc aatggttcca caaactgtac gaacacggaa tgtacctgta 1500ttttcttggt cgcatcggag tgcggatatt gacaatagaa tttttcagga tagaattaat 1560caaattccgg tagtaaaggg acatacatta ggtccaggtg cttctgttat ggcaggtcct 1620ggatttacag gaggaaatat agttactaga accagtccag gtgtagtagt tttttctgga 1680gttactataa ataacgcatt atcacaaaga tatcgtgtga gaatacggta tgcttctact 1740actgacttcc gatttttctc tacactttca ggaactcgtc tttatgccac tcaggctact 1800aaaactatga ataaaggaca acaattaaca tatgaatcat ttcagtatgc aacaattaat 1860tctacattta catttgagaa tataaatgat agtttgacaa taggtgcaga tcaatttcta 1920agtggtgagc aagtctatgt agatagatat gaagtaatcc cagtggatgc aacgtttgag 1980gcggagaacg atttagaggt ggcaaagtag 201012669PRTArtificial SequenceRX002-truncated 12Met Arg Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Thr Pro Ser1 5 10 15 Arg Thr Ser Val Ser Asn Asp Ser Val Arg Tyr Pro Phe Ala Asn Glu 20 25 30 Pro Thr Thr Asp Leu Asn Asn Met Asn Tyr Lys Asp Phe Leu Lys Thr 35 40 45 Val Asn Gly Tyr Asn Thr Gly Asp Leu Ser Gly Ser Glu Ala Phe Ile 50 55 60 Ser Gln Thr Ala Ile Ser Thr Ala Gly Lys Ala Val Gly Thr Val Leu65 70 75 80 Gly Leu Leu Gly Val Pro Leu Ala Gly Ala Val Gly Pro Leu Ile Thr 85 90 95 Phe Tyr Gly Thr Ile Ala Leu Leu Phe Trp Gly Pro Gly Asp Pro Trp 100 105 110 Gln Ala Phe Met Thr Gln Val Glu Ala Leu Val Asn Gln Lys Ile Ala 115 120 125 Asp Tyr Ala Arg Ser Lys Ala Ile Ser Glu Leu Gln Gly Leu Arg Asn 130 135 140 Ile Leu Asp Leu Tyr Arg Ser Ala Leu Ile Asp Trp Gln Glu Asn Pro145 150 155 160 Thr Arg Thr Arg Ser Val Thr Asn Ile Arg Ser Gln Phe Glu Thr Val 165 170 175 Asn Asn Phe Phe Glu Tyr Gln Met Pro Ser Phe Ala Val Ala Gly Tyr 180 185 190 Glu Val Pro Leu Leu Ala Val Tyr Ala Gln Ala Ala Asn Leu His Leu 195 200 205 Ser Ile Leu Arg Asp Ala Ala Thr Phe Gly Ala Gln Trp Gly Met Ser 210 215 220 Gln Thr Ala Ile Asn Asn Ile Tyr Asp Leu Gln Gln Arg Arg Thr Ala225 230 235 240 Glu Tyr Thr Asn His Cys Val Lys Trp Tyr Asn Asn Gly Leu Asp Lys 245 250 255 Leu Arg Gly Ser Asn Ala Gly Gln Trp Val Asn Phe Asn Arg Tyr Arg 260 265 270 Arg Glu Met Thr Leu Met Val Leu Asp Ile Val Ala Ile Phe Pro Asn 275 280 285 Tyr Asp Thr Arg Thr Tyr Pro Ser Gly Ile Gly Thr Ser Val Gln Leu 290 295 300 Thr Arg Glu Val Tyr Thr Asp Pro Ile Gly Ser Thr Ala Thr Gln Gly305 310 315 320 Gly Val Ser Trp Tyr Asp Glu Ala Pro Ser Phe Thr Ala Ile Glu Ser 325 330 335 Ser Val Val Arg Pro Leu His Leu Phe Asp Leu Leu Thr Gly Val Thr 340 345 350 Val Tyr Ala Ala Ser Ser Ser Trp Asp Ser Ser His Tyr Phe Arg Phe 355 360 365 Trp Asn Gly His Lys Val Asp Thr Lys Gly Ile Asn Ser Ser Ile Gln 370 375 380 Tyr Ser Asn Val Tyr Gly Ser Thr Ser Asn Ala Val Ser Thr Thr Thr385 390 395 400 Ile Ser Phe Ser Gly Phe Glu Val Phe Lys Thr Ile Ser Ile Ala Gly 405 410 415 Val Leu Phe Ala Trp Thr Thr Arg Tyr Phe Gly Val Pro Lys Val Leu 420 425 430 Phe Ser Lys Ile Asp Pro Ile Ser Gly Ile Gly Arg Asp Ser Glu Phe 435 440 445 Ser Glu Arg Tyr Ala Gly Ile Gly Glu Gln Ile Lys Asn Ser Leu Glu 450 455 460 Glu Leu Pro Leu Gln Thr Glu Asp Glu Pro Asp Tyr Lys Ser Tyr Ser465 470 475 480 His Lys Leu Asn His Ile Ser Met Val Pro Gln Thr Val Arg Thr Arg 485 490 495 Asn Val Pro Val Phe Ser Trp Ser His Arg Ser Ala Asp Ile Asp Asn 500 505 510 Arg Ile Phe Gln Asp Arg Ile Asn Gln Ile Pro Val Val Lys Gly His 515 520 525 Thr Leu Gly Pro Gly Ala Ser Val Met Ala Gly Pro Gly Phe Thr Gly 530 535 540 Gly Asn Ile Val Thr Arg Thr Ser Pro Gly Val Val Val Phe Ser Gly545 550 555 560 Val Thr Ile Asn Asn Ala Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 565 570 575 Tyr Ala Ser Thr Thr Asp Phe Arg Phe Phe Ser Thr Leu Ser Gly Thr 580 585 590 Arg Leu Tyr Ala Thr Gln Ala Thr Lys Thr Met Asn Lys Gly Gln Gln 595 600 605 Leu Thr Tyr Glu Ser Phe Gln Tyr Ala Thr Ile Asn Ser Thr Phe Thr 610 615 620 Phe Glu Asn Ile Asn Asp Ser Leu Thr Ile Gly Ala Asp Gln Phe Leu625 630 635 640 Ser Gly Glu Gln Val Tyr Val Asp Arg Tyr Glu Val Ile Pro Val Asp 645 650 655 Ala Thr Phe Glu Ala Glu Asn Asp Leu Glu Val Ala Lys 660 665 131974DNAArtificial SequenceCry1Bd-4c6-truncated 13atgacttcaa ataggaaaaa tgagaatgaa attataaatg ccttatcgat tccagctgta 60tcgaatcatt ccgcacaaat ggatctatcg ctagatgctc gtattgagga ttctttgtgt 120atagccgagg ggaataatat caatccactt gttagcgcat caacagtcca aacgggtata 180aacatagctg gtagaatatt gggcgtatta ggtgtgccgt ttgctggaca actagctagt 240ttttatagtt ttcttgttgg ggaattatgg cctagtggca gagatccatg ggaaattttc 300ctggaacatg tagaacaact tataagacaa caagtaacag aaaatactag gaatacggct 360attgctcgat tagaaggtct aggaaaaggc tatagatctt accagcaggc tcttgaaact 420tggttagata accgaaatga tgcaagatca agaagcatta ttcttgagcg ctatgttgct 480ttagaacttg acattactac tgctataccg cttttcagaa tacgaaatga agaagttcca 540ttattaatgg tatatgctca agctgcaaat ttacacctat tattattgag agacgcatcc 600ctttttggta gtgaatgggg gatggcatct tccgatgtta accaatatta ccaagaacaa 660atcaggtata cagaggaata ttctaaccat tgcgtacaat ggtataatac agggctaaat 720aacttaagag ggacaaatgc tgaaagttgg ctgcggtata atcaattccg tagagaccta 780acgttagggg tattagattt agtagcccta ttcccaagct atgatactcg cacttatcca 840atcaatacga gtgctcagtt aacaagagaa atttatacag atccaattgg gagaacaaat 900gcaccttcag gatttgcaag tacgaattgg tttaataata atgcaccatc gttttctgcc 960atagaggctg ccattttcag gcctccgcat ctacttgatt ttccagaaca acttacaatt 1020tacagtgcat caagccgttg gagtagcact caacatatga attattgggt gggacatagg 1080cttaacttcc gcccaatagg agggacatta aatacctcaa cacaaggact tactaataat 1140acttcaatta atcctgtaac attacagttt acgtctcgtg acgtttatag aacagaatca 1200aatgcaggga caaatatact atttactact cctgtgaatg gagtaccttg ggctagattt 1260aattttataa accctcagaa tatttatgaa agaggcgcca ctacctacag tcaaccgtat 1320cagggagttg ggattcaatt atttgattca gaaactgaat taccaccaga aacaacagaa 1380cgaccaaatt atgaatcata tagtcataga ttatctcata taggactaat cataggaaac 1440actttgagag caccagtcta ttcttggacg catcgtagtg cagatcgtac gaatacgatt 1500ggaccaaata gaattactca aattcctgca gtgaagggaa gatttctttt taatggttct 1560gtaatttcag gaccaggatt tactggtgga gacgtagtta gattgaatag gaataatggt 1620aatattcaaa atagagggta tattgaagtt ccaattcaat tcacgtcgac atctaccaga 1680tatcgagttc gagtacgtta tgcttctgta acctcgattg agctcaatgt taatttgggc 1740aattcatcaa tttttacgaa cacattacca gcaacagctg catcattaga taatctacaa 1800tcaggggatt ttggttatgt tgaaatcaac aatgctttta catccgcaac aggtaatata 1860gtaggtgcta gaaattttag tgcaaatgca gaagtaataa tagacagatt tgaatttatc 1920ccagttactg caaccttcga ggcagaatat gatttagaaa gagcacaaaa gtga 197414657PRTArtificial SequenceCry1Bd-4c6-truncated 14Met Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser1 5 10 15 Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40 45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser65 70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr Arg Asn Thr Ala Ile Ala Arg Leu Glu Gly Leu Gly 115 120 125 Arg Gly Tyr Arg Ser Tyr Gln Gln Ala Leu Glu Thr Trp Leu Asp Asn 130 135 140 Arg Asn Asp Ala Arg Ser Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala145 150 155 160 Leu Glu Leu Asp Ile Thr Thr Ala Ile Pro Leu Phe Arg Ile Arg Asn 165 170 175 Glu Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly Met 195 200 205 Ala Ser Ser Asp Val Asn Gln Tyr Tyr Gln Glu Gln Ile Arg Tyr Thr 210 215 220 Glu Glu Tyr Ser Asn His Cys Val Gln Trp Tyr Asn Thr Gly Leu Asn225 230 235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr Gln Gly Leu Thr Asn Asn Thr Ser Ile Asn 370 375 380 Pro Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser385 390 395 400 Asn Ala Gly Thr Asn Ile Leu Phe Thr Thr Pro Val Asn Gly Val Pro 405 410 415 Trp Ala Arg Phe Asn Phe Ile Asn Pro Gln Asn Ile Tyr Glu Arg Gly 420 425 430 Ala Thr Thr Tyr Ser Gln Pro Tyr Gln Gly Val Gly Ile Gln Leu Phe 435 440 445 Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr 450 455 460 Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Leu Ile Ile Gly Asn465 470 475 480 Thr Leu Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg 485 490 495 Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gln Ile Pro Ala Val Lys 500 505 510 Gly Arg Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr 515 520 525 Gly Gly Asp Val Val Arg Leu Asn Arg Asn Asn Gly Asn Ile Gln Asn 530 535 540 Arg Gly Tyr Ile Glu Val Pro Ile Gln Phe Thr Ser Thr Ser Thr Arg545 550 555 560 Tyr Arg Val Arg Val Arg Tyr Ala Ser Val Thr Ser Ile Glu Leu Asn 565 570 575 Val Asn Leu Gly Asn Ser Ser Ile Phe Thr Asn Thr Leu Pro Ala Thr 580 585 590 Ala Ala Ser Leu Asp Asn Leu Gln Ser Gly Asp Phe Gly Tyr Val Glu 595 600 605 Ile Asn Asn Ala Phe Thr Ser Ala Thr Gly Asn Ile Val Gly Ala Arg 610 615 620 Asn Phe Ser Ala Asn Ala Glu Val Ile Ile Asp Arg Phe Glu Phe Ile625 630 635 640 Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln 645 650 655 Lys153570DNAArtificial SequenceNusA-IP3-1misc_feature(1)...(1485)NusAmisc_feature(1486)...(1638)- Linkermisc_feature(1639)...(3570)IP3-1 15atgaacaaag aaattttggc tgtagttgaa gccgtatcca atgaaaaggc gctacctcgc 60gagaagattt tcgaagcatt ggaaagcgcg ctggcgacag caacaaagaa aaaatatgaa 120caagagatcg acgtccgcgt acagatcgat cgcaaaagcg gtgattttga cactttccgt 180cgctggttag ttgttgatga agtcacccag ccgaccaagg aaatcaccct tgaagccgca 240cgttatgaag atgaaagcct gaacctgggc gattacgttg aagatcagat tgagtctgtt 300acctttgacc gtatcactac ccagacggca aaacaggtta tcgtgcagaa agtgcgtgaa 360gccgaacgtg cgatggtggt tgatcagttc cgtgaacacg aaggtgaaat catcaccggc 420gtggtgaaaa aagtaaaccg cgacaacatc tctctggatc tgggcaacaa cgctgaagcc 480gtgatcctgc gcgaagatat gctgccgcgt gaaaacttcc gccctggcga ccgcgttcgt 540ggcgtgctct attccgttcg cccggaagcg cgtggcgcgc aactgttcgt cactcgttcc 600aagccggaaa tgctgatcga actgttccgt attgaagtgc cagaaatcgg cgaagaagtg 660attgaaatta aagcagcggc tcgcgatccg ggttctcgtg cgaaaatcgc ggtgaaaacc 720aacgataaac gtatcgatcc ggtaggtgct tgcgtaggta tgcgtggcgc gcgtgttcag 780gcggtgtcta ctgaactggg tggcgagcgt atcgatatcg tcctgtggga tgataacccg 840gcgcagttcg tgattaacgc aatggcaccg gcagacgttg cttctatcgt ggtggatgaa 900gataaacaca ccatggacat cgccgttgaa gccggtaatc tggcgcaggc gattggccgt 960aacggtcaga acgtgcgtct ggcttcgcaa ctgagcggtt gggaactcaa cgtgatgacc 1020gttgacgacc tgcaagctaa gcatcaggcg gaagcgcacg cagcgatcga caccttcacc 1080aaatatctcg acatcgacga agacttcgcg actgttctgg tagaagaagg cttctcgacg 1140ctggaagaat tggcctatgt gccgatgaaa gagctgttgg aaatcgaagg ccttgatgag 1200ccgaccgttg aagcactgcg cgagcgtgct aaaaatgcac tggccaccat tgcacaggcc 1260caggaagaaa gcctcggtga taacaaaccg gctgacgatc tgctgaacct tgaaggggta 1320gatcgtgatt tggcattcaa actggccgcc cgtggcgttt gtacgctgga agatctcgcc 1380gaacagggca ttgatgatct ggctgatatc gaagggttga ccgacgaaaa agccggagca 1440ctgattatgg ctgcccgtaa tatttgctgg ttcggtgacg aagcgactag tggttctggt 1500catcaccatc accatcactc cgcgggtaaa gaaaccgctg ctgcgaaatt tgaacgccag 1560cacatggact cgccaccgcc aactggtctg

