Patent application title: Preparation of Deallergenized Proteins and Permuteins
Inventors:
Murtaza E. Alibhai (Chesterfield, MO, US)
James D. Astwood (Eureka, MO, US)
Charles A. Mcwherter (Chesterfield, MO, US)
Hugh A. Sampson (Larchmont, NY, US)
IPC8 Class: AG01N3353FI
USPC Class:
435 71
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay
Publication date: 2008-10-16
Patent application number: 20080254484
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Patent application title: Preparation of Deallergenized Proteins and Permuteins
Inventors:
Hugh A. Sampson
Murtaza E. Alibhai
James D. Astwood
Charles A. McWherter
Agents:
HOWREY LLP
Assignees:
Origin: FALLS CHURCH, VA US
IPC8 Class: AG01N3353FI
USPC Class:
435 71
Abstract:
Modified proteins are disclosed that maintain enzymatic and insecticidal
activity while displaying reduced or eliminated allergenicity. Epitopes
which bind to anti-patatin antibodies were identified, and removed via
site directed mutagenesis. Tyrosines were observed to generally
contribute to the allergenic properties of patatin proteins. Removal of
glycosylation sites was observed to reduce or eliminate antibody binding.
Permuteins are also disclosed which have a rearranged amino acid sequence
while retaining enzymatic activity.
Deallergenized proteins and permuteins can be used as insecticidal
materials, as nutritional supplements, and as immunotherapeutic agents.Claims:
1-53. (canceled)
54. A method for decreasing allergen eliciting properties of a native protein comprising the steps of:a) identifying a patient exhibiting an allergic sensitivity to said native protein and obtaining serum from said patient;b) exposing synthetic overlapping peptides representative of said native protein to said patient serum to identify peptides exhibiting epitopes which bind IgE present within said patient serum;c) producing variant peptides exhibiting alanine scanning or rational scanning amino acid substitutions based on peptides from step (b), wherein said variant peptides exhibit decreased IgE binding compared to peptides from step (b), said amino acid substitutions comprising result effective substitutions;d) modifying the amino acid sequence of said native protein to contain one or more of said result effective substitutions, thereby obtaining a modified protein; ande) isolating and purifying the modified protein comprising said one or more result effective amino acid substitutions;wherein the modified protein exhibits decreased allergen eliciting properties of said native protein as determined by reduced binding of IgE when exposed to said patient serum when compared with said native protein.
55. The method according to claim 54, wherein said native protein is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, and SEQ ID NO:293.
56. A method for producing a modified acyl lipid hydrolase protein comprising:a) identifying a patient exhibiting an allergic sensitivity to a native acyl lipid hydrolase protein and obtaining serum from said patient;b) exposing synthetic overlapping peptides representative of said native acyl lipid hydrolase protein to said patient serum to identify peptides exhibiting epitopes which bind immunoglobulins present within said patient serum, said immunoglobulins exhibiting a binding specificity for said native acyl lipid hydrolase protein;c) producing variant peptides exhibiting alanine scanning or rational scanning amino acid substitutions based on peptides from step (b), wherein said variant peptides exhibit decreased immunoglobulin binding compared to peptides from step (b), said amino acid substitutions comprising result effective substitutions;d) modifying the amino acid sequence of said native acyl lipid hydrolase protein to contain one or more of said result effective substitutions, thereby obtaining a modified acyl lipid hydrolase protein; ande) isolating and purifying the modified acyl lipid hydrolase protein comprising said one or more result effective amino acid substitutions;wherein the modified acyl lipid hydrolase protein exhibits reduced binding of immunoglobulins when exposed to said patient serum when compared with said native acyl lipid hydrolase protein.
57-60. (canceled)
61. The method according to claim 56, wherein said native acyl lipid hydrolase protein is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, and SEQ ID NO:293.
62. The method according to claim 54, wherein said synthetic overlapping peptides are selected from the group consisting of SEQ ID NOs:16-104.
63. The method according to claim 56, wherein said synthetic overlapping peptides are selected from the group consisting of SEQ ID NOs:16-104.
64. The method according to claim 54, wherein said epitopes contain tyrosine and said result effective substitutions include substitutions of alanine or phenylalanine for tyrosine.
65. The method according to claim 56, wherein said epitopes contain tyrosine and said result effective substitutions include substitutions of alanine or phenylalanine for tyrosine.
66. The method for decreasing allergen eliciting properties of a native protein according to claim 54, wherein said modified protein is used in immunotherapy.
67. The method for producing a modified acyl lipid hydrolase protein according to claim 56, wherein said modified acyl lipid hydrolase protein is used in immunotherapy.
Description:
FIELD OF THE INVENTION
[0001]The invention relates generally to non-naturally occurring novel proteins which have insecticidal properties, and more specifically to the design, preparation, and use of proteins that have been deallergenized while maintaining their insecticidal properties. Deallergenized patatin proteins include variants that have had allergenic sequences modified, and permuteins that have had their amino acid sequences rearranged at one or more breakpoints.
BACKGROUND OF THE INVENTION
[0002]Insecticidal Proteins
[0003]The use of natural products, including proteins, is a well known method of controlling many insect, fungal, viral, bacterial, and nematode pathogens. For example, endotoxins of Bacillus thuringiensis (B.t.) are used to control both lepidopteran and coleopteran insect pests. Genes producing these endotoxins have been introduced into and expressed by various plants, including cotton, tobacco, and tomato. There are, however, several economically important insect pests such as boll weevil (BWV), Anthonomus grandis, and corn rootworm (CRW), Diabrotica spp. that are not as susceptible to B.t. endotoxins as are insects such as lepidopterans. In addition, having other, different gene products for control of insects which are susceptible to B.t. endotoxins is important, if not vital, for resistance management.
[0004]It has been recently discovered that the major storage protein of potato tubers, patatins (Gaillaird, T., Biochem. J. 121: 379-390, 1971; Racusen, D., Can. J. Bot., 62: 1640-1644, 1984; Andrews, D. L., et al., Biochem. J, 252: 199-206, 1988), will control various insects, including western rootworm (WCRW, Diabrotica virigifera), southern corn rootworm (SCRW, Diabrotica undecimpunctata), and boll weevil (BWV, Anthonomus grandis) (U.S. Pat. No. 5,743,477). Patatins are lethal to some larvae and will stunt the growth of survivors so that maturation is prevented or severely delayed, resulting in no reproduction. These proteins, have nonspecific lipid acyl hydrolase activity and studies have shown that the enzyme activity is essential for its insecticidal activity (Strickland, J. A., et al., Plant Physiol., 109: 667-674, 1995; U.S. Pat. No. 5,743,477). Patatins can be applied directly to the plants or introduced in other ways well known in the art, such as through the application of plant-colonizing microorganisms, which have been transformed to produce the enzymes, or by the plants themselves after similar transformation.
[0005]In potato, the patatins are found predominantly in tubers, but also at much lower levels in other plant organs (Hofgen, R. and Willmitzer, L., Plant Science, 66: 221-230, 1990). Genes that encode patatins have been previously isolated by Mignery, G. A., et al. (Nucleic Acids Research, 12: 7987-8000, 1984; Mignery, G. A., et al., Gene, 62: 27-44, 1988; Stiekema, et al., Plant Mol. Biol., 11: 255-269, 1988) and others. Patatins are found in other plants, particularly solanaceous species (Ganal, et al., Mol. Gen. Genetics, 225: 501-509, 1991; Vancanneyt, et al., Plant Cell, 1: 533-540, 1989) and recently Zea mays (WO 96/37615). Rosahl, et al. (EMBO J., 6: 1155-1159, 1987) transferred it to tobacco plants, and observed expression of patatin, demonstrating that the patatin genes can be heterologously expressed by plants.
[0006]Patatin is an attractive for use in planta as an insect control agent, but unfortunately a small segment of the population displays allergic reactions to patatin proteins, and in particular to potato patatin, as described below.
[0007]Food Allergens
[0008]There are a variety of proteins that cause allergic reactions. Proteins that have been identified as causing an allergic reaction in hypersensitive patients occur in many plant and animal derived foods, pollens, fungal spores, insect venoms, insect feces, and animal dander and urine (King, H. C., Ear Nose Throat J., 73(4): 237-241, 1994; Astwood, J. D., et al., Clin. Exp. Allergy, 25: 66-72, 1995; Astwood, J. D. and Fuchs R. L., Monographs in allergy Vol. 32: Highlights in food allergy, pp. 105-120, 1996; Metcalfe, D. D., et al., Critical Reviews in Food Science and Nutrition, 36S: 165-186, 1996). The offending proteins of many major sources of allergens have been characterized by clinical and molecular methods. The functions of allergenic proteins in vivo are diverse, ranging from enzymes to regulators of the cell cytoskeleton.
[0009]To understand the molecular basis of allergic disease, the important IgE binding epitopes of many allergen proteins have been mapped (Elsayed, S. and Apold, J., Allergy 38(7): 449-459, 1983; Elsayed, S. et al., Scand J. Clin. Lab. Invest. Suppl. 204: 17-31 1991; Zhang, L., et al., Mol. Immunol. 29(11): 1383-1389, 1992). The optimal peptide length for IgE binding has been reported to be between 8 and 12 amino acids. Conservation of epitope sequences is observed in homologous allergens of disparate species (Astwood, J. D., et al., Clin. Exp. Allergy, 25: 66-72, 1995). Indeed, conservative substitutions introduced by site-directed mutagenesis reduce IgE binding of known epitopes when presented as peptides.
[0010]Food allergy occurs in 2-6% of the population. Eight foods or food groups (milk, eggs, fish, crustacea, wheat, peanuts, soybeans, and tree nuts) account for 90% of allergies to foods. Nevertheless, over 160 different foods have been reported to cause adverse reactions, including potato (Hefle, S., et al., Crit. Rev. in Food Sci. Nutr., 36S: 69-90, 1996).
[0011]Mode of Action of Allergens
[0012]Regardless of the identity of the allergen, it is theorized that the underlying mechanism of allergen response is the same. Immediate hypersensitivity (or anaphylactic response) is a form of allergic reaction which develops very quickly, i.e., within seconds or minutes of exposure of the patient to the causative allergen, and is mediated by B lymphocyte IgE antibody production. Allergic patients exhibit elevated levels of IgE, mediating hypersensitivity by priming mast cells which are abundant in the skin, lymphoid organs, in the membranes of the eye, nose and mouth, and in the respiratory tree and intestines. The IgE in allergy-suffering patients becomes bound to the IgE receptors of mast cells. When this bound IgE is subsequently contacted by the appropriate allergen, the mast cell is caused to degranulate and release various substances such as histamine into the surrounding tissue (Church et al. In: Kay, A. B. ed., Allergy and Allergic Diseases, Oxford, Blackwell Science, pp. 149-197, 1997).
[0013]It is the release of these substances which is responsible for the clinical symptoms typical of immediate hypersensitivity, namely contraction of smooth muscle in the airways or in the intestine, the dilation of small blood vessels, and the increase in their permeability to water and plasma proteins, the secretion of thick sticky mucus, and (in the skin) the stimulation of nerve endings that result in itching or pain. Immediate hypersensitivity is, at best, a nuisance to the suffer; at worst it can present very serious problems and can in rare cases even result in death.
[0014]Allergic Reactions to Potato
[0015]Food allergy to potato is considered rare in the general population (Castells, M. C., et al., Allergy Clin. Immunol., 8: 1110-1114, 1986; Hannuksela, M., et al., Contact Dermatitis, 3: 79-84, 1977; Golbert, T. M., et al., Journal of Allergy, 44: 96-107, 1969). Approximately 200 individuals have participated in published clinical accounts of potato allergy (Hefle, S. et al., Critical Reviews in Food Science and Nutrition, 36S: 69-90, 1996). A number of IgE binding proteins have been identified in potato tuber extracts (see Table 1), however the amino acid sequence and function of these proteins has not been determined (Wahl, R., et al., Intl. Arch. Allergy Appl. Immunol., 92: 168-174, 1990).
TABLE-US-00001 TABLE 1 Studies of potato tuber IgE-binding proteins (allergens) Study Protein Characteristics (Castells, M. C. et al. J. Allergy Clin. Unknown 14 to 40 kDa Immunol. 78, 1110-1114, 1986) (Wahl, R. et al. Int. Arch. Allergy Appl. Unknown 42/43 kDa Immunol. 92: 168-174, 1990) Unknown 65 kDa Unknown 26 kDa Unknown 20 kDa Unknown 14 kDa Unknown <14 kDa (~5 kDa) (Ebner, C. et al. in: Wuthrich, Unknown 42/43 kDa B. & Ortolani, C. (eds.), Highlights in food allergy. Monographs in Allergy, Volume 32 Basil, Karger, pp. 73-77, 1996) Unknown 23 kDa Unknown ~16 kDa Unknown <14 kDa (~5 kDa)
[0016]Improved Safety from the Use of Hypoallergenic Proteins
[0017]Patatin has been identified as an allergenic protein (Seppala, U. et al., J. Allergy Clin. Immunol. 103:165-171, 1999). Accordingly, potato allergic subjects may not be able to safely consume products containing unmodified patatin protein, such as crops to which foliar applications of patatins have been applied, or crops which have been engineered to express patatin. In addition, proliferation of food allergens in the food supply is considered hazardous (Metcalfe, D. D., et al., Critical Reviews and Food Science and Nutrition, 36S: 165-186, 1996). There are additional concerns regarding the use of potentially allergenic food proteins where workers might be exposed to airborne particulates, initiating a new allergic response (Moneret-Vautrin, D. A., et al., Rev. Med. Interne., 17(7): 551-557, 1996).
[0018]Permuteins
[0019]Novel proteins generated by the method of sequence transposition resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al. Proc. Natl. Sci., U.S.A., 76: 3218-3222, 1979; Teather, et al., J. Bacteriol., 172: 3837-3841, 1990; Schimming, et al., Eur. J. Biochem., 204: 13-19, 1992; Yamiuchi, et al., FEBS Lett., 260: 127-130, 1991; MacGregor, et al., FEBS. Lett., 378: 263-266, 1996). The first in vitro application of sequence rearrangement to proteins was described by Goldenberg and Creighton (Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion or sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain. This approach has been applied to proteins which range in size from 58 to 462 amino acids and represent a broad range of structural classes (Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983; Li and Coffino, Mol. Cell. Biol., 13: 2377-2383, 1993; Zhang, et al., Nature Struct. Biol., 1: 434-438, 1995; Buchwalder, et al., Biochemistry, 31: 1621-1630, 1994; Protasova, et al., Prot. Eng., 7: 1373-1377, 1995; Mullins, et al., J. Am. Chem. Soc., 116: 5529-5533, 1994; Garrett, et al., Protein Science, 5: 204-211, 1996; Hahn, et al., Proc. Natl. Acad. Sci. U.S.A., 91: 10417-10421, 1994; Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984, 1993; Luger, et al., Science, 243: 206-210, 1989; Luger, et al., Prot. Eng., 3: 249-258, 1990; Lin, et al., Protein Science, 4: 159-166, 1995; Vignais, et al., Protein Science, 4: 994-1000, 1995; Ritco-Vonsovici, et al., Biochemistry, 34: 16543-16551, 1995; Horlick, et al., Protein Eng., 5: 427-431, 1992; Kreitman, et al., Cytokine, 7: 311-318, 1995; Viguera, et al., Mol. Biol., 247: 670-681, 1995; Koebnik and Kramer, J. Mol. Biol., 250: 617-626, 1995; Kreitman, et al., Proc. Natl. Acad. Sci., 91: 6889-6893, 1994).
[0020]There exists a need for the development of plant expressible insecticidal proteins which possess minimal or no allergenic properties.
SUMMARY OF THE INVENTION
[0021]Novel protein sequences, and nucleic acid sequences encoding them are disclosed. The proteins maintain desirable enzymatic and insecticidal properties while displaying reduced or eliminated allergenicity.
[0022]Allergenic epitopes are identified by scanning overlapping peptide sequences with an immunoreactivity assay. Alanine scanning and `rational substitution` is performed on identified peptide sequences to determine specific amino acids which contribute to antibody binding, and presumably, to the allergenic properties of the whole protein. Individual mutations are introduced into the whole protein sequence by methods such as site directed mutagenesis of the encoding nucleic acid sequence to delete or modify the allergenic sequences.
[0023]Glycosylation target residues are identified within amino acid sequences of proteins which have demonstrated allergy eliciting properties. Glycosylation target amino acid residues are rationally substituted with other amino acid residues to eliminate glycosylation and to provide a variant deglycosylated protein. The variant protein may then exhibit reduced allergen eliciting properties and may also exhibit reduced binding to IgE within serum of patients observed to be allergic to said glycosylated protein.
[0024]Permuteins of the deallergenized protein sequences can be constructed to further reduce or eliminate allergic reactions. The encoding nucleic acid sequence is modified to produce a non-naturally occurring protein having a linear amino acid sequence different from the naturally occurring protein sequence, while maintaining enzymatic and insecticidal properties. The permutein is preferably produced in plant cells, and more preferably produced at a concentration which is toxic to insects ingesting the plant cells.
[0025]Methods for reducing, eliminating, or decreasing allergen eliciting properties of a protein are specifically contemplated herein. Such methods comprise steps including identifying one or more patients exhibiting an allergic sensitivity to an allergen eliciting protein and obtaining a sample of serum from the patient; exposing the patient serum to a first set of synthetic overlapping peptides which represent the allergen eliciting protein in order to identify such peptides which exhibit epitopes which bind to IgE present within the allergic patients' serum and wherein the IgE present in the serum has a specific affinity for the said allergen eliciting protein; producing a second set of peptides which are variant peptides based on the first set of peptides which were identified to bind specifically to IgE present in patient serum, wherein the second set variant peptides exhibit alanine scanning or rational scanning amino acid substitutions which exhibit reduced, decreased, or eliminated IgE binding when compared to the first set non-variant peptides, and wherein such substitutions which reduce, eliminate or decrease IgE binding are identified as result effective substitutions; and modifying the amino acid sequence of the allergen eliciting protein to contain one or more of said result effective substitutions, wherein the modified protein is a variant of the allergen eliciting protein which lacks allergen eliciting protein or exhibits reduced allergen eliciting properties, and wherein the variant of the allergen eliciting protein comprising one or more result effective substitutions exhibits reduced, decreased, or totally eliminated binding of IgE present within said patients' serum.
[0026]The novel proteins can be used in controlling insects, as nutritional supplements, in immunotherapy protocols, and in other potential applications. Transgenic plant cells and plants containing the encoding nucleic acid sequence can be particularly beneficial in the control of insects, and as a nutritional/immunotherapy material.
DESCRIPTION OF THE FIGURES
[0027]The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0028]FIG. 1 illustrates the alignment of potato patatin PatA (acyl lipid hydrolase) with patatin (acyl lipid hydrolase) homologs and related amino acid sequences, the homologs and related sequences being from both dicot and monocot plant species.
[0029]FIG. 2 illustrates IgE binding to overlapping peptide sequences.
[0030]FIG. 3 illustrates construction of nucleic acid sequences encoding patatin permutein proteins, and in this figure for illustrative purposes a breakpoint at position 247 is shown.