gtcccccggg gcagcgcggg ttctggtacg 1620attgatgacg acgacaagat gaaccctaac aacaggtcag agcacgacac gatcaagaca 1680accgagaaca acgaggtgcc gacgaaccac gtccagtacc cactcgccga aacaccgaac 1740cctaccctgg aggacctcaa ctacaaggag ttcctgagga tgacagcgga caacaacacc 1800gaagccctcg actctagcac gaccaaggac gtgattcaga agggcatctc tgtggtcggc 1860gacctgcttg gcgttgtcgg gttcccgttc ggcggcgctc tcgtcagctt ctacacgaac 1920ttcttgaaca ccatctggcc gtccgaagac cccctcaagg cgttcatgga gcaggtggag 1980gccctcatcg accagaagat cgctgactat gccaagaaca aggctctggc ggagctgcaa 2040ggcctgcaga acaacttcga ggactatgtg agcgccctca gctcttggca gaagaaccca 2100gtcagctccc gcaacccgca cagccagggc cgcatcaggg agctgttcag ccaggccgag 2160agccacttcc gcaactccat gccgagcttc gccgtgagcg gctacgaggt cctcttcctg 2220actacctatg cccaggctgc caacacccac ttgcttctcc tgaaggacgc ccagatctac 2280ggcgaagagt ggggctacga gaaggaggac atcgccgagt tctaccacag gcagctcaag 2340ctgacgcagg agtacaccga ccactgcgtg aagtggtaca acgtcggcct tgacaagctg 2400agagggtcta catacgagag ctgggtcaac ttcaaccgct ataggcgcga gatgacactt 2460accgtcctcg acctgatcgc gctcttccct ctgtacgacg tcagacttta ctcgaagggt 2520gtcaagaccg agttgaccag ggacgtgctt accgacccaa tcgtcggcgt caacaacctg 2580cgcggctacg ggaccacctt cagcaacatc gagaactaca tcaggaagcc gcacctgttc 2640gactacctgc accgcattca gttccacacc aggctgcagc ccggctacta cggcaacgac 2700agcttcaact actggagcgg caactacgtg tctaccagac ccagcatcgg ctccaacgac 2760atcatcacgt ccccgttcta cggcaacaag agcagcgagc cggtccagaa cctggagttc 2820aacggcgaga aggtctatcg cgctgtcgcc aacaccaacc tcgcggtctg gccgagcgcg 2880gtgtacagcg gcgtcactaa ggtcgagttc agccagtaca acgaccagac cgacgaggcg 2940tccacgcaga cctacgacag caagaggaac gttggcgccg tctcctggga cagcatcgac 3000cagctcccgc cagagaccac tgacgagcca cttgagaagg cttatagcca ccagctgaac 3060tacgtcatgt gcttcctcat gcaaggctct cgcggcacca ttccggtgtt cacctggaca 3120cacaagagcg ttgacttctt caacaccatc gacagcaaga agatcaccca gctcccgctt 3180gtgaaggcct acaagctcca gagcggcgcg agcgtggtcg ccgggcctgg tttcacaggc 3240ggcgacatca ttcagtgtac ggagaacggc agcgcggcca ccatctacgt cacgccggac 3300gtgagctact cccagaagta cagagcccgc atccactacg cctccacgag ccagatcacc 3360ttcaccctga gcctcgacgg cgcgcccttc aaccagtact acttcgacaa gaccatgaac 3420aagggcgaca cactgaccta caacagcttc aacctggcta gcttctctac cccattcgag 3480ctgagcggca acaacctgca gatcggcgtc acaggcctca gcgccggcga caaggtgtac 3540atcgacaaga ttgagttcat cccggtctga 3570161189PRTArtificial SequenceNusA-IP3-1DOMAIN(1)...(495)NusADOMAIN(496)...(546)LinkerDOMAIN(54- 7)...(1189)IP3-1 16Met Asn Lys Glu Ile Leu Ala Val Val Glu Ala Val Ser Asn Glu Lys1 5 10 15 Ala Leu Pro Arg Glu Lys Ile Phe Glu Ala Leu Glu Ser Ala Leu Ala 20 25 30 Thr Ala Thr Lys Lys Lys Tyr Glu Gln Glu Ile Asp Val Arg Val Gln 35 40 45 Ile Asp Arg Lys Ser Gly Asp Phe Asp Thr Phe Arg Arg Trp Leu Val 50 55 60 Val Asp Glu Val Thr Gln Pro Thr Lys Glu Ile Thr Leu Glu Ala Ala65 70 75 80 Arg Tyr Glu Asp Glu Ser Leu Asn Leu Gly Asp Tyr Val Glu Asp Gln 85 90 95 Ile Glu Ser Val Thr Phe Asp Arg Ile Thr Thr Gln Thr Ala Lys Gln 100 105 110 Val Ile Val Gln Lys Val Arg Glu Ala Glu Arg Ala Met Val Val Asp 115 120 125 Gln Phe Arg Glu His Glu Gly Glu Ile Ile Thr Gly Val Val Lys Lys 130 135 140 Val Asn Arg Asp Asn Ile Ser Leu Asp Leu Gly Asn Asn Ala Glu Ala145 150 155 160 Val Ile Leu Arg Glu Asp Met Leu Pro Arg Glu Asn Phe Arg Pro Gly 165 170 175 Asp Arg Val Arg Gly Val Leu Tyr Ser Val Arg Pro Glu Ala Arg Gly 180 185 190 Ala Gln Leu Phe Val Thr Arg Ser Lys Pro Glu Met Leu Ile Glu Leu 195 200 205 Phe Arg Ile Glu Val Pro Glu Ile Gly Glu Glu Val Ile Glu Ile Lys 210 215 220 Ala Ala Ala Arg Asp Pro Gly Ser Arg Ala Lys Ile Ala Val Lys Thr225 230 235 240 Asn Asp Lys Arg Ile Asp Pro Val Gly Ala Cys Val Gly Met Arg Gly 245 250 255 Ala Arg Val Gln Ala Val Ser Thr Glu Leu Gly Gly Glu Arg Ile Asp 260 265 270 Ile Val Leu Trp Asp Asp Asn Pro Ala Gln Phe Val Ile Asn Ala Met 275 280 285 Ala Pro Ala Asp Val Ala Ser Ile Val Val Asp Glu Asp Lys His Thr 290 295 300 Met Asp Ile Ala Val Glu Ala Gly Asn Leu Ala Gln Ala Ile Gly Arg305 310 315 320 Asn Gly Gln Asn Val Arg Leu Ala Ser Gln Leu Ser Gly Trp Glu Leu 325 330 335 Asn Val Met Thr Val Asp Asp Leu Gln Ala Lys His Gln Ala Glu Ala 340 345 350 His Ala Ala Ile Asp Thr Phe Thr Lys Tyr Leu Asp Ile Asp Glu Asp 355 360 365 Phe Ala Thr Val Leu Val Glu Glu Gly Phe Ser Thr Leu Glu Glu Leu 370 375 380 Ala Tyr Val Pro Met Lys Glu Leu Leu Glu Ile Glu Gly Leu Asp Glu385 390 395 400 Pro Thr Val Glu Ala Leu Arg Glu Arg Ala Lys Asn Ala Leu Ala Thr 405 410 415 Ile Ala Gln Ala Gln Glu Glu Ser Leu Gly Asp Asn Lys Pro Ala Asp 420 425 430 Asp Leu Leu Asn Leu Glu Gly Val Asp Arg Asp Leu Ala Phe Lys Leu 435 440 445 Ala Ala Arg Gly Val Cys Thr Leu Glu Asp Leu Ala Glu Gln Gly Ile 450 455 460 Asp Asp Leu Ala Asp Ile Glu Gly Leu Thr Asp Glu Lys Ala Gly Ala465 470 475 480 Leu Ile Met Ala Ala Arg Asn Ile Cys Trp Phe Gly Asp Glu Ala Thr 485 490 495 Ser Gly Ser Gly His His His His His His Ser Ala Gly Lys Glu Thr 500 505 510 Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser Pro Pro Pro Thr 515 520 525 Gly Leu Val Pro Arg Gly Ser Ala Gly Ser Gly Thr Ile Asp Asp Asp 530 535 540 Asp Lys Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Thr545 550 555 560 Thr Glu Asn Asn Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu Ala 565 570 575 Glu Thr Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe Leu 580 585 590 Arg Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr 595 600 605 Lys Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly 610 615 620 Val Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn625 630 635 640 Phe Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Leu Lys Ala Phe Met 645 650 655 Glu Gln Val Glu Ala Leu Ile Asp Gln Lys Ile Ala Asp Tyr Ala Lys 660 665 670 Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp 675 680 685 Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg 690 695 700 Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu705 710 715 720 Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Gly Tyr Glu 725 730 735 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 740 745 750 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys 755 760 765 Glu Asp Ile Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Glu 770 775 780 Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu785 790 795 800 Arg Gly Ser Thr Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg 805 810 815 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 820 825 830 Asp Val Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp 835 840 845 Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly 850 855 860 Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe865 870 875 880 Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Leu Gln Pro Gly Tyr 885 890 895 Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 900 905 910 Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly 915 920 925 Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys 930 935 940 Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala945 950 955 960 Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 965 970 975 Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly 980 985 990 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp 995 1000 1005 Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Val Met Cys 1010 1015 1020 Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Phe Thr Trp Thr1025 1030 1035 1040 His Lys Ser Val Asp Phe Phe Asn Thr Ile Asp Ser Lys Lys Ile Thr 1045 1050 1055 Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val 1060 1065 1070 Val Ala Gly Pro Gly Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu 1075 1080 1085 Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser 1090 1095 1100 Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr1105 1110 1115 1120 Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp 1125 1130 1135 Lys Thr Met Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu 1140 1145 1150 Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile 1155 1160 1165 Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile 1170 1175 1180 Glu Phe Ile Pro Val1185 172406DNAArtificial SequenceTrxA-IP3-1misc_feature(1)...(327)TrxAmisc_feature(328)...(474)Lin- kermisc_feature(475)...(2406)IP3-1 17atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420ttcgaacgcc agcacatgga cagcccagat ctgggtaccg atgacgacga caagatgaac 480cctaacaaca ggtcagagca cgacacgatc aagacaaccg agaacaacga ggtgccgacg 540aaccacgtcc agtacccact cgccgaaaca ccgaacccta ccctggagga cctcaactac 600aaggagttcc tgaggatgac agcggacaac aacaccgaag ccctcgactc tagcacgacc 660aaggacgtga ttcagaaggg catctctgtg gtcggcgacc tgcttggcgt tgtcgggttc 720ccgttcggcg gcgctctcgt cagcttctac acgaacttct tgaacaccat ctggccgtcc 780gaagaccccc tcaaggcgtt catggagcag gtggaggccc tcatcgacca gaagatcgct 840gactatgcca agaacaaggc tctggcggag ctgcaaggcc tgcagaacaa cttcgaggac 900tatgtgagcg ccctcagctc ttggcagaag aacccagtca gctcccgcaa cccgcacagc 960cagggccgca tcagggagct gttcagccag gccgagagcc acttccgcaa ctccatgccg 1020agcttcgccg tgagcggcta cgaggtcctc ttcctgacta cctatgccca ggctgccaac 1080acccacttgc ttctcctgaa ggacgcccag atctacggcg aagagtgggg ctacgagaag 1140gaggacatcg ccgagttcta ccacaggcag ctcaagctga cgcaggagta caccgaccac 1200tgcgtgaagt ggtacaacgt cggccttgac aagctgagag ggtctacata cgagagctgg 1260gtcaacttca accgctatag gcgcgagatg acacttaccg tcctcgacct gatcgcgctc 1320ttccctctgt acgacgtcag actttactcg aagggtgtca agaccgagtt gaccagggac 1380gtgcttaccg acccaatcgt cggcgtcaac aacctgcgcg gctacgggac caccttcagc 1440aacatcgaga actacatcag gaagccgcac ctgttcgact acctgcaccg cattcagttc 1500cacaccaggc tgcagcccgg ctactacggc aacgacagct tcaactactg gagcggcaac 1560tacgtgtcta ccagacccag catcggctcc aacgacatca tcacgtcccc gttctacggc 1620aacaagagca gcgagccggt ccagaacctg gagttcaacg gcgagaaggt ctatcgcgct 1680gtcgccaaca ccaacctcgc ggtctggccg agcgcggtgt acagcggcgt cactaaggtc 1740gagttcagcc agtacaacga ccagaccgac gaggcgtcca cgcagaccta cgacagcaag 1800aggaacgttg gcgccgtctc ctgggacagc atcgaccagc tcccgccaga gaccactgac 1860gagccacttg agaaggctta tagccaccag ctgaactacg tcatgtgctt cctcatgcaa 1920ggctctcgcg gcaccattcc ggtgttcacc tggacacaca agagcgttga cttcttcaac 1980accatcgaca gcaagaagat cacccagctc ccgcttgtga aggcctacaa gctccagagc 2040ggcgcgagcg tggtcgccgg gcctggtttc acaggcggcg acatcattca gtgtacggag 2100aacggcagcg cggccaccat ctacgtcacg ccggacgtga gctactccca gaagtacaga 2160gcccgcatcc actacgcctc cacgagccag atcaccttca ccctgagcct cgacggcgcg 2220cccttcaacc agtactactt cgacaagacc atgaacaagg gcgacacact gacctacaac 2280agcttcaacc tggctagctt ctctacccca ttcgagctga gcggcaacaa cctgcagatc 2340ggcgtcacag gcctcagcgc cggcgacaag gtgtacatcg acaagattga gttcatcccg 2400gtctga 240618801PRTArtificial SequenceTrxA-IP3-1DOMAIN(1)...(109)TrxADOMAIN(110)...(158)LinkerDOMAIN(15- 9)...(801)IP3-1 18Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro 115 120 125 Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln 130 135 140 His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Met Asn145 150 155 160 Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Thr Thr Glu Asn Asn 165 170 175 Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu Ala Glu Thr Pro Asn 180 185 190 Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe Leu Arg Met Thr Ala 195 200 205 Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys Asp Val Ile 210 215 220 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly Phe225 230 235 240 Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 245 250 255 Ile Trp Pro Ser Glu Asp Pro Leu Lys Ala Phe Met Glu Gln Val Glu 260 265 270 Ala Leu Ile Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu 275 280 285 Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val Ser Ala 290 295 300 Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro His Ser305 310 315 320 Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg 325 330 335 Asn Ser Met Pro Ser Phe Ala Val Ser Gly Tyr Glu Val Leu Phe Leu 340 345 350 Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu Leu Lys Asp 355 360 365 Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala 370 375 380

Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp His385 390 395 400 Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser Thr 405 410 415 Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu 420 425 430 Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg Leu 435 440 445 Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr Asp 450 455 460 Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser465 470 475 480 Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu His 485 490 495 Arg Ile Gln Phe His Thr Arg Leu Gln Pro Gly Tyr Tyr Gly Asn Asp 500 505 510 Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser Ile 515 520 525 Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser Ser 530 535 540 Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala545 550 555 560 Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser Gly 565 570 575 Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala 580 585 590 Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp 595 600 605 Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu 610 615 620 Lys Ala Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu Met Gln625 630 635 640 Gly Ser Arg Gly Thr Ile Pro Val Phe Thr Trp Thr His Lys Ser Val 645 650 655 Asp Phe Phe Asn Thr Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu 660 665 670 Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala Gly Pro 675 680 685 Gly Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly Ser Ala 690 695 700 Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys Tyr Arg705 710 715 720 Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr Leu Ser 725 730 735 Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr Met Asn 740 745 750 Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser Phe Ser 755 760 765 Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val Thr Gly 770 775 780 Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe Ile Pro785 790 795 800 Val196647DNAArtificial SequencepMAL-SA vectormisc_feature(158)...(2631)MBPmisc_feature(2632)...(2688)SA Linker 19ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 60gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 120gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 180cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 240aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 300acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 360tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 420ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 480ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 540cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 600tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 660cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 720cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 780atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 840tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcagta gtgggatacg 900acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 960gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1020agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1080cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1140cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 1200gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 1260tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 1320tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 1380ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 1440attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga 1500gcacttcacc aacaaggacc atagcatatg gacaaaatcg aagaaggtaa actggtaatc 1560tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa 1620gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag 1680gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc 1740tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg 1800tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct 1860gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg 1920gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc 1980aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc 2040aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa 2100gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat 2160tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg 2220tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc 2280ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc 2340agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt 2400ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa 2460gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc 2520atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac 2580gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac tcgtatcacc 2640aagagcatgt ctgcatctgc atctgcatct gcatctgcat ctgcacgcat gcatcattag 2700gatcctctag agtcgacctg caggcaagct tggcactggc cgtcgtttta caacgtcgtg 2760actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 2820gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 2880atggcgaatg gcagcttggc tgttttggcg gatgagataa gattttcagc ctgatacaga 2940ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 3000tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 3060tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 3120tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 3180acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca 3240ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc 3300ctttttgcgt ttctacaaac tctttttgtt tatttttcta aatacattca aatatgtatc 3360cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 3420gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 3480ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 3540tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 3600aacgttctcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg 3660ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 3720agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 3780gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 3840gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 3900gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 3960tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 4020ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 4080cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 4140gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 4200cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 4260tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttac 4320cccggttgat aatcagaaaa gccccaaaaa caggaagatt gtataagcaa atatttaaat 4380tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 4440taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 4500gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 4560caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat cacccaaatc 4620aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 4680atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 4740aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 4800cgccgcgctt aatgcgccgc tacagggcgc gtaaaaggat ctaggtgaag atcctttttg 4860ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 4920tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 4980aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 5040tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 5100agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 5160taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 5220caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 5280agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 5340aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 5400gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 5460tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 5520gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 5580ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 5640ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 5700aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 5760accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta 5820tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc 5880gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 5940gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag 6000ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac agatgtctgc ctgttcatcc 6060gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc 6120atgttaaggg cggttttttc ctgtttggtc acttgatgcc tccgtgtaag ggggaatttc 6180tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg 6240atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc 6300ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag 6360gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg 6420gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg 6480ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg 6540attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga 6600gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgaaatt 6647203111DNAArtificial SequenceMBP-IP3-1 20atggacaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac 1140aacaacctcg ggatcgaggg aaggatttca gaattaggca tgaaccctaa caacaggtca 1200gagcacgaca cgatcaagac aaccgagaac aacgaggtgc cgacgaacca cgtccagtac 1260ccactcgccg aaacaccgaa ccctaccctg gaggacctca actacaagga gttcctgagg 1320atgacagcgg acaacaacac cgaagccctc gactctagca cgaccaagga cgtgattcag 1380aagggcatct ctgtggtcgg cgacctgctt ggcgttgtcg ggttcccgtt cggcggcgct 1440ctcgtcagct tctacacgaa cttcttgaac accatctggc cgtccgaaga ccccctcaag 1500gcgttcatgg agcaggtgga ggccctcatc gaccagaaga tcgctgacta tgccaagaac 1560aaggctctgg cggagctgca aggcctgcag aacaacttcg aggactatgt gagcgccctc 1620agctcttggc agaagaaccc agtcagctcc cgcaacccgc acagccaggg ccgcatcagg 1680gagctgttca gccaggccga gagccacttc cgcaactcca tgccgagctt cgccgtgagc 1740ggctacgagg tcctcttcct gactacctat gcccaggctg ccaacaccca cttgcttctc 1800ctgaaggacg cccagatcta cggcgaagag tggggctacg agaaggagga catcgccgag 1860ttctaccaca ggcagctcaa gctgacgcag gagtacaccg accactgcgt gaagtggtac 1920aacgtcggcc ttgacaagct gagagggtct acatacgaga gctgggtcaa cttcaaccgc 1980tataggcgcg agatgacact taccgtcctc gacctgatcg cgctcttccc tctgtacgac 2040gtcagacttt actcgaaggg tgtcaagacc gagttgacca gggacgtgct taccgaccca 2100atcgtcggcg tcaacaacct gcgcggctac gggaccacct tcagcaacat cgagaactac 2160atcaggaagc cgcacctgtt cgactacctg caccgcattc agttccacac caggctgcag 2220cccggctact acggcaacga cagcttcaac tactggagcg gcaactacgt gtctaccaga 2280cccagcatcg gctccaacga catcatcacg tccccgttct acggcaacaa gagcagcgag 2340ccggtccaga acctggagtt caacggcgag aaggtctatc gcgctgtcgc caacaccaac 2400ctcgcggtct ggccgagcgc ggtgtacagc ggcgtcacta aggtcgagtt cagccagtac 2460aacgaccaga ccgacgaggc gtccacgcag acctacgaca gcaagaggaa cgttggcgcc 2520gtctcctggg acagcatcga ccagctcccg ccagagacca ctgacgagcc acttgagaag 2580gcttatagcc accagctgaa ctacgtcatg tgcttcctca tgcaaggctc tcgcggcacc 2640attccggtgt tcacctggac acacaagagc gttgacttct tcaacaccat cgacagcaag 2700aagatcaccc agctcccgct tgtgaaggcc tacaagctcc agagcggcgc gagcgtggtc 2760gccgggcctg gtttcacagg cggcgacatc attcagtgta cggagaacgg cagcgcggcc 2820accatctacg tcacgccgga cgtgagctac tcccagaagt acagagcccg catccactac 2880gcctccacga gccagatcac cttcaccctg agcctcgacg gcgcgccctt caaccagtac 2940tacttcgaca agaccatgaa caagggcgac acactgacct acaacagctt caacctggct 3000agcttctcta ccccattcga gctgagcggc aacaacctgc agatcggcgt cacaggcctc 3060agcgccggcg acaaggtgta catcgacaag attgagttca tcccggtctg a 3111211036PRTArtificial SequenceMBP-IP3-1DOMAIN(1)...(367)MBPDOMAIN(368)...(393)LinkerDOMAIN(394)- ...(1036)IP3-1 21Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250 255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu

Ala Leu Lys Asp Ala Gln Thr 355 360 365 Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly 370 375 380 Ile Glu Gly Arg Ile Ser Glu Leu Gly Met Asn Pro Asn Asn Arg Ser385 390 395 400 Glu His Asp Thr Ile Lys Thr Thr Glu Asn Asn Glu Val Pro Thr Asn 405 410 415 His Val Gln Tyr Pro Leu Ala Glu Thr Pro Asn Pro Thr Leu Glu Asp 420 425 430 Leu Asn Tyr Lys Glu Phe Leu Arg Met Thr Ala Asp Asn Asn Thr Glu 435 440 445 Ala Leu Asp Ser Ser Thr Thr Lys Asp Val Ile Gln Lys Gly Ile Ser 450 455 460 Val Val Gly Asp Leu Leu Gly Val Val Gly Phe Pro Phe Gly Gly Ala465 470 475 480 Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr Ile Trp Pro Ser Glu 485 490 495 Asp Pro Leu Lys Ala Phe Met Glu Gln Val Glu Ala Leu Ile Asp Gln 500 505 510 Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu Ala Glu Leu Gln Gly 515 520 525 Leu Gln Asn Asn Phe Glu Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln 530 535 540 Lys Asn Pro Val Ser Ser Arg Asn Pro His Ser Gln Gly Arg Ile Arg545 550 555 560 Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser 565 570 575 Phe Ala Val Ser Gly Tyr Glu Val Leu Phe Leu Thr Thr Tyr Ala Gln 580 585 590 Ala Ala Asn Thr His Leu Leu Leu Leu Lys Asp Ala Gln Ile Tyr Gly 595 600 605 Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala Glu Phe Tyr His Arg 610 615 620 Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp His Cys Val Lys Trp Tyr625 630 635 640 Asn Val Gly Leu Asp Lys Leu Arg Gly Ser Thr Tyr Glu Ser Trp Val 645 650 655 Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu Thr Val Leu Asp Leu 660 665 670 Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg Leu Tyr Ser Lys Gly Val 675 680 685 Lys Thr Glu Leu Thr Arg Asp Val Leu Thr Asp Pro Ile Val Gly Val 690 695 700 Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser Asn Ile Glu Asn Tyr705 710 715 720 Ile Arg Lys Pro His Leu Phe Asp Tyr Leu His Arg Ile Gln Phe His 725 730 735 Thr Arg Leu Gln Pro Gly Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr Trp 740 745 750 Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser Ile Gly Ser Asn Asp Ile 755 760 765 Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser Ser Glu Pro Val Gln Asn 770 775 780 Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala Val Ala Asn Thr Asn785 790 795 800 Leu Ala Val Trp Pro Ser Ala Val Tyr Ser Gly Val Thr Lys Val Glu 805 810 815 Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala Ser Thr Gln Thr Tyr 820 825 830 Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp Asp Ser Ile Asp Gln 835 840 845 Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser His 850 855 860 Gln Leu Asn Tyr Val Met Cys Phe Leu Met Gln Gly Ser Arg Gly Thr865 870 875 880 Ile Pro Val Phe Thr Trp Thr His Lys Ser Val Asp Phe Phe Asn Thr 885 890 895 Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu Val Lys Ala Tyr Lys 900 905 910 Leu Gln Ser Gly Ala Ser Val Val Ala Gly Pro Gly Phe Thr Gly Gly 915 920 925 Asp Ile Ile Gln Cys Thr Glu Asn Gly Ser Ala Ala Thr Ile Tyr Val 930 935 940 Thr Pro Asp Val Ser Tyr Ser Gln Lys Tyr Arg Ala Arg Ile His Tyr945 950 955 960 Ala Ser Thr Ser Gln Ile Thr Phe Thr Leu Ser Leu Asp Gly Ala Pro 965 970 975 Phe Asn Gln Tyr Tyr Phe Asp Lys Thr Met Asn Lys Gly Asp Thr Leu 980 985 990 Thr Tyr Asn Ser Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu 995 1000 1005 Ser Gly Asn Asn Leu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp 1010 1015 1020 Lys Val Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val1025 1030 1035 223189DNAArtificial SequenceMBP-RX002 22atggacaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac 1140aacaacctcg ggatcgaggg aaggatttca gaattaggca tgcgtccaaa taatcaaaat 1200gaatatgaaa ttatagatac accatctcgt acgtctgtat ctaatgattc tgtcagatat 1260ccttttgcga atgaaccaac aactgattta aacaatatga attataaaga tttccttaaa 1320acagtaaatg gttacaatac tggagatctt tctggatctg aagcatttat cagtcaaaca 1380gcaattagta ctgcaggtaa agctgtgggt acagtactag ggttattggg tgttccatta 1440gccggagcag ttggcccctt aataaccttc tatggtacca tcgcactatt attctggggg 1500ccgggagatc catggcaagc ttttatgacc caagtagagg cattagttaa ccaaaaaata 1560gcagattatg caagaagtaa agcaatctca gaattacaag gattaaggaa tattctcgat 1620ttatatcgtt cagcacttat agattggcaa gagaacccaa caagaacaag atcagtaaca 1680aatatccgtt ctcaatttga aactgtaaat aattttttcg aatatcaaat gccatctttt 1740gcagtggcag gttatgaggt tccattgtta gcagtatatg cacaggcggc aaatcttcat 1800ttatcaatat taagagatgc cgcgacattc ggagcacaat ggggaatgtc tcaaactgct 1860attaataaca tatatgacct tcagcagaga agaactgctg agtatactaa tcattgtgtg 1920aaatggtata ataatggttt agataaatta agaggttcga atgctgggca atgggttaat 1980tttaatcgct accgtagaga gatgacacta atggtattgg atattgtagc gatatttcca 2040aactatgata cacgtacgta tccaagtgga attggaacta gtgtccaact tacaagagaa 2100gtatatacgg atcctattgg ttcgacagca acacaaggtg gcgtttcttg gtatgacgaa 2160gcaccttctt ttacagctat tgaaagttcc gtggttcgac cacttcactt atttgattta 2220ctaacaggcg ttacagtcta tgccgctagt agttcttggg attcaagtca ttattttaga 2280ttttggaatg ggcataaagt agacacaaag ggaattaata gttctattca atatagtaat 2340gtatatggtt ctactagtaa tgcggttagt acaactacta tatcattttc gggttttgaa 2400gtttttaaaa ccatttcaat agctggtgta ctatttgctt ggacaacaag gtattttgga 2460gtccctaaag ttcttttttc taaaatagac cctatttctg gtattggaag ggattcagaa 2520tttagcgaaa ggtatgcagg gattggagaa caaataaaga attcacttga ggaattacct 2580ttacaaacag aagatgagcc ggattataaa tcttatagtc ataaattgaa tcatatttca 2640atggttccac aaactgtacg aacacggaat gtacctgtat tttcttggtc gcatcggagt 2700gcggatattg acaatagaat ttttcaggat agaattaatc aaattccggt agtaaaggga 2760catacattag gtccaggtgc ttctgttatg gcaggtcctg gatttacagg aggaaatata 2820gttactagaa ccagtccagg tgtagtagtt ttttctggag ttactataaa taacgcatta 2880tcacaaagat atcgtgtgag aatacggtat gcttctacta ctgacttccg atttttctct 2940acactttcag gaactcgtct ttatgccact caggctacta aaactatgaa taaaggacaa 3000caattaacat atgaatcatt tcagtatgca acaattaatt ctacatttac atttgagaat 3060ataaatgata gtttgacaat aggtgcagat caatttctaa gtggtgagca agtctatgta 3120gatagatatg aagtaatccc agtggatgca acgtttgagg cggagaacga tttagaggtg 3180gcaaagtag 3189231062PRTArtificial SequenceMBP-RX002DOMAIN(1)...(368)MBPDOMAIN(369)...(393)LinkerDOMAIN(394)- ...(1062)RX002 23Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250 255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly 370 375 380 Ile Glu Gly Arg Ile Ser Glu Leu Gly Met Arg Pro Asn Asn Gln Asn385 390 395 400 Glu Tyr Glu Ile Ile Asp Thr Pro Ser Arg Thr Ser Val Ser Asn Asp 405 410 415 Ser Val Arg Tyr Pro Phe Ala Asn Glu Pro Thr Thr Asp Leu Asn Asn 420 425 430 Met Asn Tyr Lys Asp Phe Leu Lys Thr Val Asn Gly Tyr Asn Thr Gly 435 440 445 Asp Leu Ser Gly Ser Glu Ala Phe Ile Ser Gln Thr Ala Ile Ser Thr 450 455 460 Ala Gly Lys Ala Val Gly Thr Val Leu Gly Leu Leu Gly Val Pro Leu465 470 475 480 Ala Gly Ala Val Gly Pro Leu Ile Thr Phe Tyr Gly Thr Ile Ala Leu 485 490 495 Leu Phe Trp Gly Pro Gly Asp Pro Trp Gln Ala Phe Met Thr Gln Val 500 505 510 Glu Ala Leu Val Asn Gln Lys Ile Ala Asp Tyr Ala Arg Ser Lys Ala 515 520 525 Ile Ser Glu Leu Gln Gly Leu Arg Asn Ile Leu Asp Leu Tyr Arg Ser 530 535 540 Ala Leu Ile Asp Trp Gln Glu Asn Pro Thr Arg Thr Arg Ser Val Thr545 550 555 560 Asn Ile Arg Ser Gln Phe Glu Thr Val Asn Asn Phe Phe Glu Tyr Gln 565 570 575 Met Pro Ser Phe Ala Val Ala Gly Tyr Glu Val Pro Leu Leu Ala Val 580 585 590 Tyr Ala Gln Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Ala Ala 595 600 605 Thr Phe Gly Ala Gln Trp Gly Met Ser Gln Thr Ala Ile Asn Asn Ile 610 615 620 Tyr Asp Leu Gln Gln Arg Arg Thr Ala Glu Tyr Thr Asn His Cys Val625 630 635 640 Lys Trp Tyr Asn Asn Gly Leu Asp Lys Leu Arg Gly Ser Asn Ala Gly 645 650 655 Gln Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu Met Val 660 665 670 Leu Asp Ile Val Ala Ile Phe Pro Asn Tyr Asp Thr Arg Thr Tyr Pro 675 680 685 Ser Gly Ile Gly Thr Ser Val Gln Leu Thr Arg Glu Val Tyr Thr Asp 690 695 700 Pro Ile Gly Ser Thr Ala Thr Gln Gly Gly Val Ser Trp Tyr Asp Glu705 710 715 720 Ala Pro Ser Phe Thr Ala Ile Glu Ser Ser Val Val Arg Pro Leu His 725 730 735 Leu Phe Asp Leu Leu Thr Gly Val Thr Val Tyr Ala Ala Ser Ser Ser 740 745 750 Trp Asp Ser Ser His Tyr Phe Arg Phe Trp Asn Gly His Lys Val Asp 755 760 765 Thr Lys Gly Ile Asn Ser Ser Ile Gln Tyr Ser Asn Val Tyr Gly Ser 770 775 780 Thr Ser Asn Ala Val Ser Thr Thr Thr Ile Ser Phe Ser Gly Phe Glu785 790 795 800 Val Phe Lys Thr Ile Ser Ile Ala Gly Val Leu Phe Ala Trp Thr Thr 805 810 815 Arg Tyr Phe Gly Val Pro Lys Val Leu Phe Ser Lys Ile Asp Pro Ile 820 825 830 Ser Gly Ile Gly Arg Asp Ser Glu Phe Ser Glu Arg Tyr Ala Gly Ile 835 840 845 Gly Glu Gln Ile Lys Asn Ser Leu Glu Glu Leu Pro Leu Gln Thr Glu 850 855 860 Asp Glu Pro Asp Tyr Lys Ser Tyr Ser His Lys Leu Asn His Ile Ser865 870 875 880 Met Val Pro Gln Thr Val Arg Thr Arg Asn Val Pro Val Phe Ser Trp 885 890 895 Ser His Arg Ser Ala Asp Ile Asp Asn Arg Ile Phe Gln Asp Arg Ile 900 905 910 Asn Gln Ile Pro Val Val Lys Gly His Thr Leu Gly Pro Gly Ala Ser 915 920 925 Val Met Ala Gly Pro Gly Phe Thr Gly Gly Asn Ile Val Thr Arg Thr 930 935 940 Ser Pro Gly Val Val Val Phe Ser Gly Val Thr Ile Asn Asn Ala Leu945 950 955 960 Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Phe 965 970 975 Arg Phe Phe Ser Thr Leu Ser Gly Thr Arg Leu Tyr Ala Thr Gln Ala 980 985 990 Thr Lys Thr Met Asn Lys Gly Gln Gln Leu Thr Tyr Glu Ser Phe Gln 995 1000 1005 Tyr Ala Thr Ile Asn Ser Thr Phe Thr Phe Glu Asn Ile Asn Asp Ser 1010 1015 1020 Leu Thr Ile Gly Ala Asp Gln Phe Leu Ser Gly Glu Gln Val Tyr Val1025 1030 1035 1040 Asp Arg Tyr Glu Val Ile Pro Val Asp Ala Thr Phe Glu Ala Glu Asn 1045 1050 1055 Asp Leu Glu Val Ala Lys 1060 243228DNAArtificial SequenceMBP-2A-12 24atggacaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt

300tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac 1140aacaacctcg ggatcgaggg aaggatttca gaattaggca tgcgaatgag tccaaataat 1200caaaatgaat atgaaattat agatgcgaca ccttctactt ctgtatccaa tgattctaac 1260agataccctt ttgcgaatga gccaacaaat gcgctacaaa atatggatta taaagattat 1320ttaaaaatgt ctgcgggaaa tgctagtgaa taccctggtt cacctgaagt acttgtcagc 1380ggacaagatg cagctaaggc cgcaattgat atagtaggta aattactatc aggtttaggg 1440gtcccatttg ttgggccgat agtgagtctt tatactcaac ttattgatat tctgtggcct 1500tcagggcaaa agagtcaatg ggagattttt atggaacaag tagaagaact cataaatcaa 1560aaaatagcag aatatgcaag gaataaagcg ctttcggaat tagaaggatt aggtaataat 1620taccaattat atctaaccgc gcttgaagaa tggaaagaaa atccatttcg tagaggattt 1680agaagaggtg ccttacgaga tgcgcgaaat cgatttgaaa tcctggatag tttatttacg 1740caatatatgc catcttttag agtgacaaat tttgaagtac cattccttac tgtatatgca 1800atggcagcca accttcattt actgttatta aaggacgcgt caatttttgg agaagaatgg 1860ggatggtcaa caactactat taataactat tatgatcgtc aaatgaaact tactgcagaa 1920tattctgatc actgtgtaaa gtggtatgaa actggtttag caaaattaaa aggcacgagc 1980gctaaacaat gggtcgacta caaccaattc cgtagagaaa tgacactgac ggttttagat 2040gttgttgcat tattcccaaa ttatgacaca cgcacgtacc caatggaaac gaaagcacaa 2100ctaacaaggg aagtatatac agatccactg ggcgcggtaa acgtgtcttc aattggttcc 2160tggtatgaca aagcaccttc tttcggagtg atagaatcat ccgttattcg accaccccat 2220gtatttgatt atataacggg actcacagtg tatacacaat caagaagcat ttcttccgct 2280cgctatataa gacattgggc tggtcatcaa ataagctacc atcgtgtcag taggggtagt 2340aatcttcaac aaatgtatgg aactaatcaa aatctacaca gcaccagtac ctttgatttt 2400acgaattatg atatttacaa gacgttatca aaagatgcgg tgctccttga tattgttttt 2460cctggttata cgtatatatt ttttggaatg ccagaagtcg agtttttcat ggtaaaccaa 2520ttgaataata ccagaaagac gttaaagtat aatccggttt ccaaagatat tatagcgggg 2580acaagagatt cggaattaga attgcctcca gaaacttcag atcaaccaaa ttatgagtca 2640aatagccata gattatgtca tatcacaagt attcccgcga cgggtaacac taccggatta 2700gtgcctgtat tttcttggac acatcgaagt gcagatttaa acaatacaat atattcagat 2760aaaatcactc aaattccggc cgttaaatgt tgggataatt taccgtttgt tccagtggta 2820aaaggaccag gacatacagg aggggattta ttacagtata atagaagtac tggttctgta 2880ggaaccttat ttctagctcg atatggccta gcattagaaa aagcaggaaa atatcgtgta 2940agactgagat atgctactga tgcagatatt gtattgcatg taaacgatgc tcagattcag 3000atgccaaaaa caatgaaccc aggtgaggat ctgacatcta aaacttttaa agttgcagat 3060gctatcacaa cagttaattt agcaacagat agttcggttg cagttaaaca taatgttggt 3120gaagacccta attcaacagt ttctggtata gtttacgttg accgaatcga attcatccca 3180gtagatgaga catatgaagc ggaacaagat ttagaagcgg ccaaatga 3228251075PRTArtificial SequenceMBP-2A-12DOMAIN(1)...(368)MBPDOMAIN(369)...(393)LinkerDOMAIN(394)- ...(1075)2A-12 25Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250 255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly 370 375 380 Ile Glu Gly Arg Ile Ser Glu Leu Gly Met Arg Met Ser Pro Asn Asn385 390 395 400 Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro Ser Thr Ser Val Ser 405 410 415 Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu Pro Thr Asn Ala Leu 420 425 430 Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met Ser Ala Gly Asn Ala 435 440 445 Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val Ser Gly Gln Asp Ala 450 455 460 Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu Leu Ser Gly Leu Gly465 470 475 480 Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr Thr Gln Leu Ile Asp 485 490 495 Ile Leu Trp Pro Ser Gly Gln Lys Ser Gln Trp Glu Ile Phe Met Glu 500 505 510 Gln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala Glu Tyr Ala Arg Asn 515 520 525 Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn Asn Tyr Gln Leu Tyr 530 535 540 Leu Thr Ala Leu Glu Glu Trp Lys Glu Asn Pro Phe Arg Arg Gly Phe545 550 555 560 Arg Arg Gly Ala Leu Arg Asp Ala Arg Asn Arg Phe Glu Ile Leu Asp 565 570 575 Ser Leu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe Glu 580 585 590 Val Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu His Leu Leu 595 600 605 Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser Thr 610 615 620 Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr Ala Glu625 630 635 640 Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu Ala Lys Leu 645 650 655 Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn Gln Phe Arg Arg 660 665 670 Glu Met Thr Leu Thr Val Leu Asp Val Val Ala Leu Phe Pro Asn Tyr 675 680 685 Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala Gln Leu Thr Arg Glu 690 695 700 Val Tyr Thr Asp Pro Leu Gly Ala Val Asn Val Ser Ser Ile Gly Ser705 710 715 720 Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser Val Ile 725 730 735 Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly Leu Thr Val Tyr Thr 740 745 750 Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile Arg His Trp Ala Gly 755 760 765 His Gln Ile Ser Tyr His Arg Val Ser Arg Gly Ser Asn Leu Gln Gln 770 775 780 Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr Ser Thr Phe Asp Phe785 790 795 800 Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu Leu 805 810 815 Asp Ile Val Phe Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro Glu 820 825 830 Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr Leu 835 840 845 Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Gly Thr Arg Asp Ser 850 855 860 Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu Ser865 870 875 880 Asn Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr Gly Asn 885 890 895 Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His Arg Ser Ala Asp 900 905 910 Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr Gln Ile Pro Ala Val 915 920 925 Lys Cys Trp Asp Asn Leu Pro Phe Val Pro Val Val Lys Gly Pro Gly 930 935 940 His Thr Gly Gly Asp Leu Leu Gln Tyr Asn Arg Ser Thr Gly Ser Val945 950 955 960 Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala Leu Glu Lys Ala Gly 965 970 975 Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp Ala Asp Ile Val Leu 980 985 990 His Val Asn Asp Ala Gln Ile Gln Met Pro Lys Thr Met Asn Pro Gly 995 1000 1005 Glu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala Asp Ala Ile Thr Thr 1010 1015 1020 Val Asn Leu Ala Thr Asp Ser Ser Val Ala Val Lys His Asn Val Gly1025 1030 1035 1040 Glu Asp Pro Asn Ser Thr Val Ser Gly Ile Val Tyr Val Asp Arg Ile 1045 1050 1055 Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu Gln Asp Leu Glu 1060 1065 1070 Ala Ala Lys 1075263153DNAArtificial SequenceMBP-4c6 26atggacaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac 1140aacaacctcg ggatcgaggg aaggatttca gaattaggca tgacttcaaa taggaaaaat 1200gagaatgaaa ttataaatgc cttatcgatt ccagctgtat cgaatcattc cgcacaaatg 1260gatctatcgc tagatgctcg tattgaggat tctttgtgta tagccgaggg gaataatatc 1320aatccacttg ttagcgcatc aacagtccaa acgggtataa acatagctgg tagaatattg 1380ggcgtattag gtgtgccgtt tgctggacaa ctagctagtt tttatagttt tcttgttggg 1440gaattatggc ctagtggcag agatccatgg gaaattttcc tggaacatgt agaacaactt 1500ataagacaac aagtaacaga aaatactagg aatacggcta ttgctcgatt agaaggtcta 1560ggaaaaggct atagatctta ccagcaggct cttgaaactt ggttagataa ccgaaatgat 1620gcaagatcaa gaagcattat tcttgagcgc tatgttgctt tagaacttga cattactact 1680gctataccgc ttttcagaat acgaaatgaa gaagttccat tattaatggt atatgctcaa 1740gctgcaaatt tacacctatt attattgaga gacgcatccc tttttggtag tgaatggggg 1800atggcatctt ccgatgttaa ccaatattac caagaacaaa tcaggtatac agaggaatat 1860tctaaccatt gcgtacaatg gtataataca gggctaaata acttaagagg gacaaatgct 1920gaaagttggc tgcggtataa tcaattccgt agagacctaa cgttaggggt attagattta 1980gtagccctat tcccaagcta tgatactcgc acttatccaa tcaatacgag tgctcagtta 2040acaagagaaa tttatacaga tccaattggg agaacaaatg caccttcagg atttgcaagt 2100acgaattggt ttaataataa tgcaccatcg ttttctgcca tagaggctgc cattttcagg 2160cctccgcatc tacttgattt tccagaacaa cttacaattt acagtgcatc aagccgttgg 2220agtagcactc aacatatgaa ttattgggtg ggacataggc ttaacttccg cccaatagga 2280gggacattaa atacctcaac acaaggactt actaataata cttcaattaa tcctgtaaca 2340ttacagttta cgtctcgtga cgtttataga acagaatcaa atgcagggac aaatatacta 2400tttactactc ctgtgaatgg agtaccttgg gctagattta attttataaa ccctcagaat 2460atttatgaaa gaggcgccac tacctacagt caaccgtatc agggagttgg gattcaatta 2520tttgattcag aaactgaatt accaccagaa acaacagaac gaccaaatta tgaatcatat 2580agtcatagat tatctcatat aggactaatc ataggaaaca ctttgagagc accagtctat 2640tcttggacgc atcgtagtgc agatcgtacg aatacgattg gaccaaatag aattactcaa 2700attcctgcag tgaagggaag atttcttttt aatggttctg taatttcagg accaggattt 2760actggtggag acgtagttag attgaatagg aataatggta atattcaaaa tagagggtat 2820attgaagttc caattcaatt cacgtcgaca tctaccagat atcgagttcg agtacgttat 2880gcttctgtaa cctcgattga gctcaatgtt aatttgggca attcatcaat ttttacgaac 2940acattaccag caacagctgc atcattagat aatctacaat caggggattt tggttatgtt 3000gaaatcaaca atgcttttac atccgcaaca ggtaatatag taggtgctag aaattttagt 3060gcaaatgcag aagtaataat agacagattt gaatttatcc cagttactgc aaccttcgag 3120gcagaatatg atttagaaag agcacaaaag tga 3153271050PRTArtificial SequenceMBP-4c6DOMAIN(1)...(368)MBPDOMAIN(369)...(393)LinkerDOMAIN(394)..- .(1050)4c6 27Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250