DESCRIPTION OF THE SEQUENCE LISTINGS
[0031]The following description of the sequence listing forms part of the present specification and is included to further demonstrate certain aspects of the present invention. The invention can be better understood by reference to one or more of these sequences in combination with the detailed description of specific embodiments presented herein. [0032]SEQ ID NO:1 DNA sequence encoding a patatin (acyl lipid hydrolase) protein [0033]SEQ ID NO:2 potato patatin protein sequence [0034]SEQ ID NO:3 thermal amplification primer [0035]SEQ ID NO:4 thermal amplification primer [0036]SEQ ID NO:5 thermal amplification product [0037]SEQ ID NO:6 Pre-cleavage patatin protein produced in Pichia pastoris [0038]SEQ ID NO:7 Post-cleavage patatin protein produced in Pichia pastoris [0039]SEQ ID NO:8 Y106F mutagenic primer [0040]SEQ ID NO:9 Y129F mutagenic primer [0041]SEQ ID NO:10 Y185F mutagenic primer [0042]SEQ ID NO:11 Y193F mutagenic primer [0043]SEQ ID NO:12 Y185F and Y193F mutagenic primer [0044]SEQ ID NO:13 Y270F mutagenic primer [0045]SEQ ID NO:14 Y316F mutagenic primer [0046]SEQ ID NO:15 Y362F mutagenic primer [0047]SEQ ID NO:16-104 Peptide scan sequences of a patatin protein [0048]SEQ ID NO:105-241 Alanine and rational scan sequences of selected patatin peptides [0049]SEQ ID NO:242 thermal amplification primer 27 [0050]SEQ ID NO:243 thermal amplification primer 48 [0051]SEQ ID NO:244 thermal amplification primer 47 [0052]SEQ ID NO:245 thermal amplification primer 36 [0053]SEQ ID NO:246 pMON37402 sequence encoding permutein protein [0054]SEQ ID NO:247 Permutein protein encoded from pMON37402 sequence [0055]SEQ ID NO:248 thermal amplification primer 58 [0056]SEQ ID NO:249 thermal amplification primer 59 [0057]SEQ ID NO:250 pMON37405 sequence encoding permutein protein [0058]SEQ ID NO:251 Permutein protein encoded by pMON37405 sequence [0059]SEQ ID NO:252 thermal amplification primer 60 [0060]SEQ ID NO:253 thermal amplification primer 61 [0061]SEQ ID NO:254 pMON37406 sequence encoding permutein protein [0062]SEQ ID NO:255 Permutein protein encoded by pMON37406 sequence [0063]SEQ ID NO:256 thermal amplification primer 62 [0064]SEQ ID NO:257 thermal amplification primer 63 [0065]SEQ ID NO:258 pMON37407 sequence encoding permutein protein [0066]SEQ ID NO:259 Permutein protein encoded by pMON37407 sequence [0067]SEQ ID NO:260 thermal amplification primer 60 [0068]SEQ ID NO:261 thermal amplification primer 65 [0069]SEQ ID NO:262 pMON37408 sequence encoding permutein protein [0070]SEQ ID NO:263 Permutein protein encoded by pMON37408 sequence [0071]SEQ ID NO:264 pMON40701 sequence encoding permutein protein [0072]SEQ ID NO:265 Permutein protein encoded by pMON40701 sequence [0073]SEQ ID NO:266 thermal amplification primer Syn1 [0074]SEQ ID NO:267 thermal amplification primer Syn2 [0075]SEQ ID NO:268 thermal amplification primer Syn3 [0076]SEQ ID NO:269 thermal amplification primer Syn4 [0077]SEQ ID NO:270 pMON40703 sequence encoding permutein protein [0078]SEQ ID NO:271 Permutein protein encoded by pMON40703 sequence [0079]SEQ ID NO:272 thermal amplification primer Syn10 [0080]SEQ ID NO:273 thermal amplification primer Syn11 [0081]SEQ ID NO:274 pMON40705 sequence encoding permutein protein [0082]SEQ ID NO:275 Permutein protein encoded by pMON40705 sequence [0083]SEQ ID NO:276-277 Permutein linker sequences [0084]SEQ ID NO:278 Patatin isozyme PatA+ (including signal peptide) [0085]SEQ ID NO:279 Patatin isozyme PatB+ (including signal peptide) [0086]SEQ ID NO:280 Patatin isozyme PatFm (mature protein lacking signal peptide) [0087]SEQ ID NO:281 Patatin isozyme PatIm (mature protein lacking signal peptide) [0088]SEQ ID NO:282 Patatin isozyme PatL+ (including signal peptide) [0089]SEQ ID NO:283 Rational substitution peptide [0090]SEQ ID NO:284 Corn homolog peptide [0091]SEQ ID NO:285 patatin homolog Pat17 DNA coding sequence and amino acid translation [0092]SEQ ID NO:286 patatin homolog Pat17 amino acid sequence [0093]SEQ ID NO:287 dicot patatin homolog amino acid sequence pentin1_phb [0094]SEQ ID NO:288 dicot patatin homolog amino acid sequence 5c9_phb [0095]SEQ ID NO:289 maize patatin homolog amino acid sequence corn1_pep [0096]SEQ ID NO:290 maize patatin homolog amino acid sequence corn2_pep [0097]SEQ ID NO:291 maize patatin homolog amino acid sequence corn3_pep [0098]SEQ ID NO:292 maize patatin homolog amino acid sequence corn4_pep [0099]SEQ ID NO:293 maize patatin homolog amino acid sequence corn5_pep
DEFINITIONS
[0100]The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention. Some words and phrases may also be defined in other sections of the specification. No limitation should be placed on the definitions presented for the terms below, where other meanings are evidenced elsewhere in the specification in addition to those specified below.
[0101]Allergen" refers to a biological or chemical substance that induces an allergic reaction or response. An allergic response can be an immunoglobulin E-mediated response.
[0102]Amino acid codes: A (Ala)=alanine; C (Cys)=cysteine; D (Asp)=aspartic acid; E (Glu)=glutamic acid; F (Phe)=phenylalanine; G (Gly)=glycine; H (His)=histidine; I (Ile)=isoleucine; K (Lys)=lysine; L (Leu)=leucine; M (Met)=methionine; N (Asn)=asparagine; P (Pro)=proline; Q (Gln)=glutamine; R (Arg)=arginine; S (Ser)=serine; T (Thr)=threonine; V=(Val) valine; W (Trp)=tryptophan; Y (Tyr)=tyrosine.
[0103]Amplification: refers to increasing the number of copies of a desired molecule.
[0104]Coding sequence", "open reading frame", and "structural sequence" refer to the region of continuous sequential nucleic acid base pair triplets encoding a protein, polypeptide, or peptide sequence.
[0105]Codon" refers to a sequence of three nucleotides that specify a particular amino acid.
[0106]Complementarity" refers to the specific binding of adenine to thymine (or uracil in RNA) and cytosine to guanine on opposite strands of DNA or RNA.
[0107]Deallergenize" (render hypoallergenic) refers to the method of engineering or modifying a protein or the encoding DNA such that the protein has a reduced or eliminated ability to induce an allergic response with respect to the ability of the unmodified protein. A deallergenized protein can be referred to as being hypoallergenic. The degree of deallergenization of a protein can be measured in vitro by the reduced binding of IgE antibodies.
[0108]DNA segment heterologous to the promoter region" means that the coding DNA segment does not exist in nature in the same gene with the promoter to which it is now attached.
[0109]DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species.
[0110]Electroporation" refers to a method of introducing foreign DNA into cells that uses a brief, high voltage DC (direct current) charge to permeabilize the host cells, causing them to take up extra-chromosomal DNA.
[0111]Encoding DNA" refers to chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA which encodes any of the enzymes discussed herein.
[0112]Endogenous" refers to materials originating from within an organism or cell.
[0113]Endonuclease" refers to an enzyme that hydrolyzes double stranded DNA at internal locations.
[0114]Epitope" refers to a region on an allergen that interacts with the cells of the immune system. Epitopes are often further defined by the type of antibody or cell with which they interact, e.g. if the region reacts with B-cells or antibodies (IgE), it is called a B-cell epitope.
[0115]Exogenous" refers to materials originating from outside of an organism or cell. This typically applies to nucleic acid molecules used in producing transformed or transgenic host cells and plants.
[0116]Expressibly coupled" and "expressibly linked" refer to a promoter or promoter region and a coding or structural sequence in such an orientation and distance that transcription of the coding or structural sequence can be directed by the promoter or promoter region.
[0117]Expression" refers to the transcription of a gene to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product, i.e., a peptide, polypeptide, or protein.
[0118]Heterologous DNA" refers to DNA from a source different than that of the recipient cell.
[0119]Homologous DNA" refers to DNA from the same source as that of the recipient cell.
[0120]Identity" refers to the degree of similarity between two nucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
[0121]IgE" (Immunoglobulin E) refers to a specific class of immunoglobulin secreted by B cells. IgE binds to specific receptors on Mast cells. Interaction of an allergen with mast cell-bound IgE may trigger allergic symptoms.
[0122]Immunotherapy" refers to any type of treatment that targets the immune system. Allergy immunotherapy is a treatment in which a progressively increasing dose of an allergen is given in order to induce an immune response characterized by tolerance to the antigen/allergen, also known as desensitization.
[0123]In vitro" refers to "in the laboratory" and/or "outside of a living organism".
[0124]In vivo" refers to "in a living organism".
[0125]Insecticidal polypeptide" refers to a polypeptide having insecticidal properties that adversely affects the growth and development of insect pests.
[0126]Monocot" refers to plants having a single cotyledon (the first leaf of the embryo of seed plants); examples include cereals such as maize, rice, wheat, oats, and barley.
[0127]Multiple cloning site" refers to an artificially constructed collection of restriction enzyme sites in a vector that facilitates insertion of foreign DNA into the vector.
[0128]Mutation" refers to any change or alteration in a nucleic acid sequence. Several types exist, including point, frame shift, splicing, and insertion/deletions.
[0129]Native" refers to "naturally occurring in the same organism". For example, a native promoter is the promoter naturally found operatively linked to a given coding sequence in an organism. A native protein is one naturally found in nature and untouched or not otherwise manipulated by the hand of man.
[0130]Nucleic acid segment" is a nucleic acid molecule that has been isolated free of total genomic DNA of a particular species, or that has been synthesized. Included with the term "nucleic acid segment" are DNA segments, recombinant vectors, plasmids, cosmids, phagemids, phage, viruses, etcetera.
[0131]Nucleic acid" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
[0132]Nucleic acid codes: A=adenosine; C=cytosine; G=guanosine; T=thymidine; N=equimolar A, C, G, and T; I=deoxyinosine; K=equimolar G and T; R=equimolar A and G; S=equimolar C and G; W=equimolar A and T; Y=equimolar C and T.
[0133]Open reading frame (ORF)" refers to a region of DNA or RNA encoding a peptide, polypeptide, or protein or capable of being translated to protein, or a region of DNA capable of being transcribed into an RNA product.
[0134]Plasmid" refers to a circular, extrachromosomal, self-replicating piece of DNA.
[0135]Point mutation" refers to an alteration of a single nucleotide in a nucleic acid sequence.
[0136]Polymerase chain reaction (PCR)" refers to an enzymatic technique to create multiple copies of one sequence of nucleic acid. Copies of DNA sequence are prepared by shuttling a DNA polymerase between two oligonucleotides. The basis of this amplification method is multiple cycles of temperature changes to denature, then re-anneal amplimers, followed by extension to synthesize new DNA strands in the region located between the flanking amplimers. Also known as thermal amplification.
[0137]Probe" refers to a polynucleotide sequence which is complementary to a target polynucleotide sequence in the analyte. An antibody can also be used as a probe to detect the presence of an antigen. In that sense, the antigen binding domain of the antibody has some detectable affinity for the antigen and binds thereto. The binding of the antibody to the antigen can be measured by means known in the art, such as by chemiluminescence, phosphorescence, flourescence, colorimetric chemical deposition at the site of binding, or otherwise.
[0138]Promoter" or "promoter region" refers to a DNA sequence, usually found upstream (5') to a coding sequence, that controls expression of the coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site for RNA polymerase and/or other factors necessary for start of transcription at the correct site. As contemplated herein, a promoter or promoter region includes variations of promoters derived by means of ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences. The promoter region disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its ability to produce mRNA.
[0139]Recombinant DNA construct" or "recombinant vector" refers to any agent such as a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule in which one or more DNA sequences have been linked in a functionally operative manner. Such recombinant DNA constructs or vectors are capable of introducing a 5' regulatory sequence or promoter region and a DNA sequence for a selected gene product into a cell in such a manner that the DNA sequence is transcribed into a functional mRNA which is translated and therefore expressed. Recombinant DNA constructs or recombinant vectors can be constructed to be capable of expressing antisense RNAs, in order to inhibit translation of a specific RNA of interest.
[0140]Recombinant proteins", also referred to as "heterologous proteins", are proteins which are normally not produced by the host cell.
[0141]Regeneration" refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant).
[0142]Regeneration" refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant).
[0143]Regulatory sequence" refers to a nucleotide sequence located upstream (5'), within, and/or downstream (3') to a DNA sequence encoding a selected gene product whose transcription and expression is controlled by the regulatory sequence in conjunction with the protein synthetic apparatus of the cell.
[0144]Restriction enzyme" refers to an enzyme that recognizes a specific palindromic sequence of nucleotides in double stranded DNA and cleaves both strands; also called a restriction endonuclease. Cleavage typically occurs within the restriction site.
[0145]Result-effective substitution" (RES) refers to an amino acid substitution within an IgE-binding region (epitope) of a target protein which reduces or eliminates the IgE binding by that epitope. Examples herein are directed to patatin protein and homologues, however, as will be readily recognized by those skilled in the art, the method is more broadly applicable to proteins other than patatins, and in particular is applicable to any protein exhibiting allergen eliciting properties.
[0146]Selectable marker" refers to a nucleic acid sequence whose expression confers a phenotype facilitating identification of cells containing the nucleic acid sequence. Selectable markers include those which confer resistance to toxic chemicals (e.g. ampicillin resistance, kanamycin resistance), complement a nutritional deficiency (e.g. uracil, histidine, leucine), or impart a visually distinguishing characteristic (e.g. color changes or fluorescence).
[0147]Transcription" refers to the process of producing an RNA copy from a DNA template.
[0148]Transformation" refers to a process of introducing an exogenous nucleic acid sequence (e.g., a vector, recombinant nucleic acid molecule) into a cell or protoplast in which that exogenous nucleic acid is incorporated into a chromosome or is capable of autonomous replication.
[0149]Transformed cell" is a cell whose DNA has been altered by the introduction of an exogenous nucleic acid molecule into that cell.
[0150]Transgenic cell" refers to any cell derived from or regenerated from a transformed cell or derived from a transgenic cell. Exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells obtained from a transgenic plant.
[0151]Transgenic plant" refers to a plant or progeny thereof derived from a transformed plant cell or protoplast, wherein the plant DNA contains an introduced exogenous nucleic acid sequence not originally present in a native, non-transgenic plant of the same species. Alternatively, the plant DNA can contain the introduced nucleic acid sequence in a higher copy number than in the native, non-transgenic plant of the same species.
[0152]Translation" refers to the production of protein from messenger RNA.
[0153]Vector" refers to a plasmid, cosmid, bacteriophage, or virus that carries foreign DNA into a host organism.
[0154]Western blot" refers to protein or proteins that have been separated by electrophoresis, transferred and immobilized onto a solid support, then probed with an antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0155]Design of Deallergenized Patatin Proteins
[0156]Deallergenizing a protein can be accomplished by the identification of allergenic sites, followed by modification of the sites to reduce or eliminate the binding of antibodies to the sites. The IgE-binding regions of patatin were previously unreported. Mapping of the IgE epitopes was accomplished by synthesizing 10-mer peptides based on the patatin 17 protein sequence (SEQ ID NO: 2) which overlap by six amino acids. As potato proteins are denatured upon cooking potato products, it is expected that the 10-mer peptides sufficiently mimic the unfolded full length protein for antibody binding purposes. Peptides were identified based upon their ability to bind to IgE antibodies. Individual amino acids within the identified peptides were changed to reduce or eliminate binding to IgE present in sera from potato sensitive patients. These changes are termed result-effective amino acid substitutions (RES). The RES can be subsequently introduced into the full length protein by site directed mutagenesis of the encoding nucleic acid sequence or other means known in the art. Similar strategies have been employed elsewhere to determine the dominant IgE epitopes in a major peanut allergen (Stanley, J. S., et al., Arch. Biochem. Biophys., 342(2): 244-253, 1997).
[0157]Certain amino acid residues important for allergenicity of patatin are identified. Some of the designed patatin peptides wherein single amino acid residues were replaced with alanine or phenylalanine, showed significantly reduced or no binding to sera from potato sensitive patients.
[0158]A "deallergenized patatin" refers to a patatin protein differing in at least one of the amino acid residues as defined by the result effective substitutions resulting in the patatin protein having reduced reactivity towards sera from potato sensitive patients. The deallergenized patatin preferably maintains insecticidal properties, and preferably maintains its characteristic enzymatic profile.
[0159]Summary of Method to Deallergenize a Patatin Protein [0160]Mapping of IgE epitopes by immunoassay of synthetic overlapping peptides using sera from potato sensitive patients; [0161]Identification of result-effective substitutions by alanine scanning and/or rational scanning; [0162]Modification of the amino acid sequence of patatin by site-directed mutagenesis of the encoding nucleic acid sequence; [0163]Evaluation of enzyme activity (esterase) and/or insecticidal activity of the modified protein(s); and [0164]Evaluation of the new protein(s) for allergenicity by IgE immunoassay.
[0165]Nucleic acid sequences encoding patatin have been cloned by several investigators (e.g. Mignery, et al., Nucleic Acids Research, 12: 7987-8000, 1984; Mignery, et al., Gene, 62: 27-44, 1988; WO 94/21805; Canadian Patent Application No. 2090552). These nucleic acid sequences can then be manipulated using site directed mutagenesis to encode a hypoallergenic patatin. These nucleic acid sequences can than be used to transform bacterial, yeast or plant cells, resulting in the production of hypoallergenic patatin protein.
[0166]Deallergenized Patatin Proteins
[0167]For simplicity, individual amino acids are referred to by their single letter codes. Correlation between the single letter codes, three letter codes, and full amino acid names is presented in the definitions section above.
[0168]One embodiment of the invention is an isolated deallergenized patatin protein. The protein is modified relative to the wild-type protein sequence such that they exhibit reduced binding to anti-patatin antibodies such as those obtained from humans or animals allergic to potatoes. The reduced binding is measured relative to the binding of the unmodified patatin protein to the anti-patatin antibodies.
[0169]The deallergenized patatin protein can comprise SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
[0170]Preferably, the deallergenized patatin protein comprises SEQ ID NO:2 modified by one or more changes, or SEQ ID NO:7 modified by one or more changes. SEQ ID NO:7 differs from wild type SEQ ID NO:2 in that the first 22 amino acids of SEQ ID NO:2 are replaced with EAE (Glu-Ala-Glu). For example, the changes to SEQ ID NO:2 or SEQ ID NO:7 can be: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
[0171]More preferably, SEQ ID NO:2 is modified by the following changes or SEQ ID NO:7 is modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 Of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0172]Most preferably, SEQ ID NO:2 is modified by the following changes or SEQ ID NO:7 is modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0173]Nucleic Acids
[0174]An additional embodiment of the invention is an isolated nucleic acid molecule segment comprising a structural nucleic acid sequence which encodes a deallergenized patatin protein.
[0175]The structural nucleic acid sequence can generally encode any deallergenized patatin protein. The structural nucleic acid sequence preferably encodes a deallergenized patatin protein comprising SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7),184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
[0176]Alternatively, the structural nucleic acid sequence encodes SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
[0177]More preferably, the structural nucleic acid sequence encodes SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y Corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0178]Most preferably, the structural nucleic acid sequence encodes SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0179]Recombinant Vectors
[0180]An additional embodiment is directed towards recombinant vectors comprising a structural nucleic acid sequence which encodes a deallergenized patatin protein. The recombinant vector comprises operatively linked in the 5' to 3' orientation: a promoter that directs transcription of a structural nucleic acid sequence; a structural nucleic acid sequence, and a 3' transcription terminator.
[0181]The structural nucleic acid sequence can encode SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
[0182]Alternatively, the recombinant vector comprises operatively linked in the 5' to 3' orientation: a promoter that directs transcription of a structural nucleic acid sequence; a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 of position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A; and a 3' transcription terminator.
[0183]More preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0184]Most preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0185]Recombinant Host Cells
[0186]A further embodiment of the invention is directed towards recombinant host cells comprising a structural nucleic acid sequence encoding a deallergenized patatin protein. The recombinant host cell preferably produces a deallergenized patatin protein. More preferably, the recombinant host cell produces a deallergenized patatin protein in a concentration sufficient to inhibit growth or to kill an insect which ingests the recombinant host cell. The recombinant host cell can generally comprise any structural nucleic acid sequence encoding a deallergenized patatin protein.
[0187]The recombinant host cell can comprise a structural nucleic acid sequence encoding SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
[0188]Alternatively, the recombinant host cell comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
[0189]More preferably, the recombinant host cell comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0190]Most preferably, the recombinant host cell comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0191]The recombinant host cell can generally be any type of host cell, and preferably is a bacterial, fungal, or plant cell. The bacterial cell is preferably an Escherichia coli bacterial cell. The fungal cell is preferably a Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris fungal cell. The plant cell can be a monocot, dicot, or conifer plant cell. The plant cell is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant cell. The recombinant host cell preferably further comprises operatively linked to the structural nucleic acid sequence a promoter that directs transcription of the structural nucleic acid sequence. The recombinant host cell preferably further comprises operatively linked to the structural nucleic acid sequence a 3' transcription terminator and a polyadenylation site.
[0192]Recombinant Plants
[0193]An additional embodiment of the invention is a recombinant plant comprising a structural nucleic acid sequence encoding a deallergenized patatin protein. The recombinant plant preferably produces a deallergenized patatin protein. More preferably, the recombinant plant produces a deallergenized patatin protein in a concentration sufficient to inhibit growth or to kill an insect which ingests plant tissue from the recombinant plant.
[0194]The recombinant plant can comprise a structural nucleic acid sequence encoding SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
[0195]Alternatively, the recombinant plant can comprise a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
[0196]More preferably, the recombinant plant comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0197]Most preferably, the recombinant plant comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0198]The recombinant plant can generally be any type of plant. The plant can be a monocot, dicot, or conifer plant. The plant is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.
[0199]The recombinant plant preferably further comprises operatively linked to the structural nucleic acid sequence a promoter that directs transcription of the structural nucleic acid sequence. The recombinant plant preferably further comprises operatively linked to the structural nucleic acid sequence a 3' transcription terminator and a polyadenylation site.
[0200]Methods of Preparation
[0201]Embodiments of the invention are further directed towards methods of preparing recombinant host cells and recombinant plants useful for the production of deallergenized patatin proteins.
[0202]A method of preparing a recombinant host cell useful for the production of deallergenized patatin proteins can comprise selecting a host cell; transforming the host cell with a recombinant vector; and obtaining recombinant host cells.
[0203]The recombinant vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
[0204]Alternatively, the recombinant vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
[0205]More preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0206]Most preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID N0:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0207]The method can generally be used to prepare any type of recombinant host cell. Preferably, the method can be used to prepare a recombinant bacterial cell, a recombinant fungal cell, or a recombinant plant cell. The bacterial cell is preferably an Escherichia coli bacterial cell. The fungal cell is preferably a Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris fungal cell. The plant cell can be a monocot, dicot, or conifer plant cell. The plant cell is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant cell.
[0208]An additional embodiment is directed towards methods for the preparation of recombinant plants useful for the production of deallergenized patatin proteins. The method can comprise selecting a host plant cell; transforming the host plant cell with a recombinant vector; obtaining recombinant host cells; and regenerating a recombinant plant from the recombinant host plant cells.
[0209]The recombinant vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
[0210]Alternatively, the recombinant vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
[0211]More preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0212]Most preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
[0213]The recombinant plant can generally be any type of plant. The plant can be a monocot, dicot, or conifer plant. The plant is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.
[0214]Deallergenized patatin proteins can be prepared by isolating the deallergenized patatin protein from any one of the above described host cells or plants.
[0215]Deglycosylation
[0216]The examples herein provide evidence that glycosylation of can contribute to the allergenicity of a protein. Accordingly, rational substitution of amino acid residues likely to be the targets of glycosylation within a subject allergen protein may reduce or eliminate the allergenic properties of the protein without adversely affecting the enzymatic, insecticidal, antifungal or other functional properties of the protein.
[0217]Glycosylation commonly occurs as either N-linked or O-linked forms. N-linked glycosylation usually occurs at the motif Asn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro (Kasturi, L. et al., Biochem J. 323: 415-519, 1997; Melquist, J. L. et al., Biochemistry 37: 6833-6837, 1998). O-linked glycosylation occurs between the hydroxyl group of serine or threonine and an amino sugar.
[0218]Site directed mutagenesis of selected asparagine, serine, or threonine may be used to reduce or eliminate the glycosylation of patatin proteins. A search of SEQ ID NO:2 for the Asn-Xaa-Ser/Thr motif reveals one occurrence at amino acid positions 202-204. Mutagenization of the nucleic acid sequence encoding this region may result in a reduced allergenicity of the encoded protein.
[0219]In order to test this conceptual approach to reducing allergenicity of patatin proteins, two sets of experiments were performed: a) production of patatin proteins in Escherichia coli, which do not glycosylate proteins; and b) production of patatin proteins with an N202Q site directed mutation.
[0220]Antibodies obtained from patients HS-07 and G15-MON (not potato allergic) did not show specific binding to wild type patatin, patatin produced in E. coli, or the N202Q variant. Antibodies obtained from patient HS-01 (potato allergic) bound to wild type patatin, but not to patatin produced in E. coli or the N202Q variant. Antibodies obtained from patient HS-05 (potato allergic) bound strongly to wild type patatin, but extremely weakly to patatin produced in E. coli, and binding to the N202Q variant resembled vector controls. Antibodies obtained from patient HS-03 (potato allergic) bound to wild type patatin, but not to patatin produced in E. coli or the N202Q variant. Antibodies obtained from patient HS-05 (potato allergic) bound to wild type patatin, but very weakly to patatin produced in E. coli and the N202Q variant. Antibodies obtained from patient HS-06 (potato allergic) strongly bound wild type patatin, the N202Q variant, and to patatin produced in E. coli. These results strongly suggest that glycosylation is at least partially responsible for the antigenic properties of patatin proteins, and that site directed mutagenesis may be used to reduce or eliminate specific antibody binding. Mutagenesis at position 202 of SEQ ID NO:2 may be useful for reducing or eliminating specific antibody binding.
[0221]Permuteins
[0222]The positions of the internal breakpoints described in the following Examples are found on the protein surface, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle. Breakpoints occurring below the protein surface can additionally be selected. The rearranged two subunits can be joined by a peptide linker. A preferred embodiment involves the linking of the N-terminal and C-terminal subunits by a three amino acid linker, although linkers of various sizes can be used. Additionally, the N-terminal and C-terminal subunits can be joined lacking a linker sequence. Furthermore, a portion of the C-terminal subunit can be deleted and the connection made from the truncated C-terminal subunit to the original N-terminal subunit and vice versa as previously described (Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984, 1993; Viguera, et al., Mol. Biol., 247: 670-681, 1995; Protasova, et al., Prot. Eng., 7: 1373-1377, 1994).
[0223]The novel insecticidal proteins of the present invention can be represented by the formula:
X1-(L)a-X2
[0224]wherein; [0225]a is 0 or 1, and if a is 0, then the permutein does not contain a linker sequence; [0226]X1 is a polypeptide sequence corresponding to amino acids n+1 through J; [0227]X2 is a polypeptide corresponding to amino acids 1 through n; [0228]n is an integer ranging from 1 to J-1; [0229]J is an integer greater than n+1; and [0230]L is a linker.
[0231]In the formula above, the constituent amino acid residues of the novel insecticidal protein are numbered sequentially 1 through J from the original amino terminus to the original carboxyl terminus. A pair of adjacent amino acids within this protein can be numbered n and n+1 respectively where n is an integer ranging from 1 to J-1. The residue n+1 becomes the new N-terminus of the novel insecticidal protein and the residue n becomes the new C-terminus of the novel insecticidal protein.
[0232]For example, a parent protein sequence consisting of 120 amino acids can be selected as a starting point for designing a permutein (J=120). If the breakpoint is selected as being between position 40 and position 41, then n=40. If a linker is selected to join the two subunits, the resulting permutein will have the formula: (amino acids 41-120)-L-(amino acids 1-40). If a linker was not used, the resulting permutein will have the formula: (amino acids 41-120)-(amino acids 1-40).
[0233]The length of the amino acid sequence of the linker can be selected empirically, by using structural information, or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be made whose length can span a range of 0 to 50 A and whose sequence is chosen in order to be substantially consistent with surface exposure (Hopp and Woods, Mol. Immunol., 20: 483-489, 1983; Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982; Lee and Richards, J. Mol. Biol., 55: 379-400, 1971) and the ability to adopt a conformation which does not significantly affect the overall configuration of the protein (Karplus and Schulz, Naturwissenschaften, 72: 212-213, 1985). Assuming an average length of 2.0 to 3.8 â„« per residue, this would mean the length to test would be between about 0 to about 30 residues, with 0 to about 15 residues being the preferred range. Accordingly, there are many such sequences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor excessively short (Sandhu, et al., Critical Rev. Biotech., 12: 437-467, 1992). If the linker is too long, entropy effects may destabilize the three-dimensional fold and may affect protein folding. If the linker is too short, it may destabilize the molecule due to torsional or steric strain.
[0234]Use of the distance between the chain ends, defined as the distance between the C-alpha carbons, can be used to define the length of the sequence to be used, or at least to limit the number of possibilities that can be tested in an empirical selection of linkers. Using the calculated length as a guide, linkers with a range of number of residues (calculated using 2 to 3.8 A per residue) can be selected. These linkers can be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues can be chosen to be flexible and hydrophilic as described above; or optionally the original sequence can be substituted for using a series of linkers, one example being Gly-Pro-Gly (SEQ ID NO:277); or optionally a combination of the original sequence and new sequence having the appropriate total length can be used. An alternative short, flexible linker sequence is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276).
[0235]Selection of Permutein Breakpoints
[0236]Sequences of novel patatin analogs capable of folding to biologically active molecules can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while optionally using a linker sequence as described above. Amino and carboxyl termini can be selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel amino acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases, the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus. However, selections of termini anywhere within the region may result in a functional protein, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.
[0237]The primary amino acid sequence of a protein dictates folding to the three-dimensional structure beneficial for expression of its biological function. It is possible to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions. Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops (Kabsch and Sander, Biopolymers, 22: 2577-2637, 1983), the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, C., Ann. Rev. Biochem., 53: 537-572, 1984), and the static and dynamic distribution of conformations along the polypeptide chain (Alber and Mathews, Methods Enzymol., 154: 511-533, 1987). In some cases additional information is known about solvent exposure of residues, one example is a site of post-translational attachment of carbohydrate which is necessarily on the surface of the protein. When experimental structural information is not available, or when it is not feasible to obtain the information, methods are available to analyze the primary amino acid sequence in order to make predictions of protein secondary and tertiary structure, solvent accessibility and the occurrence of turns and loops (Fasman, G., Ed. Plenum, New York, 1989; Robson, B. and Garnier, J. Nature 361: 506, 1993).
[0238]Biochemical methods can be applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile, F. and Salvatore, G., Eur. J. Biochem., 218: 603-621, 1993). Thus, using either the experimentally derived structural information or predictive methods (Srinivasan, R. and Rose, G. D. Proteins, 22: 81-99, 1995), the parental amino acid sequence can be analyzed to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The sequences within regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so-called hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini. Regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity, can be preferred sites for new amino and carboxyl termini. Stretches of amino acid sequence that are preferred based on the above criteria can be selected as breakpoint regions.
[0239]An embodiment of the invention is directed towards patatin permutein proteins. The permutein proteins preferably maintain esterase activity and insecticidal properties. The permutein proteins preferably are less allergenic than the wild type patatin protein to individuals or animals allergic to potatoes. This can be assayed by the binding of antibodies to the wild type patatin and patatin permutein proteins.
[0240]The permutein proteins can optionally contain a linker sequence. The linker can generally be any amino acid sequence, preferably is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276) or Gly-Pro-Gly (SEQ ID NO:277), and more preferably is Gly-Pro-Gly (SEQ ID NO:277). Specific permutein proteins comprise: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2), (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2), SEQ ID NO:247, and SEQ ID NO:259.
[0241]Embodiments of the invention also include isolated nucleic acid molecule segments comprising a structural nucleic acid sequence encoding a patatin permutein protein. The encoded permutein protein can generally be any permutein protein, and preferably comprises (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2), (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2), SEQ ID NO:247, or SEQ ID NO:259. The linker can generally be any amino acid sequence, preferably is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276) or Gly-Pro-Gly (SEQ ID NO:277), and more preferably is Gly-Pro-Gly (SEQ ID NO:277). Alternatively, the encoded patatin permutein protein can lack a linker sequence. The structural nucleic acid sequence is preferably SEQ ID NO:246 or SEQ ID NO:258.
[0242]An embodiment of the invention is directed towards recombinant vectors which encode a patatin permutein protein. The vector can comprise operatively linked in the 5' to 3' orientation: a promoter that directs transcription of a structural nucleic acid sequence; a structural nucleic acid sequence encoding a protein selected from the group consisting of: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2); and (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2); and a 3' transcription terminator. The linker can comprise Gly-Pro-Gly (SEQ ID NO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively, the encoded patatin permutein protein can lack a linker sequence. The structural nucleic acid sequence can preferably be SEQ ID NO:246 or SEQ ID NO:258, and preferably encodes SEQ ID NO:247 or SEQ ID NO:259.
[0243]An additional embodiment of the invention is directed towards recombinant host cells useful for the production of a patatin permutein protein. The recombinant host cell preferably produces a patatin permutein protein. More preferably, the recombinant host cell produces a patatin permutein protein in a concentration sufficient to inhibit growth or to kill an insect which ingests the recombinant host cell. The recombinant host cell can comprise a structural nucleic acid sequence encoding a protein selected from the group consisting of: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2); and (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2). The linker can generally be any amino acid sequence, and preferably is Gly-Pro-Gly (SEQ ID NO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively, the encoded patatin permutein protein can lack a linker sequence. The structural nucleic acid sequence is preferably SEQ ID NO:246 or SEQ ID NO:258, and preferably encodes SEQ ID NO:247 or SEQ ID NO:259. The structural nucleic acid sequence can be operatively linked to a promoter sequence that directs transcription of the structural nucleic acid sequence, a 3' transcription terminator, and a 3' polyadenylation signal sequence. The recombinant host cell can generally be any type of host cell, and preferably is a bacterial, fungal, or plant host cell. The bacterial cell is preferably an Escherichia coli bacterial cell. The fungal cell is preferably a Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris fungal cell. The plant cell can be a monocot, dicot, or conifer plant cell. The plant cell is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant cell.
[0244]An additional embodiment of the invention is directed towards recombinant plants which are useful for the production of patatin permutein proteins. The recombinant plant preferably produces a patatin permutein protein. More preferably, the recombinant plant produces a patatin permutein protein in a concentration sufficient to inhibit growth or to kill an insect which ingests tissue from the recombinant plant. The recombinant plant can comprise a structural nucleic acid sequence encoding a protein selected from the group consisting of: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2); and (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2). The linker can comprise Gly-Pro-Gly (SEQ ID NO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively, the encoded protein can lack a linker sequence. The structural nucleic acid sequence is preferably SEQ ID NO:246 or SEQ ID NO:258, and preferably encodes SEQ ID NO:247 or SEQ ID NO:259. The structural nucleic acid sequence can be operatively linked to a promoter sequence that directs transcription of the structural nucleic acid sequence, a 3' transcription terminator, and a 3' polyadenylation signal sequence. The recombinant plant can generally be any type of plant, and preferably is an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.
[0245]Permutein proteins can be prepared by isolating the permutein protein from any one of the above described host cells or plants.
[0246]Immunotherapy for Potato Allergy
[0247]Immunotherapy for food allergy has been largely unsuccessful due to the lack of appropriate therapeutic reagents (Sampson, H. A., J. Allergy Clin. Immunol., 90(2): 151-152, 1992). Immunotherapy has typically involved the administration (orally or by subcutaneous injections) of increasing doses of crude protein extracts of the offending allergenic entities which usually contain variable mixes of many different proteins (Scheiner, O., Wien Klin Wochenschr., 105(22): 653-658, 1993). While there are reports of highly successful clinical applications of immunotherapy for food allergens (Romano, P. C., et al., Allergol. Immunopathol. (Madr), 12(4): 275-281, 1984), those reports are rare and the clinical literature in general recommends avoidance far more strongly than therapy (Gay, G., Allerg. Immunol. (Paris), 29(6): 169-170, 1997). One of the primary reasons for the failure of many clinical attempts to induce tolerance to allergens in general and food allergens in particular relates to anecdotal comments by numerous allergists, that patients don't tolerate the doses of allergen required to achieve tolerance. Animal studies examining the relationship of antigen dose and the induction of tolerance have demonstrated a strong positive correlation (Chen, Y., et al., Proc. Natl. Acad. Sci., U.S.A., 93: 388-391, 1996; Tokai, T., et al., Nat. Biotechnol., 15(8): 754-758, 1997). Due to the very real possibility of inducing an anaphylactic reaction in patients with native allergen, most clinical therapists are quite hesitant to use high doses therapeutically and are therefore compromising the likelihood of successful therapy.
[0248]In recent reports, recombinant technology has been used to reduce the allergenic potential of a major allergen without modifying the T cell epitopes, and allowing higher doses of protein to be used in therapy (Tokai, T., et al., Nat. Biotechnol., 15(8): 754-758, 1997). In addition, a lack of understanding about the appropriate route of administration, the uncertainty of mechanisms responsible for induction of allergy and the uncertainty of mechanisms by which immunotherapy suppresses or blocks the T cell-IgE-eosinophil/mast cell cycle have contributed to the large number of equivocal studies and clinical trials. Recent studies in animal models dealing with mechanisms, routes of administration, adjuvants and vaccine formulations have increased the likelihood that immunotherapy for allergies, including food allergies, will become a reproducibly successful clinical treatment when the appropriate therapeutic reagents are available (Sampson, H. A. and Burks, A. W., Annu. Rev. Nutr., 16: 161-177, 1996; Kaminogawa, S., Biosci. Biotechnol. Biochem., 60(11): 1749-1756, 1996; Chapman, M. D., et al., Allergy, 52: 374-379, 1997; Barbeau, W. E., Adv. Exp. Med. Biol., 415: 183-193, 1997; Cao, Y, et al., Immunology, 90(1): 46-51, 1997; Garside, P. and Mowat, A.M., Crit. Rev. Immunol., 17(2): 119-137, 1997; Rothe, M. J. and Grant-Kels, J. M., J. Am. Acad. Dermatol., 35(1): 1-13, 1996; Strobel, S., Allergy, 50(20): 18-25, 1995; Kruisbeek, A. M. and Amsen, D., Curr. Opin. Immunol., 8(2): 233-244, 1996; Herz, U., et al., Adv. Exp. Med. Biol., 409: 25-32, 1996; Litwin, A., et al., J. Allergy Clin. Immunol., 100: 30-38, 1997; Vandewalker, M. L., Mo. Med., 94(7): 311, 1997; Marshall, G. D., Jr. and Davis, F., Nat. Biotechnol., 15(8): 718-719, 1997; Van Deusen, M. A., et al., Ann. Allergy Asthma Immunol., 78: 573-580, 1997; Jacobsen, L., et al., Allergy, 52: 914-920, 1997, Scheiner, O. and Kraft, D., Allergy 50(5): 384-391, 1995).
[0249]Relative to immunotherapy, the critical aspects of the modified patatin genes described in this patent are that they can be used to synthesize purified, deallergenized-protein which can be used for patatin (potato) specific immunotherapy, with reduced potential for adverse and potentially fatal anaphylactic reactions in human or veterinary patients who have allergies to patatin or potatoes. Various strategies, including fixing or cross linking allergens, encapsulation of allergen for oral delivery, the use of small, T-cell epitope peptides and most recently, the use of engineered recombinant proteins, or modified gene vaccines are being tested in attempts to decrease the potential for anaphylactic reactions while inducing tolerance (Cao, Y., et al., Immunology, 90(1): 46-51, 1997; Chapman, M. D., et al., Allergy, 52: 374-379, 1997; Chapman, M. D., et al., Int. Arch. Allergy Immunol., 113(1-3): 102-104, 1997; Collins, S. P., et al., Clin. Exp. Allergy, 26(1): 36-42, 1996; Takai, T., et al., Mol. Immunol., 34(3): 255-261, 1997; Takai, T., et al., Nat. Biotechnol., 15(8) 754-758, 1997; Jirapongsananruk, O. and Leung, D. Y. M., Ann. Allergy Asthma Immunol., 79: 5-20, 1997; Litwin, A., et al., J. Allergy Clin. Immunol., 100: 30-38, 1997; Vandewalker, M. L., Mo. Med., 94(7): 311, 1997; Raz, E., et al., Proc. Natl. Acad. Sci., U.S.A., 93: 5141-5145, 1996; Hoyne, G. F., et al., Clin. Immunol. Immunopathol., 80: S23-30, 1996; Hoyne, G. F., et al., Int. Immunol., 9(8): 1165-1173, 1997; Vrtala, S., et al., J. Clin. Invest., 99(7): 1673-1681, 1997; Sato, Y., et al., Science, 273: 352-354, 1996; Lee, D. J., et al., Int. Arch. Allergy Immunol., 113(1-3): 227-230, 1997; Tsitoura, D. C., et al., J. Immunol., 157(5): 2160-2165, 1996; Hsu, C. H., et al., Int. Immunol., 8(9):1405-1411, 1996; Hsu, C. H., et al., Nat. Med., 2(5): 540-544, 1996).