255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly 370 375 380 Ile Glu Gly Arg Ile Ser Glu Leu Gly Met Thr Ser Asn Arg Lys Asn385 390 395 400 Glu Asn Glu Ile Ile Asn Ala Leu Ser Ile Pro Ala Val Ser Asn His 405 410 415 Ser Ala Gln Met Asp Leu Ser Leu Asp Ala Arg Ile Glu Asp Ser Leu 420 425 430 Cys Ile Ala Glu Gly Asn Asn Ile Asn Pro Leu Val Ser Ala Ser Thr 435 440 445 Val Gln Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val Leu Gly 450 455 460 Val Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly465 470 475 480 Glu Leu Trp Pro Ser Gly Arg Asp Pro Trp Glu Ile Phe Leu Glu His 485 490 495 Val Glu Gln Leu Ile Arg Gln Gln Val Thr Glu Asn Thr Arg Asn Thr 500 505 510 Ala Ile Ala Arg Leu Glu Gly Leu Gly Lys Gly Tyr Arg Ser Tyr Gln 515 520 525 Gln Ala Leu Glu Thr Trp Leu Asp Asn Arg Asn Asp Ala Arg Ser Arg 530 535 540 Ser Ile Ile Leu Glu Arg Tyr Val Ala Leu Glu Leu Asp Ile Thr Thr545 550 555 560 Ala Ile Pro Leu Phe Arg Ile Arg Asn Glu Glu Val Pro Leu Leu Met 565 570 575 Val Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala 580 585 590 Ser Leu Phe Gly Ser Glu Trp Gly Met Ala Ser Ser Asp Val Asn Gln 595 600 605 Tyr Tyr Gln Glu Gln Ile Arg Tyr Thr Glu Glu Tyr Ser Asn His Cys 610 615 620 Val Gln Trp Tyr Asn Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala625 630 635 640 Glu Ser Trp Leu Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr Leu Gly 645 650 655 Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr 660 665 670 Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Ile Tyr Thr Asp Pro 675 680 685 Ile Gly Arg Thr Asn Ala Pro Ser Gly Phe Ala Ser Thr Asn Trp Phe 690 695 700 Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ile Phe Arg705 710 715 720 Pro Pro His Leu Leu Asp Phe Pro Glu Gln Leu Thr Ile Tyr Ser Ala 725 730 735 Ser Ser Arg Trp Ser Ser Thr Gln His Met Asn Tyr Trp Val Gly His 740 745 750 Arg Leu Asn Phe Arg Pro Ile Gly Gly Thr Leu Asn Thr Ser Thr Gln 755 760 765 Gly Leu Thr Asn Asn Thr Ser Ile Asn Pro Val Thr Leu Gln Phe Thr 770 775 780 Ser Arg Asp Val Tyr Arg Thr Glu Ser Asn Ala Gly Thr Asn Ile Leu785 790 795 800 Phe Thr Thr Pro Val Asn Gly Val Pro Trp Ala Arg Phe Asn Phe Ile 805 810 815 Asn Pro Gln Asn Ile Tyr Glu Arg Gly Ala Thr Thr Tyr Ser Gln Pro 820 825 830 Tyr Gln Gly Val Gly Ile Gln Leu Phe Asp Ser Glu Thr Glu Leu Pro 835 840 845 Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu 850 855 860 Ser His Ile Gly Leu Ile Ile Gly Asn Thr Leu Arg Ala Pro Val Tyr865 870 875 880 Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro Asn 885 890 895 Arg Ile Thr Gln Ile Pro Ala Val Lys Gly Arg Phe Leu Phe Asn Gly 900 905 910 Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly Asp Val Val Arg Leu 915 920 925 Asn Arg Asn Asn Gly Asn Ile Gln Asn Arg Gly Tyr Ile Glu Val Pro 930 935 940 Ile Gln Phe Thr Ser Thr Ser Thr Arg Tyr Arg Val Arg Val Arg Tyr945 950 955 960 Ala Ser Val Thr Ser Ile Glu Leu Asn Val Asn Leu Gly Asn Ser Ser 965 970 975 Ile Phe Thr Asn Thr Leu Pro Ala Thr Ala Ala Ser Leu Asp Asn Leu 980 985 990 Gln Ser Gly Asp Phe Gly Tyr Val Glu Ile Asn Asn Ala Phe Thr Ser 995 1000 1005 Ala Thr Gly Asn Ile Val Gly Ala Arg Asn Phe Ser Ala Asn Ala Glu 1010 1015 1020 Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val Thr Ala Thr Phe Glu1025 1030 1035 1040 Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys 1045 10502825PRTArtificial SequenceNEB pMAL Linker 28Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly1 5 10 15 Ile Glu Gly Arg Ile Ser Glu Leu Gly 20 25 2919PRTArtificial SequenceSA Linker 29Arg Ile Thr Lys Ser Met Ser Ala Ser Ala Ser Ala Ser Ala Ser Ala1 5 10 15 Ser Ala Arg3051PRTArtificial SequenceNusA Linker 30Thr Ser Gly Ser Gly His His His His His His Ser Ala Gly Lys Glu1 5 10 15 Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser Pro Pro Pro 20 25 30 Thr Gly Leu Val Pro Arg Gly Ser Ala Gly Ser Gly Thr Ile Asp Asp 35 40 45 Asp Asp Lys 50 3149PRTArtificial SequenceTrxA Linker 31Gly Ser Gly Ser Gly His Met His His His His His His Ser Ser Gly1 5 10 15 Leu Val Pro Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe 20 25 30 Glu Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp 35 40 45 Lys 323174DNAArtificial SequenceMBP-SA-RX002 32atggacaaga tcgaggaggg caagctcgtg atctggatca acggcgacaa gggctacaac 60ggcctcgccg aggtcggcaa gaagttcgag aaggacaccg gcatcaaggt gaccgtcgag 120cacccggaca agctggagga gaagttcccc caggtggctg cgaccggcga cggcccggac 180attatcttct gggcccacga caggttcggc ggatacgccc agagcggcct tctcgctgag 240atcacgccag acaaggcctt ccaggacaag ctgtatccat tcacttggga cgctgtccgc 300tacaacggca agctgatcgc ctaccccatc gctgtggagg cactgtcact gatctacaac 360aaggacctcc ttcctaaccc accgaagacg tgggaggaga tcccagcgct ggacaaggag 420ctcaaggcga agggcaagtc cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480cctctgattg ccgctgacgg cggctacgcc ttcaagtacg agaacgggaa gtacgacatc 540aaggacgtcg gcgtcgacaa cgctggcgcc aaggccgggc tcacattcct tgtggacctg 600atcaagaaca agcacatgaa cgccgacacg gactactcta tcgctgaggc tgccttcaac 660aaaggggaga cagccatgac catcaacggc ccctgggcct ggagcaacat cgacaccagc 720aaggtcaact acggcgtgac cgtcctccct acgttcaagg gccagccgtc caagccattc 780gttggcgtgc tgagcgccgg catcaacgcg gcctccccca acaaggagct cgcgaaagag 840ttcctggaga actacctgct cactgacgag ggcctggagg cggtcaacaa ggacaagccg 900cttggcgccg tcgctctgaa gtcttacgag gaggagctgg ccaaggaccc taggatcgcc 960gctacgatgg agaacgccca gaagggcgag atcatgccca acatcccgca gatgtccgcg 1020ttctggtatg cggtgcgcac tgccgtcatc aacgctgcca gcggcaggca gaccgttgac 1080gaggcgctta aggacgcgca gaccaggatc acgaagtcta tgagcgcctc tgcttcagcc 1140tccgcgagcg cctccgctag gagcatgcgc ccgaacaacc agaacgagta cgagatcatc 1200gacaccccat cccgcacgtc cgtctccaac gactccgtcc gctacccgtt cgcgaacgag 1260ccgactaccg atctgaacaa catgaactac aaggacttcc tgaagaccgt gaatggctac 1320aacaccggcg acctgtccgg gtccgaggcg tttatctccc agacggcgat atctacagcc 1380ggcaaggcgg tcggcaccgt tctcggactg ctcggcgtcc cgctggctgg ggctgtcggc 1440ccgttgatca ccttctacgg cacgatcgca ctcctcttct ggggacccgg cgacccttgg 1500caggctttca tgactcaggt cgaggccctc gttaaccaga agatcgccga ttacgccagg 1560tcaaaggcca tatccgagct gcaggggctc cgtaacatct tggacctcta ccgttcagcg 1620ctcatcgact ggcaggagaa cccgacacgc accaggtccg tgaccaacat aagatctcag 1680ttcgagactg ttaataactt cttcgaatac cagatgccgt ccttcgccgt cgcggggtac 1740gaggtcccgt tgctcgcagt gtacgcgcag gccgcgaacc tccacctcag cattctgagg 1800gacgcagcca ctttcggcgc ccaatggggt atgagccaaa ccgccatcaa caacatatac 1860gacctgcagc agcgtaggac ggccgagtac accaaccact gcgtcaagtg gtacaataat 1920ggcctcgaca agctccgtgg cagcaacgcc ggacagtggg tcaacttcaa ccgctatcgc 1980agggagatga ccctcatggt cctcgacatt gtcgccattt tcccgaacta cgacacgcgg 2040acctacccgt ccgggattgg aacctctgtg cagctgacca gggaggtcta cacggacccg 2100ataggctcca ctgcgacaca aggtggtgtc tcctggtacg acgaggcccc gagcttcaca 2160gccatcgagt cctccgtggt ccggccgctt cacctgttcg accttcttac cggcgtcacc 2220gtctacgccg ccagctccag ctgggactcc agccattact tcaggttctg gaatgggcac 2280aaagtggaca ccaagggcat caactcgtcc atccagtact ccaacgttta cggctctacg 2340agcaacgctg tctccacgac caccatttcg ttcagcggtt tcgaggtgtt caagaccatc 2400tccattgccg gtgtcctgtt cgcctggacg accaggtact tcggggtccc gaaggtcctc 2460ttctccaaga tcgaccctat ctcgggaatc ggcagggact cagagttcag cgaaaggtat 2520gctgggatcg gcgagcagat taagaactca ctcgaggagc tgcccctgca aaccgaggac 2580gagccggact acaagtccta ctcccacaaa ctcaaccaca tctccatggt cccgcagaca 2640gtccgtacca ggaacgtccc ggtgttctcc tggtcccacc ggtccgccga catagacaac 2700cgtatcttcc aggacaggat caaccagatc ccggttgtca agggacacac actcggaccg 2760ggcgcttccg tcatggctgg cccgggcttc accggcggca acatagtgac taggacatct 2820ccgggcgtgg ttgtgttctc cggtgtcacc atcaataacg ctctgtctca gaggtaccgc 2880gtcaggatac gctacgccag cacgaccgac ttccgtttct tctcgactct ctccggtacc 2940aggctgtacg ccacccaggc gacgaagacg atgaacaagg gccagcagct cacgtacgaa 3000tccttccagt acgccacaat caacagtacc ttcacgttcg aaaacatcaa cgactccctc 3060actatcggcg ccgaccagtt ccttagcggc gagcaagtct acgtggaccg ctacgaggtt 3120atccccgtcg acgcgacctt cgaggccgag aacgacctgg aggtcgcgaa atga 3174331057PRTArtificial SequenceMBP-SA-RX002 33Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250 255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 Arg Ile Thr Lys Ser Met Ser Ala Ser Ala Ser Ala Ser Ala Ser Ala 370 375 380 Ser Ala Arg Ser Met Arg Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile385 390 395 400 Asp Thr Pro Ser Arg Thr Ser Val Ser Asn Asp Ser Val Arg Tyr Pro 405 410 415 Phe Ala Asn Glu Pro Thr Thr Asp Leu Asn Asn Met Asn Tyr Lys Asp 420 425 430 Phe Leu Lys Thr Val Asn Gly Tyr Asn Thr Gly Asp Leu Ser Gly Ser 435 440 445 Glu Ala Phe Ile Ser Gln Thr Ala Ile Ser Thr Ala Gly Lys Ala Val 450 455 460 Gly Thr Val Leu Gly Leu Leu Gly Val Pro Leu Ala Gly Ala Val Gly465 470 475 480 Pro Leu Ile Thr Phe Tyr Gly Thr Ile Ala Leu Leu Phe Trp Gly Pro 485 490 495 Gly Asp Pro Trp Gln Ala Phe Met Thr Gln Val Glu Ala Leu Val Asn 500 505 510 Gln Lys Ile Ala Asp Tyr Ala Arg Ser Lys Ala Ile Ser Glu Leu Gln 515 520 525 Gly Leu Arg Asn Ile Leu Asp Leu Tyr Arg Ser Ala Leu Ile Asp Trp 530 535 540 Gln Glu Asn Pro Thr Arg Thr Arg Ser Val Thr Asn Ile Arg Ser Gln545 550 555 560 Phe Glu Thr Val Asn Asn Phe Phe Glu Tyr Gln Met Pro Ser Phe Ala 565 570 575 Val Ala Gly Tyr Glu Val Pro Leu Leu Ala Val Tyr Ala Gln Ala Ala 580 585 590 Asn Leu His Leu Ser Ile Leu Arg Asp Ala Ala Thr Phe Gly Ala Gln 595 600 605 Trp Gly Met Ser Gln Thr Ala Ile Asn Asn Ile Tyr Asp Leu Gln Gln 610 615 620 Arg Arg Thr Ala Glu Tyr Thr Asn His Cys Val Lys Trp Tyr Asn Asn625 630 635 640 Gly Leu Asp Lys Leu Arg Gly Ser Asn Ala Gly Gln Trp Val Asn Phe 645 650 655 Asn Arg Tyr Arg Arg Glu Met Thr Leu Met Val Leu Asp Ile Val Ala 660 665 670 Ile Phe Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Ser Gly Ile Gly Thr 675 680 685 Ser Val Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Ile Gly Ser Thr 690 695 700 Ala Thr Gln Gly Gly Val Ser Trp Tyr Asp Glu Ala Pro Ser Phe Thr705 710 715 720 Ala Ile Glu Ser Ser Val Val Arg Pro Leu His Leu Phe Asp Leu Leu 725 730 735 Thr Gly Val Thr Val Tyr Ala Ala Ser Ser Ser Trp Asp Ser Ser His 740 745 750 Tyr Phe Arg Phe Trp Asn Gly His Lys Val Asp Thr Lys Gly Ile Asn 755 760 765 Ser Ser Ile Gln Tyr Ser Asn Val Tyr Gly Ser Thr Ser Asn Ala Val 770 775 780 Ser Thr Thr Thr Ile Ser Phe Ser Gly Phe Glu Val Phe Lys Thr Ile785 790 795 800 Ser Ile Ala Gly Val Leu Phe Ala Trp Thr Thr Arg Tyr Phe Gly Val 805 810 815 Pro Lys Val Leu Phe Ser Lys Ile Asp Pro Ile Ser Gly Ile Gly Arg 820 825 830 Asp Ser Glu Phe Ser Glu Arg Tyr Ala Gly Ile Gly Glu Gln Ile Lys 835 840 845 Asn Ser Leu Glu Glu Leu Pro Leu Gln Thr Glu Asp Glu Pro