[0250]The instant invention uses an engineered patatin protein, as expressed in any living cell, with or without post-synthesis modifications, for immunotherapy by the routes of cutaneous or subcutaneous exposure, injection, or by oral, gastro-intestinal, respiratory or nasal application, either with, or without the use of specific carriers, vehicles and adjuvants. The direct application of nucleic acid encoding recombinant patatin as the in vivo (in the patient) expression template (gene) as RNA-, DNA- or gene-vaccines is also the intended use of the engineered genetic materials defined here, coding for patatin, but with modified IgE binding sites. It is also the intent of this patent to cover the use of these modified genes described here including insertion into various DNA vectors including adenovirus, retrovirus, pox virus and replicating or non-replicating eukaryotic expression plasmids (Lee, D. J., et al., Int. Arch. Allergy 1 mmol., 113(1-3): 227-230, 1997) with various promoters and regulatory sequences, which can be inserted into the patient's somatic cells (dendritic cells, epithelial cells, muscle fiber-cells, fibroblasts, etc.) for the purpose of expressing the recombinant gene product to alter the patient's immune response to the patatin proteins (Lee D. J., et al., Int. Arch. Allergy Immunol., 113(1-3): 227-230, 1997). Potential routes of administration foreseen in this application include previously described methods of encapsulation, emulsion, receptor or membrane fusion mediated uptake and methods of direct permeabilization or insertion of the DNA or corresponding RNA into the host cells.
[0251]The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES
Example 1
Identification of Patatin as an Allergen
[0252]Since patatin is commonly obtained from an allergenic source (potato), it was hypothesized that patatins in fact encode an important class of offending potato allergens (patatin was reported as allergenic by Seppala, U. et al., J. Allergy Clin. Immunol. 103: 165-171, 1999). Assessment of potential allergens preferably include appropriate in vitro testing for IgE binding, in this case with potato allergic sera (Fuchs, R. L. and Astwood, J. D., Food Technology, 50: 83-88, 1996; Astwood, J. D., et al., Monographs in allergy Vol. 32: Highlights in food allergy, pp. 105-120, 1996, Metcalfe, D. D., et al., Critical Reviews in Food Science and Nutrition, 36S: 165-186, 1996). It is the recommendation of a working group organized by the IFBC and the ILSI Allergy and Immunology Institute that proteins encoded by nucleic acid sequences from allergenic sources such as potato (a "less-commonly" allergenic source) should be examined for their ability to react with IgEs of potato-allergic patients using a minimum of five individual patient sera (Metcalfe, D. D., et al., Critical Reviews in Food Science and Nutrition, 36S: 165-186, 1996). Patatin-17 protein was tested for IgE binding using standard in vitro testing with serum taken from patients with bona fide well defined clinically displayed potato allergy as described below.
[0253]Clinical Characterization of Potato Allergic Subjects (Serum donors)
[0254]Patients who suffer from potato allergy were identified at Johns Hopkins Clinic (Baltimore, Md.) and were evaluated for potato allergy using clinical criteria outlined in Table 2.
[0255]Serum was obtained from patients with convincing clinical history of potato allergy. The convincing history was defined as being one or more of the following: a) positive potato allergic as evaluated by double-blind placebo-control food challenge b) anaphylaix and/or hospitalization due to the consumption of potatoes or c) dramatic skin test results.
TABLE-US-00002 TABLE 2 Clinical patient data Flare/Wheal Patient Clinical History (Skin prick test) DBPCFC (potato) HS01 Most recent hospitalization: Oct. 19, 1993 7/19, 4/14, 7/17 Not performed AD, A, AR, FH, MFS, IgE = 1397 KIAUa/L HS02 Most recent hospitalization: June 1994 20/26 Not performed AD, FH, Latex (+) RAST, MFS, IgE = 7544K/L HS03 Most recent hospitalization: Jul. 27, 1995 5/13 Yes AD, A, FH, MFS, IgE = N/A HS05 Most recent hospitalization May 30, 1995 4/9 Yes AD, A, FH, MFS, IgE = 12341 ng/ml HS06 Most recent hospitalization Jun. 13, 1995 5/20, 4/13, 5/12 Yes AD, A FH, MFS IgE = N/A HS07 Not potato allergic, allergic to egg, milk, High IgE control serum, peanuts, seafood. AD, A, AR, FH, MFS not allergic to potato. HS08 Non-atopic (normal) Low IgE control serum AD = Atopic dermatitis; FH = Food hypersensitivity; AR = Allergic rhinitis; A = Asthma; MFS = Multiple food sensitivity; N/A = not available.
Example 2
Western Blotting of Patatin Proteins
[0256]Western blotting experiments were performed using patatin protein purified to near homogeneity from corn plants genetically engineered to produce patatin, patatin producing crude genetically engineered corn leaf extracts, crude potato tuber extracts, and non-transgenic corn leaf samples.
[0257]Protein samples were electrophoresed by SDS-PAGE (Laemmli, U.K., Nature 227: 680-685, 1970) and were electroblotted onto nitrocellulose. Protein blots were processed by standard Western blotting (immunoblotting) techniques and were incubated in potato allergic serum diluted 1:5 in PBS buffer for 1 hour. After washing the blots 3 times with PBS, the blots were incubated in biotinylated anti-IgE (Johns Hopkins Hospital, Baltimore, Md.) for 1 hour, followed by a 30 minute incubation in HRP-linked avidin(Promega, New York, N.Y.). IgE-reactive protein bands were visualized by DAB staining (3,3 diaminobenzidine). The blots were dried and photographed. Individual blots are labeled according to patient serum used. As a control, one blot was incubated in anti-IgE only.
[0258]Patatins were shown to be an allergen of potato by examining the reactivity of purified patatin to sera obtained from patients allergic to potato. Sera from five potato allergic subjects were tested by Western blotting techniques. All five sera reacted with purified patatin protein.
[0259]Patatin isozymes (SEQ ID NOS:278-282, FIG. 1) were tested for IgE binding by Western blotting. Isozymes of patatin were cloned into a yeast expression system and purified prior to analysis. The isozymes were subjected to IgE western blotting as described above with the exception that all five patient sera were pooled. The resulting Western blot of the yeast-expressed isozymes showed that all five isozymes bound IgE in a manner similar to patatin 17, and that all isozymes of patatin tested are also allergens.
Example 3
Western Blotting of Patatin Proteins
[0260]Eighty-nine 10-mer peptides were synthesized using the Genosys SPOTs system, each consecutive 10-mer overlapping by 6 amino acids based on the amino acid sequence of patatin 17 (SEQ ID NO:2). The peptides were evaluated for IgE binding with five different potato allergic patient sera using the same incubation procedures as described above. The results are summarized graphically in FIG. 2, showing major and minor allergenic epitopes. Interestingly, many of the immunogenic epitopes contain tyrosine. The peptide numbers, sequences, and immunoreactivity is detailed in Table 3.
TABLE-US-00003 TABLE 3 Peptide scan of patatin 17 Peptide # (SEQ ID Peptide Cumulative NO) Sequence HS01 HS02 HS03 HS05 HS06 Total 1 (16) QLGEMVTVLS 0.47 0.33 0.02 0.05 0.06 0.93 2 (17) MVTVLSIDGG 0.53 0.33 0.02 0.07 0.05 1 3 (18) LSIDGGGIRG 0.52 0.38 0.07 0.08 0.09 1.14 4 (19) GGGIRGIIPA 0.53 0.19 0.06 0.19 0.23 1.2 5 (20) RGIIPATILE 0.46 0.28 0.04 0.09 0.05 0.92 6 (21) PATILEFLEG 0.49 0.31 0.05 0.09 0.07 1.01 7 (22) LEFLEGQLQE 0.36 0.24 0.04 0.1 0.06 0.8 8 (23) EGQLQEMDNN 0.29 0.19 0.02 0.09 0.05 0.64 9 (24) QEMDNNADAR 0.22 0.13 0.01 0.05 0.04 0.45 10 (25) NNADARLADY 0.21 0.17 0.03 0.05 0.07 0.53 11 (26) ARLADYFDVI 0.54 0.31 0.16 0.15 0.25 1.41 12 (27) DYFDVIGGTS 0.61 0.34 0.46 0.06 0.15 1.62 13 (28) VIGGTSTGGL 0.63 0.72 0.05 0.15 0.09 1.64 14 (29) TSTGGLLTAM 0.3 0.17 0.03 0.06 0.09 0.65 15 (30) GLLTAMISTP 0.63 0.41 0.05 0.24 0.12 1.45 16 (31) AMISTPNENN 0.34 0.18 0.02 0.07 0.02 0.63 17 (32) TPNENNRPFA 0.46 0.22 0.03 0.19 0.07 0.97 18 (33) NNRPFAAAKE 0.37 0.21 0.05 0.07 0.06 0.76 19 (34) FAAAKEIVPF 0.52 0.29 0.08 0.11 0.08 1.08 20 (35) KEIVPFYFEH 0.29 0.14 0.28 0.29 0.23 1.23 21 (36) PFYFEHGPQI 0.65 0.06 1.08 0.51 0.17 2.47 22 (37) EHGPQIFNPS 0.34 0.15 0.03 0.05 0.06 0.63 23 (38) QIFNPSGQIL 0.33 0.29 0.02 0.07 0.07 0.78 24 (39) PSGQILGPKY 0 0 0.02 0 0.05 0.07 25 (40) ILGPKYDGKY 0 0 0.07 0 0.02 0.09 26 (41) KYDGKYLMQV 0.02 0 0.11 0.01 0.04 0.18 27 (42) KYLMQVLQEK 0.12 0.04 1.08 0.07 0.79 2.1 28 (43) QVLQEKLGET 0.46 0.16 0.01 0.07 0.02 0.72 29 (44) EKLGETRVHQ 0.5 0.12 0.01 0.07 0.04 0.74 30 (45) ETRVHQALTE 0.42 0.16 0.03 0.05 0.03 0.69 31 (46) HQALTEVVIS 0.43 0.21 0.04 0.1 0.05 0.83 32 (47) TEVVISSFDI 0.44 0.25 0.05 0.08 0.04 0.86 33 (48) ISSFDIKTNK 0.1 0.02 0.04 0.06 0.13 0.35 34 (49) DIKTNKPVIF 0.57 0.22 0.04 0.18 0.28 1.29 35 (50) NKPVIFTKSN 0 0.01 0.02 0.07 0.24 0.34 36 (51) IFTKSNLANS 0 0 0.03 0.06 0.17 0.26 37 (52) SNLANSPELD 0.43 0.96 0.01 0.09 0.02 1.51 38 (53) NSPELDAKMY 0.18 0.12 0.01 0.05 0.05 0.41 39 (54) LDAKMYDISY 0.54 0.26 0.19 0.15 0.23 1.37 40 (55) MYDISYSTAA 0.92 0.08 0.52 0.04 0.22 1.78 41 (56) SYSTAAAPTY 1.15 0.25 1.04 0.33 0.55 3.32 42 (57) AAAPTYFPPH 1.02 0.52 1.12 0.81 0.86 4.33 43 (58) TYFPPHYFVT 0.02 0.01 0.54 0.03 0.24 0.84 44 (59) PHYFVTNTSN 0.03 0.01 1.17 0.13 0.44 1.78 45 (60) VTNTSNGDEY 0.23 0.15 0.04 0.03 0.03 0.48 46 (61) SNGDEYEFNL 0.33 0.25 0.08 0.1 0.11 0.87 47 (62) EYEFNLVDGA 0.34 0.25 0.07 0.1 0.2 0.96 48 (63) NLVDGAVATV 0.3 0.18 0.02 0.06 0.05 0.61 49 (64) GAVATVADPA 0.45 0.54 0.01 0.07 0.02 1.09 50 (65) TVADPALLSI 0.48 0.29 0.01 0.07 0.03 0.88 51 (66) PALLSISVAT 0.65 0.33 0.02 0.1 0.01 1.11 52 (67) SISVATRLAQ 0.61 0.23 0.14 0.53 0.53 2.04 53 (68) ATRLAQKDPA 0.87 0.34 0.05 0.29 0.22 1.77 54 (69) AQKDPAFASI 0.86 0.32 0.04 0.12 0.03 1.37 55 (70) PAFASIRSLN 0.81 0.15 0.05 0.51 0.59 2.11 56 (71) SIRSLNYKKM 0.07 0.01 0.17 0.07 0.11 0.43 57 (72) LNYKKMLLLS 0.05 0.01 0.35 0.08 0.39 0.88 58 (73) KMLLLSLGTG 1.15 0.15 0.04 0.38 0.71 2.43 59 (74) LSLGTGTTSE 0.34 0.23 0.02 0.04 0.03 0.66 60 (75) TGTTSEFDKT 0.92 0.39 0.6 0.1 0.09 2.1 61 (76) SEFDKTYTAK 1.33 1.35 1.41 0.12 0.28 4.49 62 (77) KTYTAKLEAAT 1.36 0.94 1.11 0.76 0.4 4.57 63 (78) AKEAATWTAV 0.45 0.15 0.01 0.2 0.04 0.85 64 (79) ATWTAVHWML 0.1 0.02 0.01 0.08 0.06 0.27 65 (80) AVHWMLVIQK 0.69 0.05 0.03 0.43 0.62 1.82 66 (81) MLVIQKMTDA 0.32 0.15 0.02 0.15 0.03 0.67 67 (82) QKMTDYYLST 0.26 0.125 0.03 0.21 0.05 0.675 68 (83) DAASSYMTDY 0.2 0.14 0.08 0.08 0.1 0.6 69 (84) SYMTDYYLST 0.5 0.03 0.32 0.06 0.11 1.02 70 (85) DYYLSTAFQA 0.14 0 0.22 0.03 0.13 0.52 71 (86) STAFQALDSK 0.4 0.3 0.04 0.06 0.08 0.88 72 (87) QALDSKNNYL 0.44 0.46 0.28 0.26 0.43 1.87 73 (88) SKNNYLRVQE 0.44 0.05 1.31 0.07 0.21 2.08 74 (89) YLRVQENALT 1.38 0.03 1.31 0.11 0.2 3.03 75 (90) QENALTGTTT 0.47 0.25 0 0.06 0 0.78 76 (91) LTGTTTEMDD 0.41 0.24 0 0.06 0 0.71 77 (92) TTEMDDASEA 0.38 0.3 0 0.05 0 0.73 78 (93) DDASEANMEL 0.44 0.24 0 0.06 0 0.74 79 (94) EANMELLVQV 0.42 0.27 0 0.04 0 0.73 80 (95) ELLVQVGENL 0.4 0.25 0 0.05 0 0.7 81 (96) QVGENLLKKP 0.44 0.14 0 0.07 0 0.65 82 (97) NLLKKPVSED 0.47 0.2 0 0.03 0 0.7 83 (98) KPVSEDNPET 0.27 0.21 0 0.03 0 0.51 84 (99) EDNPETYEEA 0.13 0.11 0 0.01 0 0.25 85 (100) ETYEEALKRF 1.26 1.2 1.36 0.53 0.71 5.06 86 (101) EALKRFAKLL 1.38 0.04 0 1.06 1.12 3.6 87 (102) RFAKLLSDRK 0.98 0.05 0 0.84 0.94 2.81 88 (103) LLSDRKKLRA 0.2 0.01 0 0.37 0.51 1.09 89 (104) RKKLRANKAS 0.28 0 0 0.31 0.64 1.23 Patient 41.84 20.565 18.1 14.17 16.55 Cumulative Totals
Example 4
Identification of Result Effective Substitutions
[0261]For each major and several minor allergenic epitopes of patatin, result effective substitutions were identified by synthesizing peptides that were altered by individually substituting an alanine residue at each non-alanine position in the epitope. Similarly, the reported nucleic acid sequence encoding corn patatin (U.S. Pat. No. 5,882,668; clone 5c9) was evaluated for IgE binding by producing peptides at corresponding positions to the potato patatin protein.
[0262]For example, Epitope 41 was analyzed by alanine scanning and rational substitution as follows.
TABLE-US-00004 Epitope 41 SEFDKTYTAK (SEQ ID NO: 76) Alanine scan AEFDKTYTAK (SEQ ID NO: 165) SAFDKTYTAK (SEQ ID NO: 166) SEADKTYTAK (SEQ ID NO: 167) SEFAKTYTAK (SEQ ID NO: 168) SEFDATYTAK (SEQ ID NO: 169) SEFDKAYTAK (SEQ ID NO: 170) SEFDKTATAK (SEQ ID NO: 171) SEFDKTYAAK (SEQ ID NO: 172) SEFDKTYTAA (SEQ ID NO: 173) Rational substitution AFFDKTYTAK (SEQ ID NO: 283) SEFDKTFTAK (SEQ ID NO: 176) Corn homolog CIFDSTYTAK (SEQ ID NO: 284)
[0263]Selected epitopes were analyzed by alanine scanning and rational substitution. Immunoassay with potato-allergic serum was used as described above. Table 4 summarizes the results of these experiments to identify result effective substitutions for patatin. Blank spaces in the table indicate that binding of the peptide to patient IgE was not detectable.
TABLE-US-00005 TABLE 4 Scanning of patatin for result effective substitutions Binding of modified pep- tides by patient IgE as measured by OD Sequence SEQ ID NO HS03 HS06 HS01 HS02 DYFDVIGGTS 105 0.12 0.16 0.36 DYFDVIAGTS 106 0.14 0.17 0.4 VIGGTSTGGL 107 0.04 VIAGTSTGAL 108 AFYFEHGPQI 109 0.96 0.5 0.78 PAYFEHGPQI 110 0.75 0.41 0.69 PFAFEHGPQI 111 PFYAEHGPQI 112 0.7 0.43 0.79 PFYFAHGPQI 113 0.93 1.07 0.59 1.44 PFYFEAGPQI 114 0.08 0.93 0.65 1.34 PFYEEHAPQI 115 0.75 0.54 1.11 PFYFEHGAQI 116 0.63 0.29 0.6 PFYFEHGPAI 117 0.63 0.25 0.56 PFYFEHGPQA 118 0.27 0.16 0.33 TFYLENGPKI 119 0.05 0.48 0.68 1.07 PFFFEHGPQI 120 AYLMQVLQEK 121 0.26 0.11 0.53 KALMQVLQEK 122 KYAMQVLQEK 123 0.11 0.43 0.1 1.25 KYLAQVLQEK 124 0.22 0.48 0.11 1.34 KYLMAVLQEK 125 0.22 0.83 0.16 1.33 KYLMQALQEK 126 0.11 0.6 0.15 0.95 KYLMQVAQEK 127 0.53 0.15 0.81 KYLMQVLAEK 128 0.06 0.69 0.11 1.34 KYLMQVLQAK 129 0.74 0.79 0.05 0.58 KYLMQVLQEA 130 0.28 0.27 0.37 VFLHDKIKSL 131 0.06 0.26 0.41 AYSTAAAPTY 132 0.1 0.12 0.12 SASTAAAPTY 133 SYATAAAPTY 134 0.16 0.13 0.37 SYSAAAAPTY 135 0.13 0.12 0.32 SYSTAAAATY 136 0.15 0.13 0.34 SYSTAAAPAY 137 0.15 0.14 0.29 SYSTAAAPTA 138 0.55 0.54 1.13 CISTSAAPTY 139 0.4 SYSTAAAPAF 140 0.39 1.02 0.65 1.42 AFAAAAAPTY 141 0.07 SYSTAAAPTF 142 0.15 0.97 0.48 1.09 STSAAPTYFP 143 0.21 0.23 0.39 STSAAPTFFP 144 0.23 STSAAPTAFP 145 0.08 STAAAPTFFP 146 0.12 0.28 AAAATYFPPH 147 0.13 0.1 0.05 AAAPAYFPPH 148 0.07 0.04 AAAPTAFPPH 149 AAAPTYAPPH 150 0.23 0.14 0.21 AAAPTYFAPH 151 0.45 0.18 0.44 AAAPTYFPAH 152 0.15 0.07 0.18 AAAPTYFPPA 153 0.1 0.06 0.31 SAAPTYFPAH 154 0.77 0.73 0.96 AAAPAFFPPH 155 AAAPPFFPPH 156 AAAPTFFPPH 157 SISVATRLAQ 158 0.26 0.26 AMSMLTKEVH 159 PAFASIRSLN 160 PNFNAGSPTE 161 KMLLLSLGTG 162 NYLIISVGTG 163 0.49 1.08 0.64 1.48 KMLLLSLGAG 164 0.13 AEFDKTYTAK 165 0.09 0.22 1.34 SAFDKTYTAK 166 0.66 0.71 0.06 1.42 SEADKTYTAK 167 0.99 SEFAKTYTAK 168 0.5 0.57 0.91 SEFDATYTAK 169 0.17 SEFDKAYTAK 170 0.1 0.24 1.38 SEFDKTATAK 171 0.81 SEFDKTYAAK 172 0.2 0.35 1.39 SEFDKTYTAA 173 0.1 1.18 KQAEKYTAEQ 174 0.08 0.24 SEFDAAFAAA 175 SEFDKTFTAK 176 0.09 0.16 0.07 1.45 AEKYTAEQCA 177 ATYTAKEAAT 178 0.24 0.18 KAYTAKEAAT 179 0.28 0.33 KTATAKEAAT 180 KTYAAKEAAT 181 0.1 0.32 0.73 KTYTAAEAAT 182 0.35 KTYTAKAAAT 183 0.4 0.59 0.82 KTYTAKEAAA 184 0.36 EKYTAEQCAK 185 AAFAAAEAAT 186 KTFTAKEAAT 187 QALHCEKKYL 188 QALDSKAAYL 189 QALDSKNNFL 190 QALHCENNFL 191 CEKKYLRIQD 192 1.01 0.16 SKNNFLRVQE 193 SENNYLRVQE 194 0.31 0.96 0.42 1 ALRVQENALT 195 YARVQENALT 196 1.06 1.02 0.05 0.54 YLAVQENALT 197 0.37 1.04 0.11 1.06 YLRAQENALT 198 1.1 1 0.06 1.26 YLRVAENALT 199 1.03 0.92 0.08 1.26 YLRVQANALT 200 1.05 0.92 0.06 1.24 YLRVQEAALT 201 0.93 0.92 0.07 1.11 YLRVQENAAT 202 0.94 0.93 0.04 1.24 YLRVQENALA 203 1.05 0.96 0.43 1.16 YLRIQDDTLT 204 1.07 0.85 0.39 1.12 YLTVAAAALT 205 1.05 0.86 0.28 1.33 FLRVQENALT 206 NNYLRVQENA 207 0.23 0.88 0.5 1.17 KKYLRIQDDT 208 0.26 0.09 0.37 NNFLRVQENA 209 NAYLRVQENA 210 0.17 1.02 0.53 1.06 ATYEEAKLRF 211 0.26 1.03 0.65 EAYEEALKRF 212 0.06 0.43 0.33 ETAEEALKRF 213 1.04 ETYAEALKRF 214 0.62 1.02 1.15 ETYEAALKRF 215 1.06 0.38 0.89 ETYEEAAKRF 216 0.08 0.1 0.9 ETYEEALARF 217 0.11 ETYEEALKAF 218 0.1 ETYEEALKRA 219 0.1 GTNAQSLADF 220 ETYEAALAAF 221 0.07 0.78 0.33 0.77 ETFEEALKRF 222 YEEALKTFAK 223 1.08 0.85 0.14 1.46 AEEALKRFAK 224 0.46 0.72 0.67 AALKRFAKLL 225 0.15 0.17
EAAKRFAKLL 226 0.08 0.33 0.05 EALARFAKLL 227 0.09 EALKAFAKLL 228 EALKRAAKLL 229 0.08 0.07 EALKRFAALL 230 EALKRFAKAL 231 0.06 0.09 0.1 EALKRFAKLA 232 0.06 0.1 QSLADFAKQL 233 AALAAFAKLL 234 LADFAKQLSD 235 DFAKQLSDER 236 0.17 AFAALLSDRK 237
[0264]Result effective substitutions were identified by a reduction in IgE binding ability with respect to the non-substituted peptide sequence. Table 5 shows the identified result effective substitutions. Blank spaces in the table indicate that binding of the peptide to patient IgE was not detectable. Many substitutions of alanine or phenylalanine for the original tyrosine resulted in reduced or eliminated antibody binding.
TABLE-US-00006 TABLE 5 Result effective substitutions of patatin Location (SEQ (SEQ ID ID NO) Peptide NO) HS03 HS06 HS01 HS02 Minor PFYFEHGPQI (36) 1.08 0.17 0.65 0.06 Epitope ::A::::::: (111) 21 ::F::::::: (r) (120) :::::::::A (118) 0.27 0.16 0.33 Minor KYLMQVLQEK (42) 1.08 0.79 0.12 0.04 Epitope :A:::::::: (122) 27 :::::::::A (130) 0.28 0.27 0.37 VFLHDKIKSL (c) (131) 0.06 0.26 0.41 Major SYSTAAAPTY (56) 1.04 0.55 1.15 0.25 Epitope A::::::::: (132) 0.1 0.12 0.12 41 :A:::::::: (133) AFAA:::::: (r) (141) 0.007 CI::S::::: (c) (139) 0.04 Overlap STAAAPTYFP (238) Epitope ::S::::A:: (r) (145) 0.08 41/42 Major AAAPTYFPPH (57) 1.12 0.86 1.02 0.52 Epitope ::::A::::: (148) 0.07 0.04 42 (57) :::::A:::: (149) ::::AF:::: (r) (155) ::::PF:::: (r) (156) :::::F:::: (r) (157) Major SEFDKTYTAK (76) 0.12 0.28 1.33 1.35 Epitope ::::A::::: (169) 0.17 61 KQAE:YTAEQ (c) (174) 0.08 0.24 ::::AAFA:A (r) (175) Major KTYTAKEAAT (77) 1.11 0.04 1.36 0.94 Epitope A::::::::: (178) 0.24 0.18 62 ::A::::::: (180) :::::A:::: (182) 0.35 AAFA:A:::: (r) (186) ::F::::::: (r) (187) EK:::EQC:K (c) (185) Minor QALDSKNNYL (87) 0.28 0.43 0.44 0.46 Epitope :::HCEKK:: (c) (188) 72 ::::::AA:: (r) (189) ::::::::F: (r) (190) :::::E::F: (r) (240) Minor SKNNYLRVQE (88) 1.31 0.21 0.44 0.05 epitope ::::F::::: (r) (193) 73 Minor YLRVQENALT (87) 1.31 0.2 1.38 0.03 epitope A::::::::: (195) 74 F::::::::: (r) (206) Overlap NNYLRVQENA (207) 0.23 0.88 0.5 1.17 epitope ::F::::::: (r) (209) 73/74 Major ETYEEALKRF (100) 1.36 0.71 1.26 1.2 epitope :::::::A:: (217) 0.11 85 ::::::::A: (218) 0.1 :::::::::A (219) 0.1 ::F::::::: (r) (222) G:NAQS:AD: (c) (220) Major EALKRFAKLL (101) 0 1.12 1.38 0.04 Epitope :::A:::::: (227) 0.09 86 ::::A::::: (228) :::::A:::: (229) 0.08 0.07 :::::::A:: (230) ::::::::A: (231) 0.06 0.09 :::::::::A (232) 0.06 SD:AD:::Q: (c) (241) A::AA::::: (r) (234) Epitope LKRFAKLLSD (239) overlap (NO BINDING) 86/87 Major RFAKLLSDRK (102) 0 0.94 0.98 0.05 Epitope D:::Q:::ER (c) (236) 0.17 87 A::A:::::: (r) (237) (r) = rational; (c) = corn.
Example 5
Site Directed Mutagenesis
[0265]To introduce site specific mutations, the cloned DNA sequence of patatin (SEQ ID NO:1 encoding patatin protein SEQ ID NO:2; pMON 26820) was subjected to PCR with primers SEQ ID NO:3 and SEQ ID NO:4 to incorporate part of the α-factor signal sequence (Pichia expression manual, Invitrogen, Carlsbad, Calif.), and EcoRI and XhoI restriction sites to facilitate cloning into the Pichia pastoris yeast secretion vector pPIC9 (GenBank accession number Z46233; Invitrogen, Carlsbad, Calif.). Typical PCR conditions are 25 cycles 94° C. denaturation for 1 minute, 45° C. annealing for one minute and 72° C. extension for 2 minutes; plus one cycle 72° C. extension for 10 minutes. A 50 μL reaction contains 30 pmol of each primer and 1 μg of template DNA; and 1×PCR buffer with MgCl2, 200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 2.5 units of Pwo DNA polymerase. PCR reactions are performed in RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, Calif.).
[0266]The amplified fragment SEQ ID NO:5 was digested with restriction enzymes XhoI and EcoRI and cloned into the pBluescript vector (Stratagene, La Jolla, Calif.), digested with the same two restriction enzymes. The resulting plasmid (pMON 26869) was used for oligonucleotide-directed mutagenesis using the Bio-Rad mutagenesis kit based on the method of Kunkel (Proc. Natl. Acad. Sci. U.S.A., 82: 477-492, 1985). Briefly, single-stranded pMON26869 was used as template for mutagenesis and was prepared by superinfection of plasmid containing cells with M13K07 (Gorman, et al., DNA Prot. Eng. Techniques, 2: 3-10, 1990). The mutagenic oligonucleotides are SEQ ID NOS:8-15 (reverse complement). DNA purified from transformed DH5α E. coli colonies was used for sequence determination. Sequencing was performed using the ABI PRISM sequencing kit (Perkin Elmer Biosystems, Foster City, Calif.). The resulting plasmid containing the mutation in the patatin gene was digested with restriction enzymes XhoI and EcoRI.
[0267]The patatin nucleic acid fragment was then ligated into the pPIC9 vector (Invitrogen, Carlsbad, Calif.), digested with the same two restriction enzymes to afford plasmid pMON37401. Pichia pastoris KM71 cells were electroporated with pMON37401 containing the appropriate mutation. The resulting transformed cells were used to produce protein in Pichia pastoris using the procedure supplied by the manufacturer (Invitrogen, Carlsbad, Calif.). The encoded protein contains an alpha factor signal cleavage site. Plasmid pMON37401 encodes SEQ ID NO:6 which is cleaved to afford SEQ ID NO:7, having four amino acids added at the N-terminus of amino acids 24-386 of SEQ ID NO:2. Position four of SEQ ID NO:7 therefore corresponds to position 23 of SEQ ID NO:2.
[0268]The concentration of patatin in the culture was determined using a patatin ELISA assay and the enzyme activity was measured using the method of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). The variants containing multiple mutations were further purified using Mono Q and hydrophobic interaction chromatography (HIC). Each culture was purified by first sizing on Amicon YM10 membranes (Millipore, Bedford, Mass.) to a >10 kDa fraction, followed by chromatography on the Mono Q HR 10/10 column (Pharmacia, Piscataway, N.J.). For chromatography on the Mono Q column, the samples were loaded on the column in 25 mM Tris pH 7.5 and eluted with a gradient of 1.0 M KCl in 25 mM Tris pH 7.5. Fractions containing patatin protein were determined using SDS-PAGE. For chromatography on the HIC column, the appropriate fractions were pooled and dialyzed into 1 M ammonium sulfate in 25 mM Tris pH 7.5. The dialyzed sample was then loaded on 16/10 phenyl Sepharose column (Pharmacia, Piscataway, N.J.) and eluted with a gradient of 25 mM Tris pH7.5.
[0269]The protein concentration was determined using the Bradford method, using BSA as a standard. SDS-PAGE analysis showed that these proteins were essentially pure. The esterase activity of the newly formed variants are shown in Table 6. The activity was determined using p-nitrophenyl caprate substrate as described by Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990).
TABLE-US-00007 TABLE 6 Esterase activity of patatin mutants Variant Activity (mOD min-1μg-1) Wild type 93.2 Y106F 51.1 Y129F 74.7 Y185F 85.6 Y193F 82.2 Y185F/Y193F 99.4 Y270F 163.4 Y316F 94.88 Y362F 130.7 Y106F/Y129F/Y185F/Y193F/ 57.1 Y270F/Y316F/Y362F Y185F/Y193F/Y270F/Y316F/Y362F 161.5
[0270]Patatin proteins having a phenylalanine substitution at each of the amino acid positions 106, 129, 185, 193, 270, 316 and 362 (numbers correspond to positions in SEQ ID NO:2) of expressed SEQ ID NO:7 exhibit full enzyme activity. Proteins having multiple substitutions also displayed full enzyme activity.
[0271]In addition to nucleotide sequences encoding conservative amino acid changes within the fundamental polypeptide sequence, biologically functional equivalent nucleotide sequences include nucleotide sequences containing other base substitutions, additions, or deletions. These include nucleic acids containing the same inherent genetic information as that contained in the cDNA which encode peptides, polypeptides, or proteins conferring pathogen resistance the same as or similar to that of pathogen upon host cells and plants. Such nucleotide sequences can be referred to as "genetically equivalent modified forms" of the cDNA, and can be identified by the methods described herein.
[0272]Mutations made in the cDNA, plasmid DNA, genomic DNA, synthetic DNA, or other nucleic acid encoding the deallergenized gene preferably preserve the reading frame of the coding sequence. Furthermore, these mutations preferably do not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, that would adversely affect mRNA translation.
[0273]Although mutation sites can be predetermined, it is not necessary that the nature of the mutations per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis can be conducted at the target codon.
[0274]Alternatively, mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native cDNA sequence. Following ligation, the resulting reconstructed nucleotide sequence encodes a derivative form having the desired amino acid insertion, substitution, or deletion.
Example 6
Construction of Permutein Sequences
[0275]Nucleic acid sequences encoding permutein proteins having rearranged N-terminus/C-terminus protein sequences can be made by following the general method described by Mullins et al. (J. Am. Chem. Soc. 116: 5529-5533, 1994). The steps are shown in FIG. 3. The Figure and the following Examples involve the design and use of a linker region separating the original C-terminus and N-terminus, but the use of a linker is not a critical or required element of permutein design.
[0276]Two sets of oligonucleotide primers are used in the construction of a nucleic acid sequence encoding a permutein protein. In the first step, oligonucleotide primers "new N-termini" and "linker start" are used in a PCR reaction to create amplified nucleic acid molecule "new N-termini fragment" that contains the nucleic acid sequence encoding the new N-terminal portion of the permutein protein, followed by the polypeptide linker that connects the C-terminal and N-terminal ends of the original protein. In the second step, oligonucleotide primers "new C-termini" and "linker end" are used in a PCR reaction to create amplified nucleic acid molecule "new C-termini fragment" that contains the nucleic acid sequence encoding the same linker as used above, followed by the new C-termini portion of the permutein protein. The "new N-termini" and "new C-termini" oligonucleotide primers are designed to include appropriate restriction enzyme recognition sites which assist in the cloning of the nucleic acid sequence encoding the permutein protein into plasmids.
[0277]Any suitable PCR conditions and polymerase can be used. It is desirable to use a thermostable DNA polymerase with high fidelity to reduce or eliminate the introduction of sequence errors. Typical PCR conditions are 25 cycles 94° C. denaturation for 1 minute, 45° C. annealing for one minute and 72° C. extension for 2 minutes; plus one cycle 72° C. extension for 10 minutes. A 50 μL reaction contains 30 pmol of each primer and 1 μg of template DNA; and 1×PCR buffer with MgCl2, 200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 2.5 units of Pwo DNA polymerase. PCR reactions are performed in RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, Calif.).
[0278]The amplified "new N-termini fragment" and "new C-termini fragment" are annealed to form a template in a third PCR reaction to amplify the full-length nucleic acid sequence encoding the permutein protein. The DNA fragments "new N-termini fragment" and "new C-termini fragment" are resolved on a 1% TAE gel, stained with ethidium bromide, and isolated using the QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif.). These fragments are combined in equimolar quantities with oligonucleotide primers "new N-termini" and "new C-termini" in the third PCR reaction. The conditions for the PCR are the same as used previously. PCR reaction products can be purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.).
[0279]Alternatively, a linker sequence can be designed containing a restriction site, allowing direct ligation of the two amplified PCR products.
Example 7
Construction of Plasmid pMON 37402
[0280]The patatin protein contains a trypsin protease sensitive site at the arginine amino acid at position 246, as determined by electrophoresis of a trypsin digest reaction. In order to determine if the exposed protease site is an antigenic epitope, a permutein was constructed using positions 246-247 as a breakpoint.
[0281]The nucleic acid sequence encoding the permutein protein in plasmid pMON 37402 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule "new N-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 27 (SEQ ID NO:242) and 48 (SEQ ID NO:243). Nucleic acid molecule "new C-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 36 (SEQ ID NO:245). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments "new N-termini fragment" and "new C-termini fragment" using oligonucleotide primers 27 (SEQ ID NO:242) and 36 (SEQ ID NO:245).
[0282]The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
[0283]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON 37402 (containing SEQ ID NO:246, encoding protein sequence SEQ ID NO:247).
Example 8
Construction of Plasmid pMON 37405
[0284]Amino acids 201-202, near tyrosine 193, were chosen as a breakpoint for the construction of a permutein protein.
[0285]The nucleic acid sequence encoding the permutein protein in plasmid pMON 37405 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule "New N-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243) and 58 (SEQ ID NO:249). Nucleic acid molecule "New C-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 59 (SEQ ID NO:249). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments "New N-termini fragment" and "New C-termini fragment" using oligonucleotide primers 58 (SEQ ID NO:248) and 59 (SEQ ID NO:249).
[0286]The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
[0287]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON 37405 (containing SEQ ID NO:250, encoding protein sequence SEQ ID NO:251).
Example 9
Construction of Plasmid pMON 37406
[0288]Amino acids 183-184, adjacent to tyrosine 185, were chosen as a breakpoint for the construction of a permutein protein.
[0289]The nucleic acid sequence encoding the permutein protein in plasmid pMON 37406 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule "New N-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243) and 60 (SEQ ID NO:252). Nucleic acid molecule "New C-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 61 (SEQ ID NO:253). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments "New N-termini fragment" and "New C-termini fragment" using oligonucleotide primers 60 (SEQ ID NO:252) and 61 (SEQ ID NO:253).
[0290]The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
[0291]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON37406 (containing SEQ ID NO:254, encoding protein sequence SEQ ID NO:255).
Example 10
Construction of Plasmid pMON 37407
[0292]Amino acids 268-269, adjacent to tyrosine 270, were chosen as a breakpoint for the construction of a permutein protein.
[0293]The nucleic acid sequence encoding the permutein protein in plasmid pMON 37407 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule "New N-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243) and 62 (SEQ ID NO:256). Nucleic acid molecule "New C-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 63 (SEQ ID NO:257). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments "New N-termini fragment" and "New C-termini fragment" using oligonucleotide primers 62 (SEQ ID NO:256) and 63 (SEQ ID NO:257).
[0294]The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
[0295]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON37407 (containing SEQ ID NO:258, encoding protein sequence SEQ ID NO:259).
Example 11
Construction of Plasmid pMON 37408
[0296]Amino acids 321-322, near tyrosine 216, were chosen as a breakpoint for the construction of a permutein protein.
[0297]The nucleic acid sequence encoding the permutein protein in plasmid pMON 37408 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule "New N-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243) and 64 (SEQ ID NO:260). Nucleic acid molecule "New C-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 65 (SEQ ID NO:261). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments "New N-termini fragment" and "New C-termini fragment" using oligonucleotide primers 64 (SEQ ID NO:260) and 65 (SEQ ID NO:261).
[0298]The resulting amplified nucleic acid molecule was digested with restriction endonucleases NhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
[0299]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON37408 (containing SEQ ID NO:262, encoding protein sequence SEQ ID NO:263).
Example 12
Production of Permutein Proteins in Pichia pastoris
[0300]Plasmids pMON37402, pMON37405, pMON37406, pMON37407, and pMON37408 were individually used to electroporate KM71 cells from Pichia pastoris according to the procedure supplied by the manufacturer (Invitrogen, Carlsbad, Calif.). The resulting transformed cells were used to produce protein in Pichia pastoris following the procedure supplied by the manufacturer (Invitrogen, Carlsbad, Calif.).
[0301]The concentration of patatin in the culture was determined using a patatin ELISA assay and the enzyme activity was measured using the method of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). The variants containing multiple mutations were further purified using Mono Q and hydrophobic interaction chromatography (HIC). Each culture was purified by first sizing on YM10 membranes (Amicon, MA) to a [>10 kDa] fraction, followed by chromatography on the Mono Q HR 10/10 column (Pharmacia, NJ). For chromatography on the Mono Q column, the samples were loaded on the column in 25 mM Tris pH 7.5 and eluted with a gradient of 1.0 M KCl in 25 mM Tris pH 7.5. Fractions containing patatin protein were determined using SDS-PAGE. For chromatography on the HIC column, the appropriate fractions were pooled and dialyzed into 1 M ammonium sulfate in 25 mM Tris pH 7.5. The dialyzed sample was then loaded on 16/10 phenyl Sepharose column (Pharmacia, NJ) and eluted with a gradient of 25 mM Tris pH7.5.