Asp Tyr 850 855 860 Lys Ser Tyr Ser His Lys Leu Asn His Ile Ser Met Val Pro Gln Thr865 870 875 880 Val Arg Thr Arg Asn Val Pro Val Phe Ser Trp Ser His Arg Ser Ala 885 890 895 Asp Ile Asp Asn Arg Ile Phe Gln Asp Arg Ile Asn Gln Ile Pro Val 900 905 910 Val Lys Gly His Thr Leu Gly Pro Gly Ala Ser Val Met Ala Gly Pro 915 920 925 Gly Phe Thr Gly Gly Asn Ile Val Thr Arg Thr Ser Pro Gly Val Val 930 935 940 Val Phe Ser Gly Val Thr Ile Asn Asn Ala Leu Ser Gln Arg Tyr Arg945 950 955 960 Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Phe Arg Phe Phe Ser Thr 965 970 975 Leu Ser Gly Thr Arg Leu Tyr Ala Thr Gln Ala Thr Lys Thr Met Asn 980 985 990 Lys Gly Gln Gln Leu Thr Tyr Glu Ser Phe Gln Tyr Ala Thr Ile Asn 995 1000 1005 Ser Thr Phe Thr Phe Glu Asn Ile Asn Asp Ser Leu Thr Ile Gly Ala 1010 1015 1020 Asp Gln Phe Leu Ser Gly Glu Gln Val Tyr Val Asp Arg Tyr Glu Val1025 1030 1035 1040 Ile Pro Val Asp Ala Thr Phe Glu Ala Glu Asn Asp Leu Glu Val Ala 1045 1050 1055 Lys34368PRTEscherichia coliDOMAIN(1)...(368)MBP 34Met Asp Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp1 5 10 15 Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu65 70 75 80 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp 85 90 95 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val 100 105 110 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro 115 120 125 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys 130 135 140 Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp145 150 155 160 Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 165 170 175 Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200 205 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr 210 215 220 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser225 230 235 240 Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro 245 250 255 Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala305 310 315 320 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro 325 330 335 Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala 340 345 350 Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 35495PRTEscherichia coliDOMAIN(1)...(495)NusA 35Met Asn Lys Glu Ile Leu Ala Val Val Glu Ala Val Ser Asn Glu Lys1 5 10 15 Ala Leu Pro Arg Glu Lys Ile Phe Glu Ala Leu Glu Ser Ala Leu Ala 20 25 30 Thr Ala Thr Lys Lys Lys Tyr Glu Gln Glu Ile Asp Val Arg Val Gln 35 40 45 Ile Asp Arg Lys Ser Gly Asp Phe Asp Thr Phe Arg Arg Trp Leu Val 50 55 60 Val Asp Glu Val Thr Gln Pro Thr Lys Glu Ile Thr Leu Glu Ala Ala65 70 75 80 Arg Tyr Glu Asp Glu Ser Leu Asn Leu Gly Asp Tyr Val Glu Asp Gln 85 90 95 Ile Glu Ser Val Thr Phe Asp Arg Ile Thr Thr Gln Thr Ala Lys Gln 100 105 110 Val Ile Val Gln Lys Val Arg Glu Ala Glu Arg Ala Met Val Val Asp 115 120 125 Gln Phe Arg Glu His Glu Gly Glu Ile Ile Thr Gly Val Val Lys Lys 130 135 140 Val Asn Arg Asp Asn Ile Ser Leu Asp Leu Gly Asn Asn Ala Glu Ala145 150 155 160 Val Ile Leu Arg Glu Asp Met Leu Pro Arg Glu Asn Phe Arg Pro Gly 165 170 175 Asp Arg Val Arg Gly Val Leu Tyr Ser Val Arg Pro Glu Ala Arg Gly 180 185 190 Ala Gln Leu Phe Val Thr Arg Ser Lys Pro Glu Met Leu Ile Glu Leu 195 200 205 Phe Arg Ile Glu Val Pro Glu Ile Gly Glu Glu Val Ile Glu Ile Lys 210 215 220 Ala Ala Ala Arg Asp Pro Gly Ser Arg Ala Lys Ile Ala Val Lys Thr225 230 235 240 Asn Asp Lys Arg Ile Asp Pro Val Gly Ala Cys Val Gly Met Arg Gly 245 250 255 Ala Arg Val Gln Ala Val Ser Thr Glu Leu Gly Gly Glu Arg Ile Asp 260 265 270 Ile Val Leu Trp Asp Asp Asn Pro Ala Gln Phe Val Ile Asn Ala Met 275 280 285 Ala Pro Ala Asp Val Ala Ser Ile Val Val Asp Glu Asp Lys His Thr 290 295 300 Met Asp Ile Ala Val Glu Ala Gly Asn Leu Ala Gln Ala Ile Gly Arg305 310 315 320 Asn Gly Gln Asn Val Arg Leu Ala Ser Gln Leu Ser Gly Trp Glu Leu 325 330 335 Asn Val Met Thr Val Asp Asp Leu Gln Ala Lys His Gln Ala Glu Ala 340 345 350 His Ala Ala Ile Asp Thr Phe Thr Lys Tyr Leu Asp Ile Asp Glu Asp 355 360 365 Phe Ala Thr Val Leu Val Glu Glu Gly Phe Ser Thr Leu Glu Glu Leu 370 375 380 Ala Tyr Val Pro Met Lys Glu Leu Leu Glu Ile Glu Gly Leu Asp Glu385 390 395 400 Pro Thr Val Glu Ala Leu Arg Glu Arg Ala Lys Asn Ala Leu Ala Thr 405 410 415 Ile Ala Gln Ala Gln Glu Glu Ser Leu Gly Asp Asn Lys Pro Ala Asp 420 425 430 Asp Leu Leu Asn Leu Glu Gly Val Asp Arg Asp Leu Ala Phe Lys Leu 435 440 445 Ala Ala Arg Gly Val Cys Thr Leu Glu Asp Leu Ala Glu Gln Gly Ile 450 455 460 Asp Asp Leu Ala Asp Ile Glu Gly Leu Thr Asp Glu Lys Ala Gly Ala465 470 475 480 Leu Ile Met Ala Ala Arg Asn Ile Cys Trp Phe Gly Asp Glu Ala 485 490 495 36109PRTEscherichia coliDOMAIN(1)...(109)TrxA 36Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala 100 105