[0302]The protein concentration was determined using the Bradford method, using BSA as a standard. SDS-PAGE analysis showed that these proteins were essentially pure. The esterase activity of the variants are shown in Table 7.
TABLE-US-00008 TABLE 7 Activity of permuteins pMON Breakpoint Activity (ΔOD min-1μg-1) Native enzyme 83.21 pMON37402 246/247 66.7 pMON37405 201/202 No expression pMON37406 183/184 No expression pMON37407 268/269 12.1 pMON37408 321/322 No expression
[0303]The activity was determined using p-nitrophenyl caprate substrate as described by Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990).
Example 13
Insect Bioefficacy Assays
[0304]Assays for activity against larvae of SCRW are carried out by overlaying the test sample on an agar diet similar to that described by Marrone (J. Econ. Entom. 78: 290-293, 1985). Test samples were prepared in 25 mM Tris, pH 7.5 buffer. Neonate larvae are allowed to feed on the treated diet at 26° C., and mortality and growth stunting were evaluated after 5 or 6 days. The results of this assay are shown in Table 8.
TABLE-US-00009 TABLE 8 Insect bioefficacy assay Protein (200 ppm) Mean Survival Weight % Weight Reduction Tris buffer (control) 1.26 ± 0.3 -- Wild Type 0.21 ± 0.02 83 pMON37402 0.21 ± 0.03 83 pMON37407 0.32 ± 0.04 75
[0305]These data demonstrate that the growth of the SCRW larvae is similarly reduced upon ingestion of the proteins encoded by pMON37402 and pMON37407 as compared to the wild type patatin protein.
Example 14
Permutein Sequences Improved for Monocot Expression
[0306]Modification of coding sequences has been demonstrated above to improve expression of insecticidal proteins. A modified coding sequence was thus designed to improve expression in plants, especially corn (SEQ ID NO:264).
Example 15
Construction of pMON40701 for Monocot Expression
[0307]Plasmid pMON19767 was digested with restriction endonucleases NcoI and EcoRI and the 1100 bp gene fragment was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON33719 was digested with restriction endonucleases NcoI and EcoRI, and gel purified, resulting in an approximately 3900 base pair vector fragment. The two purified restriction fragments were combined and ligated using T4 DNA ligase.
[0308]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON40700. Plasmid pMON40700 was digested with restriction endonuclease NotI and the resulting 2200 bp DNA fragment was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested with restriction endonuclease NotI, and gel purified, resulting in an approximately 4200 base pair vector fragment. The two purified restriction fragments were combined and ligated using T4 DNA ligase.
[0309]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on kanamycin-containing plates. The resulting plasmid was designated pMON40701 (containing SEQ ID NO:264, encoding protein sequence SEQ ID NO:265).
Example 16
Construction of pMON40703 for Monocot Expression
[0310]The nucleic acid sequence encoding the permutein protein in plasmid pMON40703 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule "New N-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON19767 using oligonucleotide primers Syn1 (SEQ ID NO:266) and Syn2 (SEQ ID NO:267). Nucleic acid molecule "New C-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:268) and Syn4 (SEQ ID NO:269). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments "New N-termini fragment" and "New C-termini fragment" using oligonucleotide primers Syn1 (SEQ ID NO:266) and Syn4 (SEQ ID NO:269).
[0311]The resulting amplified nucleic acid molecule was digested with restriction endonucleases NcoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON33719 was digested with restriction endonucleases NcoI and EcoRI, and gel purified, resulting in an approximately 3900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
[0312]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON40702. Plasmid pMON40702 was digested with NotI, and the resulting 2200 bp DNA fragment was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested with restriction endonuclease NotI, and gel purified, resulting in an approximately 4200 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
[0313]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on kanamycin-containing plates. The resulting plasmid was designated pMON40703 (containing SEQ ID NO:270, encoding protein sequence SEQ ID NO:271). Plasmid pMON40703 encodes a permutein protein with a "breakpoint" at positions 246/247 of the wild type patatin protein sequence (SEQ ID NO:2). The first 23 amino acids of SEQ ID NO:2 are a signal peptide sequence which is cleaved in the mature protein.
Example 17
Construction of pMON40705 for Monocot Expression
[0314]The nucleic acid sequence encoding the permutein protein in plasmid pMON40705 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule "New N-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON19767 using oligonucleotide primers Syn10 (SEQ ID NO:272) and Syn2 (SEQ ID NO:267). Nucleic acid molecule "New C-termini fragment" was created and amplified from the sequence encoding patatin in plasmid pMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:268) and Syn11 (SEQ ID NO:273). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments "New N-termini fragment" and "New C-termini fragment" using oligonucleotide primers Syn10 (SEQ ID NO:272) and Syn11 (SEQ ID NO:273).
[0315]The resulting amplified nucleic acid molecule was digested with restriction endonucleases NcoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON33719 was digested with restriction endonucleases NcoI and EcoRI, and gel purified, resulting in an approximately 3900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
[0316]The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON40704. Plasmid pMON40704 was digested with restriction endonuclease NotI, and the resulting 2200 bp DNA fragment was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested with restriction endonuclease NotI, and gel purified, resulting in an approximately 4200 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase. The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on plates containing kanamycin. The resulting plasmid was designated pMON40705 (containing SEQ ID NO:274, encoding protein sequence SEQ ID NO:275). Plasmid pMON40705 encodes a permutein protein with a "breakpoint" at positions 268/269 of the wild type patatin protein sequence (SEQ ID NO:2). The first 23 amino acids of SEQ ID NO:2 are a signal peptide sequence which is cleaved in the mature protein.
Example 18
Transient Expression of Protein in Corn Leaf Protoplasts
[0317]Plasmids pMON40701, pMON40703, and pMON40705 (all containing the native signal sequence for vacuolar targeting) were separately electroporated into corn leaf protoplasts as described by Sheen (Plant Cell 3: 225-245, 1991). Protein was extracted with glass beads and the supernatant was assayed for protein expression using ELISA for patatin and NPTII. Expression of protein by the transformed corn protoplasts was confirmed by Western blot analysis. Expression results are shown in Table 9.
TABLE-US-00010 TABLE 9 ELISA data Normalized Expression Patatin ELISA NPTII ELISA (Patatin ELISA/NPTII Sample (μg/mL) (μg/mL) ELISA) pMON40701 1.1 0.6 1.8 pMON40703 2.1 0.3 7.0 pMON40705 1.3 0.6 2.2
[0318]The results indicate that the permutein encoded by plasmid pMON40703 surprisingly shows approximately 4-fold higher expression compared to the wild type enzyme.
Example 19
Deglycosylation of Protein Sequences
[0319]This example provides evidence that glycosylation of can contribute to the allergenicity of a protein. Accordingly, rational substitution of amino acid residues likely to be the targets of glycosylation within a subject allergen protein may reduce or eliminate the allergenic properties of the protein without adversely affecting the enzymatic, insecticidal, antifungal or other functional properties of the protein.
[0320]Glycosylation commonly occurs as either N-linked or O-linked forms. N-linked glycosylation usually occurs at the motif Asn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro (Kasturi, L. et al., Biochem J. 323: 415-519, 1997; Melquist, J. L. et al., Biochemistry 37: 6833-6837, 1998). O-linked glycosylation occurs between the hydroxyl group of serine or threonine and an amino sugar.
[0321]Site directed mutagenesis of selected asparagine, serine, or threonine may be used to reduce or eliminate the glycosylation of patatin proteins. A search of SEQ ID NO:2 for the Asn-Xaa-Ser/Thr motif reveals one occurrence at amino acid positions 202-204. Mutagenization of the nucleic acid sequence encoding this region results in a reduced allergenicity of the encoded protein.
[0322]In order to test this approach to reducing allergenicity of patatin proteins, two sets of experiments were performed: a) production of patatin proteins in Escherichia coli, which do not glycosylate proteins; and b) production of patatin proteins with an N202Q site directed mutation.
[0323]Antibodies obtained from patients HS-07 and G15-MON (not potato allergic) did not show specific binding to wild type patatin, patatin produced in E. coli, or the N202Q variant. Antibodies obtained from patient HS-01 (potato allergic) bound to wild type patatin, but not to patatin produced in E. coli or the N202Q variant. Antibodies obtained from patient HS-02 (potato allergic) bound strongly to wild type patatin, but extremely weakly to patatin produced in E. coli, and binding to the N202Q variant resembled vector controls. Antibodies obtained from patient HS-03 (potato allergic) bound to wild type patatin, but not to patatin produced in E. coli or the N202Q variant. Antibodies obtained from patient HS-05 (potato allergic) bound to wild type patatin, but very weakly to patatin produced in E. coli and the N202Q variant. Antibodies obtained from patient HS-06 (potato allergic) strongly bound wild type patatin, the N202Q variant, and to patatin produced in E. coli. These results strongly suggest that glycosylation is at least partially responsible for the antigenic properties of patatin proteins, and that site directed mutagenesis may be used to reduce or eliminate specific antibody binding. Mutagenesis at position 202 of SEQ ID NO:2 may be useful for reducing or eliminating specific antibody binding.
[0324]The deglycosylation approach was also tested using a patatin homolog, Pat17. As demonstrated above, patatin epitopes exhibiting IgE binding were identified, and each contained a Tyr residue. Substitution of these Tyr residues within each epitope led to loss of IgE binding. Site-directed mutagenesis was used to produce variants with individual and multiple Tyr substitutions in the protein, which was expressed in Pichia pastoris and assessed for enzyme activity. All the variants were found to have enzymatic activity no less than the wild type protein. A single variant with all 5 tyrosine residues substituted with phenylalinine was found to have insecticidal activity no less than the unsubstituted protein and was expressed in E. coli to produce the non-glycosylated version. The E. coli 5-"Tyr to Phe" variant was assessed for IgE binding. An isozyme of patatin, designated Pat17, was also expressed in corn to produce a plant glycoprotein and in E. coli to produce a nonglycosylated protein. Sera of seven patients (five exhibiting potato allergy and one exhibiting other allergies but no allergy to potatoes) were used to assay Pat17 or Pat17 variant binding by immunoblot assay. Four of the five sera from patients exhibiting potato allergy showed either very weak or no binding to wild type patatin expressed in E. coli but did bind to the 5-Tyr variant. Serum from one patient exhibiting potato allergy showed strong binding to recombinant wild type patatin protein expressed in E. coli but weak binding to the 5-Tyr variant. Sera from all five patients exhibiting potato allergy bound strongly to patatin expressed in corn and native patatin present in potatoes. Serum from a control patient allergic to eggs, milk, peanuts and seafood, but exhibiting no allergy to potatoes showed no binding to patatin expressed in E. coli but did bind to patatin expressed in corn. Immunoblot results suggested that the sugar moiety in patatin is a non-specific IgE binding epitope and the polypeptide portion of patatin also contains immunogenic IgE epitopes.
[0325]Patients who suffer from potato allergy were identified at Johns Hopkins Clinic (Baltimore, Md.) and were evaluated for potato allergy using clinical criteria outlined in Table 2.
[0326]Serum was obtained from patients with convincing clinical history of potato allergy. The convincing history was defined as being one or more of the following: a) positive potato allergic reaction as evaluated by double-blind placebo-control food challenge b) anaphylaix and/or hospitalization due to the consumption of potatoes or c) dramatic skin test results.
Peptide Synthesis
[0327]Peptides were synthesized on cellulose membranes using the SPOTS system (Genosys Biotechnologies, TX). Membranes were stored at -20° C. until use.
Site Directed Mutagenesis
[0328]Site specific mutations were introduced into patatin by first incorporating part of the a-factor signal sequence (Pichia expression manual, Invitrogen, Carlsbad, Calif.) to the patatin gene using PCR. Primers used for the PCR were GGAGCTCGAGAAAAGAGAGGCTGAAGCTCAGTTGGGAGAAATGGTGACTGT TCT (XhoI site in italics) and GGTCTAGAG GAATTCTCATTAATAAGAAG (EcoRI site in italics). The primers contained restriction sites to facilitate cloning into Pichia pastoris yeast secretion vector pPIC9 (GenBank accession number Z46233; Invitrogen, Carlsbad, Calif.). Typical PCR conditions are 25 cycles 94° C. denaturation for 1 minute, 45° C. annealing for one minute and 72° C. extension for 2 minutes; plus one cycle 72° C. extension for 10 minutes. A 50 mL reaction contained 30 pmol of each primer and 1 mg of template DNA; and 1×PCR buffer with MgCl2, 200 mM dGTP, 200 mM dATP, 200 mM dTTP, 200 mM dCTP, 2.5 units of Pwo DNA polymerase. PCR reactions are performed in RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, Calif.).
[0329]The amplified patatin gene was digested with restriction enzymes XhoI and EcoRI and cloned into the pBluescript vector (Stratagene, La Jolla, Calif.), digested with the same two restriction enzymes. The template plasmid DNA used for the PCR was pMON26820. The resulting plasmid (pMON 26869) was used for oligonucleotide-directed mutagenesis using the Bio-Rad mutagenesis kit based on the method of Kunkel et al., Proc Natl Acad Sci USA 82, 477-92 (1985). Briefly, single-stranded pMON26869 was used as template for mutagenesis and was prepared by superinfection of plasmid containing cells with M13K07 (Gorman et al., DNA and Protein Engineering techniques 2, 3-10 (1990)). DNA purified from transformed DH5αa E. coli colonies was used for sequence determination. Sequencing was performed using the ABI PRISM sequencing kit (Perkin Elmer Biosystems, Foster City, Calif.).
Protein Expression in Pichia pastoris
[0330]Plasmids containing the mutations in the patatin gene were digested with restriction enzymes XhoI and EcoRI. The patatin nucleic acid fragment was then ligated into the pPIC9 vector (Invitrogen, Carlsbad, Calif.), digested with the same two restriction enzymes to afford plasmid pMON37401. Pichia pastoris KM71 cells were electroporated with pMON37401 containing the appropriate mutation. The resulting transformed cells were used to produce protein in Pichia pastoris using the procedure supplied by the manufacturer (Invitrogen, Carlsbad, Calif.). The proteins were purified in the same way as the proteins expressed in E. coli (see below).
Western Blotting of Proteins
[0331]Protein samples were electrophoresed by SDS-PAGE and electroblotted onto PVDF membrane (Millipore, Bedford Mass.). Protein blots were processed by standard Western blotting (immunoblotting) techniques and were incubated in potato allergic serum diluted 1:5 in PBS buffer for 1 hour. After washing the blots 3 times with PBS, the blots were incubated in biotinylated anti-IgE (Johns Hopkins Hospital, Baltimore Md.) for 1 hour, followed by a 30 minute incubation in HRP-linked avidin (Promega, New York, N.Y.). IgE-reactive protein bands were visualized by using the ECL system (Amersham Pharmacia Biotech, NJ). As a control, one blot was incubated in anti-IgE only. His-tagged glyphosate oxidase and potato extracts was prepared and provided for this study by Regulatory Sciences, Monsanto Company. The peptides were evaluated using the same incubation procedures as described above.
Expression and Purification of Patatin in Corn
[0332]An isozyme of patatin, Pat17, was generated for expression in corn using a modified plant optimized gene sequence as described by Brown et al (U.S. Pat. No. 5,689,052). All the constructs contained the native 23 amino acid signal peptide for vacuolar targeting. Corn was transformed by microprojectile bombardment (Morrish et al., in Transgenic plants. Fundamentals and Applications (ed. Hiatt, A.) 133-171 (Marcel Dekker, New York, 1993); Songstad et al., In Vitro Cell Dev Biol--Plant 32, 179-183 (1996)). Protein from the transformed corn plants was purified by first grinding the leaves in liquid nitrogen and extracting the protein using 25 mM Tris/HCl. The plant extract was further dialyzed against 25 mM Tris/HCl pH 7.5. The plant extract was then loaded onto Mono Q HR 10/10 anion-exchange column (Amersham Pharmacia, NJ) equilibrated with 25 mM Tris/HCl pH 7.5 (buffer A). The protein was eluted with 25 mM Tris/HCl pH 7.5, 1 M KCl (buffer B) using a linear gradient of 0-100% buffer B using an HPLC system (Shimadzu). Fractions containing protein were assayed for esterase activity and dialyzed against 25 mM Tris/HCl pH 7.5, 1 M Ammonium Sulfate (buffer C). The protein was purified to homogeneity by loading onto a phenyl-Sepharose 16/10 column (Amersham Pharmacia, NJ) equilibrated with buffer C. Esterase active fractions were pooled and dialyzed against 25 mM Tris/HCl pH 7.5.
Expression and Purification of Patatin in E. coli
[0333]Pat17 was expressed in E. coli using the pET expression system (Novagen, WI). The coding region of the mature Pat17 gene (without its signal peptide) was amplified by PCR using the primers 5'-GGGCCATGGCGCAGTTGGGAGAAATGGTG-3' (NcoI site in italics) and 5'-AACAAAGCTTCTTATTGAGGTGCGGCCGCTTGCATGC-3' (NotI site in italics) using standard PCR reaction conditions as described in the Gene Amp kit (Perkin-Elmer Cetus, CT) and an annealing temperature of 40° C. The template was plasmid pMON26820. The resulting DNA was digested with NcoI and NotI and cloned into a modified pET24d plasmid, designed to add an N-terminal hexa-histidine tag to the protein. The correct sequence of the PCR product was verified by sequencing, and the plasmid was transformed into E. coli BL21 (DE3), and transformants selected on LB containing 25 mg/mL kanamycin. The expression strain was grown in LB containing 25 mg/mL kanamycin and induced for 8 hrs at 28° C. with 1 mM IPTG. Cells were harvested and washed in 50 mM Tris/HCl pH 8.5, 150 mM NaCl, and lysed by French Press at 20,000 psi. His-tagged protein was recovered in the soluble fraction of lysed cells and subsequently purified using Ni-NTA resin as described in the QIAexpressionist manual (Qiagen CA). The partially purified protein was then dialyzed against 25 mM Tris/HCl pH 7.5 (buffer A) and loaded onto Mono Q HR 10/10 anion-exchange column (Amersham Pharmacia, NJ) equilibrated with buffer A. The protein was eluted with 25 mM Tris/HCl pH 7.5, 1 M KCl (buffer B) using a linear gradient of 0-100% buffer B run over 30 min at a flow rate of 4 mL/min using an HPLC system (Shimadzu). Fractions containing protein were assayed for esterase activity. Esterase active fractions were pooled, concentrated and dialyzed against 25 mM Tris/HCl pH 7.5 and stored at 4° C.
Enzyme Activity Assays
[0334]Enzyme activity was measured as described previously using p-nitrophenyl caprate (Sigma, Mo.) as a substrate, dissolved in dimethylsulfoxide (5 mM stock solution) and diluted in 4% Triton X-100, 1% SDS to a final concentration of 1 mM. For the assay, 20 mL of protein solution was added to a mixture of 25 mL of the 1 mM substrate solution and 80 mL of 50 mM Tris pH 8.5. The enzyme activity was monitored at 405 nm in 6 sec interval for a period of 10 min. Esterase activity was expressed as DOD min-1mg-1 protein.
Insect Bioassay
[0335]The protein was also assayed for activity against larvae of Diabrotica virigifera (Western corn rootworm) by overlaying the test sample on an agar diet similar to that described previously (Marrone et al., J. Econ. Entom. 78, 290-3 (1985)). Proteins to be tested were diluted in 25 mM Tris/HCl pH 7.5 and overlayed on the diet surface. Neonate larvae were allowed to feed on the diet and mortality and growth stunting were evaluated after 6 days.
IgE Binding Epitopes on Patatin
[0336]A panel of eighty-nine overlapping peptides representing the amino acid sequence of patatin were synthesized to determine the regions responsible for IgE binding. Each peptide was 10 amino acids long and consisted of 6 amino acid overlap between the consecutive peptides. The peptides were evaluated for IgE binding with five different potato allergic patient sera. Patatin has 3 major epitopes. These major IgE binding regions represent amino acids 184-193, 188-197, 269-278 and 360-369. Other minor IgE binding regions represent amino acids 104-113, 138-147 and 316-325. The amino acids essential for IgE binding in each major and minor epitopes were determined by synthesizing peptides with single amino acid changes at each position by individually substituting an alanine residue at each non-alanine position in the epitopes. The resulting alanine substituted peptides were evaluated for IgE binding. Result effective substitutions were identified by a reduction in IgE binding with respect to the non-substituted peptide sequence. It was very interesting to note that all the epitopes contained a Tyr residue and substitution of this Tyr for Ala or Phe eliminated IgE binding.
Enzyme and Bioactivity
[0337]The Tyr residues identified to be critical for IgE binding in each of the epitopes were substituted with Phe either individually or in concert using site-directed mutagenesis. All the variants were expressed in Pichia pastoris and assessed for enzyme activity and insecticidal activity. The variants included Y106F, Y129F, Y185F, Y193F, Y270F, Y316F, Y362F, Y185F/Y193F, Y185F/Y193F/Y270F/Y316F/Y362F (5-Tyr) and Y106F/Y129F/Y185F/Y193F/Y270F/Y316F/Y362F (7-Tyr). All the variants maintained enzyme activity. The 5-Tyr and 7-Tyr variants were then assessed for insecticidal activity by overlaying protein (200 ppm final concentration). The proteins caused significant stunting of the larval growth as measured by the weight of the larvae after 6 days with the 5-Tyr variant showing higher insecticidal activity compared to the 7-Tyr and wild type proteins. The 7-Tyr variant was unstable upon long term storage at 4° C. and thus was not pursued further.
Immunoblotting
[0338]In order to test if the glycan moiety on patatin was important for binding of IgE, Pat17 was expressed in E. coli to produce a nonglycosylated protein and in corn to produce a plant glycosylated protein. The 5-Tyr variant was also expressed in E. coli to assess the individual contribution of the linear epitopes without the glycan moiety on the protein. The proteins were tested for binding to IgE using sera from five patients with allergy to potatoes and sera from one patient with allergies to many things but no allergy to potatoes. Proteins from both corn and E. coli were purified to homogeneity. These proteins were transferred to PVDF membrane (Millipore, MA) and subsequently probed with sera from patients with and without allergy to potatoes. A His-tagged glyphosate oxidase control was included in all the studies to verify that the His-tag did not affect the binding of IgE. Serum obtained from patient HS-07 (no allergy to potatoes) did not bind Pat17 expressed in E. coli but showed good binding to Pat17 from corn and also a protein at the same molecular weight in potato extract. It is interesting to note that this sera also showed strong binding to another protein (>46 kDa) in the potato. Sera from patients HS-01, HS-02, HS-03, HS-05 (allergy to potatoes) shows strong binding to Pat17 expressed in corn, but very weak to no binding to Pat17 produced in E. coli. Also, the sera from patients HS-01, HS-2, HS-03 and HS-05 bound to a protein of similar molecular weight in the potato extract. Sera from patients HS-01, HS-02 and HS-03 also showed binding to another protein in potato extract of a lower molecular weight (<30 kDa). Serum obtained from patient HS-06 (allergic to potatoes) showed very strong binding to wild type patatin expressed in both corn and E. coli but weaker binding to the 5-Tyr variant expressed in E. coli. Sera from HS-06 also showed very strong binding to a protein in potato extract with similar molecular weight as patatin. The sera from all the patients showed no binding to His-tagged glyphosate oxidase indicating that the His-tag does not bind IgE. These results strongly suggest that the glycan moiety on Pat17 is responsible for IgE binding in some potato allergic patients and linear epitopes also contribute to the antigenicity of patatin.
Example 20
Alternative Nucleic Acid and Protein Sequences
[0339]For future variations of the patatin protein, sequences showing high similarity to the sequences disclosed herein could be used in producing deallergenized patatin proteins and permuteins. For example, a BLAST search (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410, 1990) can be performed to identify additional patatin sequences. Sources other than those disclosed herein can be used to obtain a patatin nucleic acid sequence, and the encoded patatin protein. Furthermore, subunit sequences from different organisms can be combined to create a novel patatin sequence incorporating structural, regulatory, and enzymatic properties from different sources.
Example 21
Nucleic Acid Mutation and Hybridization
[0340]Variations in the nucleic acid sequence encoding a patatin protein may lead to mutant patatin protein sequences that display equivalent or superior enzymatic characteristics when compared to the sequences disclosed herein. This invention accordingly encompasses nucleic acid sequences which are similar to the sequences disclosed herein, protein sequences which are similar to the sequences disclosed herein, and the nucleic acid sequences that encode them. Mutations can include deletions, insertions, truncations, substitutions, fusions, shuffling of subunit sequences, and the like.
[0341]Mutations to a nucleic acid sequence can be introduced in either a specific or random manner, both of which are well known to those of skill in the art of molecular biology. A myriad of site-directed mutagenesis techniques exist, typically using oligonucleotides to introduce mutations at specific locations in a nucleic acid sequence. Examples include single strand rescue (Kunkel, T. Proc. Natl. Acad. Sci. U.S.A., 82: 488-492, 1985), unique site elimination (Deng and Nickloff, Anal. Biochem. 200: 81, 1992), nick protection (Vandeyar, et al. Gene 65: 129-133, 1988), and PCR (Costa, et al. Methods Mol. Biol. 57: 31-44, 1996). Random or non-specific mutations can be generated by chemical agents (for a general review, see Singer and Kusmierek, Ann. Rev. Biochem. 52: 655-693, 1982) such as nitrosoguanidine (Cerda-Olmedo et al., J. Mol. Biol. 33: 705-719, 1968; Guerola, et al. Nature New Biol. 230: 122-125, 1971) and 2-aminopurine (Rogan and Bessman, J. Bacteriol. 103: 622-633, 1970), or by biological methods such as passage through mutator strains (Greener et al. Mol. Biotechnol. 7: 189-195, 1997).
[0342]Nucleic acid hybridization is a technique well known to those of skill in the art of DNA manipulation. The hybridization properties of a given pair of nucleic acids is an indication of their similarity or identity. Mutated nucleic acid sequences can be selected for their similarity to the disclosed patatin nucleic acid sequences on the basis of their hybridization to the disclosed sequences. Low stringency conditions can be used to select sequences with multiple mutations. One may wish to employ conditions such as about 0.15 M to about 0.9 M sodium chloride, at temperatures ranging from about 20° C. to about 55° C. High stringency conditions can be used to select for nucleic acid sequences with higher degrees of identity to the disclosed sequences. Conditions employed may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS and/or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at temperatures between about 50° C. and about 70° C. More preferably, high stringency conditions are 0.02 M sodium chloride, 0.5% casein, 0.02% SDS, 0.001 M sodium citrate, at a temperature of 50° C.
Example 22
Determination of Homologous and Degenerate Nucleic Acid Sequences
[0343]Modification and changes can be made in the sequence of the proteins of the present invention and the nucleic acid segments which encode them and still obtain a functional molecule that encodes a protein with desirable properties. The following is a discussion based upon changing the amino acid sequence of a protein to create an equivalent, or possibly an improved, second-generation molecule. The amino acid changes can be achieved by changing the codons of the nucleic acid sequence, according to the codons given in Table 10.
TABLE-US-00011 TABLE 10 Codon degeneracies of amino acids Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGC TGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAG Phenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine H His CAC CAT Isoleucine I Ile ATA ATC ATT Lysine K Lys AAA AAG Leucine L Leu TTA TTG CTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AAC AAT Proline P Pro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine T Thr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTT Trytophan W Trp TGG Tyrosine Y Tyr TAC TAT
[0344]Certain amino acids can be substituted for other amino acids in a protein sequence without appreciable loss of enzymatic activity. It is thus contemplated that various changes can be made in the peptide sequences of the disclosed protein sequences, or their corresponding nucleic acid sequences without appreciable loss of the biological activity.
[0345]In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
[0346]Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate/glutamine/aspartate/asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[0347]It is known in the art that certain amino acids can be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are more preferred, and those within ±0.5 are most preferred.
[0348]It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (Hopp, T. P., issued Nov. 19, 1985) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine/lysine (+3,0); aspartate/glutamate (+3.0±1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
[0349]It is understood that an amino acid can be substituted by another amino acid having a similar hydrophilicity score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are more preferred, and those within ±0.5 are most preferred.
[0350]As outlined above, amino acid substitutions are therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Changes which are not expected to be advantageous may also be used if these resulted in functional patatin proteins.
Example 23
Production of Patatin Proteins and Permuteins in Plants
[0351]Plant Vectors
[0352]In plants, transformation vectors capable of introducing nucleic acid sequences encoding patatin proteins and permuteins are easily designed, and generally contain one or more nucleic acid coding sequences of interest under the transcriptional control of 5' and 3' regulatory sequences. Such vectors generally comprise, operatively linked in sequence in the 5' to 3' direction, a promoter sequence that directs the transcription of a downstream heterologous structural nucleic acid sequence in a plant; optionally, a 5' non-translated leader sequence; a nucleic acid sequence that encodes a protein of interest; and a 3' non-translated region that encodes a polyadenylation signal which functions in plant cells to cause the termination of transcription and the addition of polyadenylate nucleotides to the 3' end of the mRNA encoding the protein. Plant transformation vectors also generally contain a selectable marker. Typical 5'-3' regulatory sequences include a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. Vectors for plant transformation have been reviewed in Rodriguez et al. (Vectors: A Survey of Molecular Coning Vectors and Their Uses, Butterworths, Boston., 1988), Glick et al. (Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., 1993), and Croy (Plant Molecular Biology Labfax, Hames and Rickwood (Eds.), BIOS Scientific Publishers Limited, Oxford, UK., 1993).
[0353]Plant Promoters
[0354]Plant promoter sequences can be constitutive or inducible, environmentally- or developmentally-regulated, or cell- or tissue-specific. Often-used constitutive promoters include the CaMV 35S promoter (Odell, J. T. et al., Nature 313: 810-812, 1985), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins et al., Nucleic Acids Res. 20: 8451-8466, 1987), the mannopine synthase (mas) promoter, the nopaline synthase (nos) promoter, and the octopine synthase (ocs) promoter. Useful inducible promoters include promoters induced by salicylic acid or polyacrylic acids (PR-1, Williams, S. W. et al, Biotechnology 10: 540-543, 1992), induced by application of safeners (substituted benzenesulfonamide herbicides, Hershey, H. P. and Stoner, T. D., Plant Mol. Biol. 17: 679-690, 1991), heat-shock promoters (Ou-Lee et al., Proc. Natl. Acad. Sci. U.S.A. 83: 6815-6819, 1986; Ainley et al., Plant Mol. Biol. 14: 949-967, 1990), a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al., Plant Mol. Biol. 17: 9-18, 1991), hormone-inducible promoters (Yamaguchi-Shinozaki, K. et al., Plant Mol. Biol. 15: 905-912, 1990; Kares et al., Plant Mol. Biol. 15: 225-236, 1990), and light-inducible promoters associated with the small subunit of RuBP carboxylase and LHCP gene families (Kuhlemeier et al., Plant Cell 1: 471, 1989; Feinbaum, R. L. et al., Mol. Gen. Genet. 226: 449-456, 1991; Weisshaar, B. et al., EMBO J. 10: 1777-1786, 1991; Lam, E. and Chua, N. H., J. Biol. Chem. 266: 17131-17135, 1990; Castresana, C. et al., EMBO J. 7: 1929-1936, 1988; Schulze-Lefert et al., EMBO J. 3: 651, 1989). Examples of useful tissue-specific, developmentally-regulated promoters include the β-conglycinin 7S promoter (Doyle, J. J. et al., J. Biol. Chem. 261: 9228-9238, 1986; Slighton and Beachy, Planta 172: 356-363, 1987), and seed-specific promoters (Knutzon, D. S. et al., Proc. Natl. Acad. Sci. U.S.A. 89: 2624-2628, 1992; Bustos, M. M. et al., EMBO J. 10: 1469-1479, 1991; Lam and Chua, Science 248: 471, 1991; Stayton et al., Aust. J. Plant. Physiol. 18: 507, 1991). Plant functional promoters useful for preferential expression in seed plastids include those from plant storage protein genes and from genes involved in fatty acid biosynthesis in oilseeds. Examples of such promoters include the 5' regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res. 1: 209-219, 1991), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, and oleosin. Seed-specific gene regulation is discussed in EP 0 255 378. Promoter hybrids can also be constructed to enhance transcriptional activity (Comai, L. and Moran, P.M., U.S. Pat. No. 5,106,739, issued Apr. 21, 1992), or to combine desired transcriptional activity and tissue specificity.
[0355]Plant Transformation and Regeneration
[0356]A variety of different methods can be employed to introduce such vectors into plant protoplasts, cells, callus tissue, leaf discs, meristems, etcetera, to generate transgenic plants, including Agrobacterium-mediated transformation, particle gun delivery, microinjection, electroporation, polyethylene glycol mediated protoplast transformation, liposome-mediated transformation, etcetera (reviewed in Potrykus, I. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205-225, 1991). In general, transgenic plants comprising cells containing and expressing DNAs encoding patatin proteins and permuteins can be produced by transforming plant cells with a DNA construct as described above via any of the foregoing methods; selecting plant cells that have been transformed on a selective medium; regenerating plant cells that have been transformed to produce differentiated plants; and selecting a transformed plant which expresses the protein-encoding nucleotide sequence.
[0357]Specific methods for transforming a wide variety of dicots and obtaining transgenic plants are well documented in the literature (Gasser and Fraley, Science 244: 1293-1299, 1989; Fisk and Dandekar, Scientia Horticulturae 55: 5-36, 1993; Christou, Agro Food Industry Hi Tech, p. 17, 1994; and the references cited therein).
[0358]Successful transformation and plant regeneration have been reported in the monocots as follows: asparagus (Asparagus officinalis; Bytebier et al., Proc. Natl. Acad. Sci. U.S.A. 84: 5345-5349, 1987); barley (Hordeum vulgarae; Wan and Lemaux, Plant Physiol. 104: 37-48, 1994); maize (Zea mays; Rhodes, C. A. et al., Science 240: 204-207, 1988; Gordon-Kamm et al., Plant Cell 2: 603-618, 1990; Fromm, M. E. et al., Bio/Technology 8: 833-839, 1990; Koziel et al., Bio/Technology 11: 194-200, 1993); oats (Avena sativa; Somers et al., Bio/Technology 10: 1589-1594, 1992); orchardgrass (Dactylis glomerata; Horn et al., Plant Cell Rep. 7: 469-472, 1988); rice (Oryza sativa, including indica and japonica varieties; Toriyama et al., Bio/Technology 6: 10, 1988; Zhang et al., Plant Cell Rep. 7: 379-384, 1988; Luo and Wu, Plant Mol. Biol. Rep. 6: 165-174, 1988; Zhang and Wu, Theor. Appl. Genet. 76: 835-840, 1988; Christou et al., Bio/Technology 9: 957-962, 1991); rye (Secale cereale; De la Pena et al., Nature 325: 274-276, 1987); sorghum (Sorghum bicolor; Casas, A. M. et al., Proc. Natl. Acad. Sci. U.S.A. 90: 11212-11216, 1993); sugar cane (Saccharum spp.; Bower and Birch, Plant J. 2: 409-416, 1992); tall fescue (Festuca arundinacea; Wang, Z. Y. et al., Bio/Technology 10: 691-696, 1992); turfgrass (Agrostis palustris; Zhong et al., Plant Cell Rep. 13: 1-6, 1993); wheat (Triticum aestivum; Vasil et al., Bio/Technology 10: 667-674, 1992; Weeks, T. et al., Plant Physiol. 102: 1077-1084, 1993; Becker et al., Plant J. 5: 299-307, 1994), and alfalfa (Masoud, S. A. et al., Transgen. Res. 5: 313, 1996); Brassica (canola/oilseed rape) (Fry, J. Plant Cell Rep. 6: 321-325, 1987); and soybean (Hinchee, M. Bio/Technol. 6: 915-922, 1988).
[0359]All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations can be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.