Claims

1. A method of enhancing pesticidal activity of a Cry endotoxin, the method comprising operably linking a first amino acid sequence of a solubility-enhancing polypeptide to a second amino acid sequence of a Cry endotoxin, whereby a chimeric pesticidal polypeptide is produced, the chimeric pesticidal polypeptide comprising the first amino acid sequence operably linked to second amino acid sequence.

2. The method of claim 1; wherein the solubility-enhancing polypeptide is selected from the group consisting of maltose-binding protein (MBP), thioredoxin, and transcription elongation factor NusA.

3. The method of claim 2, wherein the solubility-enhancing polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 4, 6, 34, 35 and 36.

4. The method of claim 1, 2 or 3, wherein the Cry endotoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 10, 12 and 14.

5. The method of claim 1, 2, 3 or 4, wherein the chimeric pesticidal polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 2, 16, 18, 21, 23, 25, 27 and 33.

6. A chimeric pesticidal polypeptide comprising a first amino acid sequence of a solubility-enhancing polypeptide operably linked to a second amino acid sequence of a Cry endotoxin, wherein the operably linked first and second amino sequences comprise an amino acid sequence selected from the group consisting of: (a) the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33; and (b) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32.

7. A nucleic acid molecule encoding the chimeric pesticidal polypeptide of claim 6.

8. An expression cassette comprising a promoter that drives expression in a host cell operably linked to the nucleic acid molecule of claim 7.

9. A vector comprising the expression cassette of claim 8.

10. A transformed plant, plant part, plant cell or seed comprising in its genome the expression cassette of claim 8.

11. A pesticidal composition comprising an effective amount of a chimeric pesticidal polypeptide of claim 6 or an active variant or fragment thereof having pesticidal activity.

12. A method of protecting a plant from an insect pest, the method comprising providing an effective amount of the pesticidal composition of claim 10 to reduce insect pest-related damage to the plant.

13. A plant comprising a polynucleotide construct stably incorporated in its genome, the polynucleotide construct comprising a nucleotide sequence operably linked to a promoter that drives expression in the plant, wherein the nucleotide sequence encodes a chimeric pesticidal polypeptide comprising a first amino acid sequence of a solubility-enhancing polypeptide operably linked to a second amino acid sequence of a Cry endotoxin, wherein the solubility-enhancing polypeptide is selected from the group consisting of maltose-binding protein (MBP), thioredoxin, and transcription elongation factor NusA.

14. The plant of claim 13, wherein the nucleotide sequence is selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32; and (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33.

15. A method of protecting a plant, plant part or plant host cell from an insect pest, the method comprising the steps of: (a) introducing into the plant, plant part or plant host cell an expression cassette of claim 7; and (b) regenerating the plant, plant part or plant host cell into a morphologically normal fertile plant, wherein the plant or part thereof comprises a chimeric pesticidal polypeptide.

16. A method of enhancing the resistance of a plant to at least one pest, the method comprising introducing into a plant or at least one plant cell a polynucleotide construct comprising a nucleotide sequence operably linked to a promoter that drives expression in the plant, wherein the nucleotide sequence encodes a chimeric pesticidal polypeptide comprising a first amino acid sequence of a solubility-enhancing polypeptide operably linked to a second amino acid sequence of a Cry endotoxin.

17. The method of claim 16, wherein the solubility-enhancing polypeptide is selected from the group consisting of maltose-binding protein (MBP), thioredoxin, and transcription elongation factor NusA.

18. The method of claim 17, wherein the nucleotide sequence comprises a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32; and (b) a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33.

Images