Sequence CWU
1
29511158DNASolanum tuberosum 1atggcaacta ctaaatcttt tttaatttta atatttatga
tattagcaac tactagttca 60acatttgctc agttgggaga aatggtgact gttcttagta
ttgatggagg tggaattaga 120gggatcattc cggctaccat tctcgaattt cttgaaggac
aacttcagga aatggacaat 180aatgcagatg caagacttgc agattacttt gatgtaattg
gaggaacaag tacaggaggt 240ttattgactg ctatgataag tactccaaat gaaaacaatc
gaccctttgc tgctgccaaa 300gaaattgtac ctttttactt cgaacatggc cctcagattt
ttaatcctag tggtcaaatt 360ttaggcccaa aatatgatgg aaaatatctt atgcaagttc
ttcaagaaaa acttggagaa 420actcgtgtgc atcaagcttt gacagaagtt gtcatctcaa
gctttgacat caaaacaaat 480aagccagtaa tattcactaa gtcaaattta gcaaactctc
cagaattgga tgctaagatg 540tatgacataa gttattccac agcagcagct ccaacatatt
ttcctccgca ttactttgtt 600actaatacta gtaatggaga tgaatatgag ttcaatcttg
ttgatggtgc tgttgctact 660gttgctgatc cggcgttatt atccattagc gttgcaacga
gacttgcaca aaaggatcca 720gcatttgctt caattaggtc attgaattac aaaaaaatgc
tgttgctctc attaggcact 780ggcactactt cagagtttga taaaacatat acagcaaaag
aggcagctac ctggactgct 840gtacattgga tgttagttat acagaaaatg actgatgcag
caagttctta catgactgat 900tattaccttt ctactgcttt tcaagctctt gattcaaaaa
acaattacct cagggttcaa 960gaaaatgcat taacaggcac aactactgaa atggatgatg
cttctgaggc taatatggaa 1020ttattagtac aagttggtga aaacttattg aagaaaccag
tttccgaaga caatcctgaa 1080acctatgagg aagctctaaa gaggtttgca aaattgctct
ctgataggaa gaaactccga 1140gcaaacaaag cttcttat
11582386PRTSolanum tuberosum 2Met Ala Thr Thr Lys
Ser Phe Leu Ile Leu Ile Phe Met Ile Leu Ala1 5
10 15Thr Thr Ser Ser Thr Phe Ala Gln Leu Gly Glu
Met Val Thr Val Leu 20 25
30Ser Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu
35 40 45Glu Phe Leu Glu Gly Gln Leu Gln
Glu Met Asp Asn Asn Ala Asp Ala 50 55
60Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly65
70 75 80Leu Leu Thr Ala Met
Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe 85
90 95Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe
Glu His Gly Pro Gln 100 105
110Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys
115 120 125Tyr Leu Met Gln Val Leu Gln
Glu Lys Leu Gly Glu Thr Arg Val His 130 135
140Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys Thr
Asn145 150 155 160Lys Pro
Val Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro Glu Leu
165 170 175Asp Ala Lys Met Tyr Asp Ile
Ser Tyr Ser Thr Ala Ala Ala Pro Thr 180 185
190Tyr Phe Pro Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly
Asp Glu 195 200 205Tyr Glu Phe Asn
Leu Val Asp Gly Ala Val Ala Thr Val Ala Asp Pro 210
215 220Ala Leu Leu Ser Ile Ser Val Ala Thr Arg Leu Ala
Gln Lys Asp Pro225 230 235
240Ala Phe Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu
245 250 255Ser Leu Gly Thr Gly
Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260
265 270Lys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met
Leu Val Ile Gln 275 280 285Lys Met
Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser 290
295 300Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn
Tyr Leu Arg Val Gln305 310 315
320Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu
325 330 335Ala Asn Met Glu
Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys 340
345 350Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu
Glu Ala Leu Lys Arg 355 360 365Phe
Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370
375 380Ser Tyr385354DNAArtificialSynthetic
construct 3ggagctcgag aaaagagagg ctgaagctca gttgggagaa atggtgactg ttct
54429DNAArtificialSynthetic construct 4ggtctagagg aattctcatt
aataagaag
2951138DNAArtificialSynthetic construct 5ggagctcgag aaaagagagg ctgaagctca
gttgggagaa atggtgactg ttcttagtat 60tgatggaggt ggaattagag ggatcattcc
ggctaccatt ctcgaatttc ttgaaggaca 120acttcaggaa atggacaata atgcagatgc
aagacttgca gattactttg atgtaattgg 180aggaacaagt acaggaggtt tattgactgc
tatgataagt actccaaatg aaaacaatcg 240accctttgct gctgccaaag aaattgtacc
tttttacttc gaacatggcc ctcagatttt 300taatcctagt ggtcaaattt taggcccaaa
atatgatgga aaatatctta tgcaagttct 360tcaagaaaaa cttggagaaa ctcgtgtgca
tcaagctttg acagaagttg tcatctcaag 420ctttgacatc aaaacaaata agccagtaat
attcactaag tcaaatttag caaactctcc 480agaattggat gctaagatgt atgacataag
ttattccaca gcagcagctc caacatattt 540tcctccgcat tactttgtta ctaatactag
taatggagat gaatatgagt tcaatcttgt 600tgatggtgct gttgctactg ttgctgatcc
ggcgttatta tccattagcg ttgcaacgag 660acttgcacaa aaggatccag catttgcttc
aattaggtca ttgaattaca aaaaaatgct 720gttgctctca ttaggcactg gcactacttc
agagtttgat aaaacatata cagcaaaaga 780ggcagctacc tggactgctg tacattggat
gttagttata cagaaaatga ctgatgcagc 840aagttcttac atgactgatt attacctttc
tactgctttt caagctcttg attcaaaaaa 900caattacctc agggttcaag aaaatgcatt
aacaggcaca actactgaaa tggatgatgc 960ttctgaggct aatatggaat tattagtaca
agttggtgaa aacttattga agaaaccagt 1020ttccgaagac aatcctgaaa cctatgagga
agctctaaag aggtttgcaa aattgctctc 1080tgataggaag aaactccgag caaacaaagc
ttcttattaa tgagaattcc tctagacc 11386452PRTArtificialSynthetic
polypeptide 6Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser
Ser1 5 10 15Ala Leu Ala
Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20
25 30Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser
Asp Leu Glu Gly Asp Phe 35 40
45Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50
55 60Phe Ile Asn Thr Thr Ile Ala Ser Ile
Ala Ala Lys Glu Glu Gly Val65 70 75
80Ser Leu Glu Lys Arg Glu Ala Glu Ala Gln Leu Gly Glu Met
Val Thr 85 90 95Val Leu
Ser Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr 100
105 110Ile Leu Glu Phe Leu Glu Gly Gln Leu
Gln Glu Met Asp Asn Asn Ala 115 120
125Asp Ala Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr
130 135 140Gly Gly Leu Leu Thr Ala Met
Ile Ser Thr Pro Asn Glu Asn Asn Arg145 150
155 160Pro Phe Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr
Phe Glu His Gly 165 170
175Pro Gln Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp
180 185 190Gly Lys Tyr Leu Met Gln
Val Leu Gln Glu Lys Leu Gly Glu Thr Arg 195 200
205Val His Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp
Ile Lys 210 215 220Thr Asn Lys Pro Val
Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro225 230
235 240Glu Leu Asp Ala Lys Met Tyr Asp Ile Ser
Tyr Ser Thr Ala Ala Ala 245 250
255Pro Thr Tyr Phe Pro Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly
260 265 270Asp Glu Tyr Glu Phe
Asn Leu Val Asp Gly Ala Val Ala Thr Val Ala 275
280 285Asp Pro Ala Leu Leu Ser Ile Ser Val Ala Thr Arg
Leu Ala Gln Lys 290 295 300Asp Pro Ala
Phe Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu305
310 315 320Leu Leu Ser Leu Gly Thr Gly
Thr Thr Ser Glu Phe Asp Lys Thr Tyr 325
330 335Thr Ala Lys Glu Ala Ala Thr Trp Thr Ala Val His
Trp Met Leu Val 340 345 350Ile
Gln Lys Met Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr 355
360 365Leu Ser Thr Ala Phe Gln Ala Leu Asp
Ser Lys Asn Asn Tyr Leu Arg 370 375
380Val Gln Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala385
390 395 400Ser Glu Ala Asn
Met Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu 405
410 415Lys Lys Pro Val Ser Glu Asp Asn Pro Glu
Thr Tyr Glu Glu Ala Leu 420 425
430Lys Arg Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn
435 440 445Lys Ala Ser Tyr
4507367PRTArtificialSynthetic polypeptide 7Glu Ala Glu Ala Gln Leu Gly
Glu Met Val Thr Val Leu Ser Ile Asp1 5 10
15Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu
Glu Phe Leu 20 25 30Glu Gly
Gln Leu Gln Glu Met Asp Asn Asn Ala Asp Ala Arg Leu Ala 35
40 45Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser
Thr Gly Gly Leu Leu Thr 50 55 60Ala
Met Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala65
70 75 80Lys Glu Ile Val Pro Phe
Tyr Phe Glu His Gly Pro Gln Ile Phe Asn 85
90 95Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly
Lys Tyr Leu Met 100 105 110Gln
Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His Gln Ala Leu 115
120 125Thr Glu Val Val Ile Ser Ser Phe Asp
Ile Lys Thr Asn Lys Pro Val 130 135
140Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro Glu Leu Asp Ala Lys145
150 155 160Met Tyr Asp Ile
Ser Tyr Ser Thr Ala Ala Ala Pro Thr Tyr Phe Pro 165
170 175Pro His Tyr Phe Val Thr Asn Thr Ser Asn
Gly Asp Glu Tyr Glu Phe 180 185
190Asn Leu Val Asp Gly Ala Val Ala Thr Val Ala Asp Pro Ala Leu Leu
195 200 205Ser Ile Ser Val Ala Thr Arg
Leu Ala Gln Lys Asp Pro Ala Phe Ala 210 215
220Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu
Gly225 230 235 240Thr Gly
Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala Lys Glu Ala
245 250 255Ala Thr Trp Thr Ala Val His
Trp Met Leu Val Ile Gln Lys Met Thr 260 265
270Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr
Ala Phe 275 280 285Gln Ala Leu Asp
Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala 290
295 300Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser
Glu Ala Asn Met305 310 315
320Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys Pro Val Ser
325 330 335Glu Asp Asn Pro Glu
Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys 340
345 350Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys
Ala Ser Tyr 355 360
365824DNAArtificialSynthetic construct 8atgttcgaag aaaaaaggta caat
24924DNAArtificialSynthetic construct
9ttgcataaga aattttccat cata
241024DNAArtificialSynthetic construct 10tgctgtggaa aaacttatgt cata
241124DNAArtificialSynthetic
construct 11cggaggaaaa aatgttggag ctgc
241251DNAArtificialSynthetic construct 12atgcggagga aaaaatgttg
gagctgctgc tgtggaaaaa cttatgtcat a
511324DNAArtificialSynthetic construct 13ttttgctgta aatgttttat caaa
241424DNAArtificialSynthetic
construct 14aaccctgagg aaattgtttt ttga
241524DNAArtificialSynthetic construct 15agcttcctca aaggtttcag
gatt
241610PRTArtificialSynthetic polypeptide 16Gln Leu Gly Glu Met Val Thr
Val Leu Ser1 5
101710PRTArtificialSynthetic polypeptide 17Met Val Thr Val Leu Ser Ile
Asp Gly Gly1 5
101810PRTArtificialSynthetic polypeptide 18Leu Ser Ile Asp Gly Gly Gly
Ile Arg Gly1 5
101910PRTArtificialSynthetic polypeptide 19Gly Gly Gly Ile Arg Gly Ile
Ile Pro Ala1 5
102010PRTArtificialSynthetic polypeptide 20Arg Gly Ile Ile Pro Ala Thr
Ile Leu Glu1 5
102110PRTArtificialSynthetic polypeptide 21Pro Ala Thr Ile Leu Glu Phe
Leu Glu Gly1 5
102210PRTArtificialSynthetic polypeptide 22Leu Glu Phe Leu Glu Gly Gln
Leu Gln Glu1 5
102310PRTArtificialSynthetic polypeptide 23Glu Gly Gln Leu Gln Glu Met
Asp Asn Asn1 5
102410PRTArtificialSynthetic polypeptide 24Gln Glu Met Asp Asn Asn Ala
Asp Ala Arg1 5
102510PRTArtificialSynthetic polypeptide 25Asn Asn Ala Asp Ala Arg Leu
Ala Asp Tyr1 5
102610PRTArtificialSynthetic polypeptide 26Ala Arg Leu Ala Asp Tyr Phe
Asp Val Ile1 5
102710PRTArtificialSynthetic polypeptide 27Asp Tyr Phe Asp Val Ile Gly
Gly Thr Ser1 5
102810PRTArtificialSynthetic polypeptide 28Val Ile Gly Gly Thr Ser Thr
Gly Gly Leu1 5
102910PRTArtificialSynthetic polypeptide 29Thr Ser Thr Gly Gly Leu Leu
Thr Ala Met1 5
103010PRTArtificialSynthetic polypeptide 30Gly Leu Leu Thr Ala Met Ile
Ser Thr Pro1 5
103110PRTArtificialSynthetic polypeptide 31Ala Met Ile Ser Thr Pro Asn
Glu Asn Asn1 5
103210PRTArtificialSynthetic polypeptide 32Thr Pro Asn Glu Asn Asn Arg
Pro Phe Ala1 5
103310PRTArtificialSynthetic polypeptide 33Asn Asn Arg Pro Phe Ala Ala
Ala Lys Glu1 5
103410PRTArtificialSynthetic polypeptide 34Phe Ala Ala Ala Lys Glu Ile
Val Pro Phe1 5
103510PRTArtificialSynthetic polypeptide 35Lys Glu Ile Val Pro Phe Tyr
Phe Glu His1 5
103610PRTArtificialSynthetic polypeptide 36Pro Phe Tyr Phe Glu His Gly
Pro Gln Ile1 5
103710PRTArtificialSynthetic polypeptide 37Glu His Gly Pro Gln Ile Phe
Asn Pro Ser1 5
103810PRTArtificialSynthetic polypeptide 38Gln Ile Phe Asn Pro Ser Gly
Gln Ile Leu1 5
103910PRTArtificialSynthetic polypeptide 39Pro Ser Gly Gln Ile Leu Gly
Pro Lys Tyr1 5
104010PRTArtificialSynthetic polypeptide 40Ile Leu Gly Pro Lys Tyr Asp
Gly Lys Tyr1 5
104110PRTArtificialSynthetic polypeptide 41Lys Tyr Asp Gly Lys Tyr Leu
Met Gln Val1 5
104210PRTArtificialSynthetic polypeptide 42Lys Tyr Leu Met Gln Val Leu
Gln Glu Lys1 5
104310PRTArtificialSynthetic polypeptide 43Gln Val Leu Gln Glu Lys Leu
Gly Glu Thr1 5
104410PRTArtificialSynthetic polypeptide 44Glu Lys Leu Gly Glu Thr Arg
Val His Gln1 5
104510PRTArtificialSynthetic polypeptide 45Glu Thr Arg Val His Gln Ala
Leu Thr Glu1 5
104610PRTArtificialSynthetic polypeptide 46His Gln Ala Leu Thr Glu Val
Val Ile Ser1 5
104710PRTArtificialSynthetic polypeptide 47Thr Glu Val Val Ile Ser Ser
Phe Asp Ile1 5
104810PRTArtificialSynthetic polypeptide 48Ile Ser Ser Phe Asp Ile Lys
Thr Asn Lys1 5
104910PRTArtificialSynthetic polypeptide 49Asp Ile Lys Thr Asn Lys Pro
Val Ile Phe1 5
105010PRTArtificialSynthetic polypeptide 50Asn Lys Pro Val Ile Phe Thr
Lys Ser Asn1 5
105110PRTArtificialSynthetic polypeptide 51Ile Phe Thr Lys Ser Asn Leu
Ala Asn Ser1 5
105210PRTArtificialSynthetic polypeptide 52Ser Asn Leu Ala Asn Ser Pro
Glu Leu Asp1 5
105310PRTArtificialSynthetic polypeptide 53Asn Ser Pro Glu Leu Asp Ala
Lys Met Tyr1 5
105410PRTArtificialSynthetic polypeptide 54Leu Asp Ala Lys Met Tyr Asp
Ile Ser Tyr1 5
105510PRTArtificialSynthetic polypeptide 55Met Tyr Asp Ile Ser Tyr Ser
Thr Ala Ala1 5
105610PRTArtificialSynthetic polypeptide 56Ser Tyr Ser Thr Ala Ala Ala
Pro Thr Tyr1 5
105710PRTArtificialSynthetic polypeptide 57Ala Ala Ala Pro Thr Tyr Phe
Pro Pro His1 5
105810PRTArtificialSynthetic polypeptide 58Thr Tyr Phe Pro Pro His Tyr
Phe Val Thr1 5
105910PRTArtificialSynthetic polypeptide 59Pro His Tyr Phe Val Thr Asn
Thr Ser Asn1 5
106010PRTArtificialSynthetic polypeptide 60Val Thr Asn Thr Ser Asn Gly
Asp Glu Tyr1 5
106110PRTArtificialSynthetic polypeptide 61Ser Asn Gly Asp Glu Tyr Glu
Phe Asn Leu1 5
106210PRTArtificialSynthetic polypeptide 62Glu Tyr Glu Phe Asn Leu Val
Asp Gly Ala1 5
106310PRTArtificialSynthetic polypeptide 63Asn Leu Val Asp Gly Ala Val
Ala Thr Val1 5
106410PRTArtificialSynthetic polypeptide 64Gly Ala Val Ala Thr Val Ala
Asp Pro Ala1 5
106510PRTArtificialSynthetic polypeptide 65Thr Val Ala Asp Pro Ala Leu
Leu Ser Ile1 5
106610PRTArtificialSynthetic polypeptide 66Pro Ala Leu Leu Ser Ile Ser
Val Ala Thr1 5
106710PRTArtificialSynthetic polypeptide 67Ser Ile Ser Val Ala Thr Arg
Leu Ala Gln1 5
106810PRTArtificialSynthetic polypeptide 68Ala Thr Arg Leu Ala Gln Lys
Asp Pro Ala1 5
106910PRTArtificialSynthetic polypeptide 69Ala Gln Lys Asp Pro Ala Phe
Ala Ser Ile1 5
107010PRTArtificialSynthetic polypeptide 70Pro Ala Phe Ala Ser Ile Arg
Ser Leu Asn1 5
107110PRTArtificialSynthetic polypeptide 71Ser Ile Arg Ser Leu Asn Tyr
Lys Lys Met1 5
107210PRTArtificialSynthetic polypeptide 72Leu Asn Tyr Lys Lys Met Leu
Leu Leu Ser1 5
107310PRTArtificialSynthetic polypeptide 73Lys Met Leu Leu Leu Ser Leu
Gly Thr Gly1 5
107410PRTArtificialSynthetic polypeptide 74Leu Ser Leu Gly Thr Gly Thr
Thr Ser Glu1 5
107510PRTArtificialSynthetic polypeptide 75Thr Gly Thr Thr Ser Glu Phe
Asp Lys Thr1 5
107610PRTArtificialSynthetic polypeptide 76Ser Glu Phe Asp Lys Thr Tyr
Thr Ala Lys1 5
107710PRTArtificialSynthetic polypeptide 77Lys Thr Tyr Thr Ala Lys Glu
Ala Ala Thr1 5
107810PRTArtificialSynthetic polypeptide 78Ala Lys Glu Ala Ala Thr Trp
Thr Ala Val1 5
107910PRTArtificialSynthetic polypeptide 79Ala Thr Trp Thr Ala Val His
Trp Met Leu1 5
108010PRTArtificialSynthetic polypeptide 80Ala Val His Trp Met Leu Val
Ile Gln Lys1 5
108110PRTArtificialSynthetic polypeptide 81Met Leu Val Ile Gln Lys Met
Thr Asp Ala1 5
108210PRTArtificialSynthetic polypeptide 82Gln Lys Met Thr Asp Tyr Tyr
Leu Ser Thr1 5
108310PRTArtificialSynthetic polypeptide 83Asp Ala Ala Ser Ser Tyr Met
Thr Asp Tyr1 5
108410PRTArtificialSynthetic polypeptide 84Ser Tyr Met Thr Asp Tyr Tyr
Leu Ser Thr1 5
108510PRTArtificialSynthetic polypeptide 85Asp Tyr Tyr Leu Ser Thr Ala
Phe Gln Ala1 5
108610PRTArtificialSynthetic polypeptide 86Ser Thr Ala Phe Gln Ala Leu
Asp Ser Lys1 5
108710PRTArtificialSynthetic polypeptide 87Gln Ala Leu Asp Ser Lys Asn
Asn Tyr Leu1 5
108810PRTArtificialSynthetic polypeptide 88Ser Lys Asn Asn Tyr Leu Arg
Val Gln Glu1 5
108910PRTArtificialSynthetic polypeptide 89Tyr Leu Arg Val Gln Glu Asn
Ala Leu Thr1 5
109010PRTArtificialSynthetic polypeptide 90Gln Glu Asn Ala Leu Thr Gly
Thr Thr Thr1 5
109110PRTArtificialSynthetic polypeptide 91Leu Thr Gly Thr Thr Thr Glu
Met Asp Asp1 5
109210PRTArtificialSynthetic polypeptide 92Thr Thr Glu Met Asp Asp Ala
Ser Glu Ala1 5
109310PRTArtificialSynthetic polypeptide 93Asp Asp Ala Ser Glu Ala Asn
Met Glu Leu1 5
109410PRTArtificialSynthetic polypeptide 94Glu Ala Asn Met Glu Leu Leu
Val Gln Val1 5
109510PRTArtificialSynthetic polypeptide 95Glu Leu Leu Val Gln Val Gly
Glu Asn Leu1 5
109610PRTArtificialSynthetic polypeptide 96Gln Val Gly Glu Asn Leu Leu
Lys Lys Pro1 5
109710PRTArtificialSynthetic polypeptide 97Asn Leu Leu Lys Lys Pro Val
Ser Glu Asp1 5
109810PRTArtificialSynthetic polypeptide 98Lys Pro Val Ser Glu Asp Asn
Pro Glu Thr1 5
109910PRTArtificialSynthetic polypeptide 99Glu Asp Asn Pro Glu Thr Tyr
Glu Glu Ala1 5
1010010PRTArtificialSynthetic polypeptide 100Glu Thr Tyr Glu Glu Ala Leu
Lys Arg Phe1 5
1010110PRTArtificialSynthetic polypeptide 101Glu Ala Leu Lys Arg Phe Ala
Lys Leu Leu1 5
1010210PRTArtificialSynthetic polypeptide 102Arg Phe Ala Lys Leu Leu Ser
Asp Arg Lys1 5
1010310PRTArtificialSynthetic polypeptide 103Leu Leu Ser Asp Arg Lys Lys
Leu Arg Ala1 5
1010410PRTArtificialSynthetic polypeptide 104Arg Lys Lys Leu Arg Ala Asn
Lys Ala Ser1 5
1010510PRTArtificialSynthetic polypeptide 105Asp Tyr Phe Asp Val Ile Gly
Gly Thr Ser1 5
1010610PRTArtificialSynthetic polypeptide 106Asp Tyr Phe Asp Val Ile Ala
Gly Thr Ser1 5
1010710PRTArtificialSynthetic polypeptide 107Val Ile Gly Gly Thr Ser Thr
Gly Gly Leu1 5
1010810PRTArtificialSynthetic polypeptide 108Val Ile Ala Gly Thr Ser Thr
Gly Ala Leu1 5
1010910PRTArtificialSynthetic polypeptide 109Ala Phe Tyr Phe Glu His Gly
Pro Gln Ile1 5
1011010PRTArtificialSynthetic polypeptide 110Pro Ala Tyr Phe Glu His Gly
Pro Gln Ile1 5
1011110PRTArtificialSynthetic polypeptide 111Pro Phe Ala Phe Glu His Gly
Pro Gln Ile1 5
1011210PRTArtificialSynthetic polypeptide 112Pro Phe Tyr Ala Glu His Gly
Pro Gln Ile1 5
1011310PRTArtificialSynthetic polypeptide 113Pro Phe Tyr Phe Ala His Gly
Pro Gln Ile1 5
1011410PRTArtificialSynthetic polypeptide 114Pro Phe Tyr Phe Glu Ala Gly
Pro Gln Ile1 5
1011510PRTArtificialSynthetic polypeptide 115Pro Phe Tyr Phe Glu His Ala
Pro Gln Ile1 5
1011610PRTArtificialSynthetic polypeptide 116Pro Phe Tyr Phe Glu His Gly
Ala Gln Ile1 5
1011710PRTArtificialSynthetic polypeptide 117Pro Phe Tyr Phe Glu His Gly
Pro Ala Ile1 5
1011810PRTArtificialSynthetic polypeptide 118Pro Phe Tyr Phe Glu His Gly
Pro Gln Ala1 5
1011910PRTArtificialSynthetic polypeptide 119Thr Phe Tyr Leu Glu Asn Gly
Pro Lys Ile1 5
1012010PRTArtificialSynthetic polypeptide 120Pro Phe Phe Phe Glu His Gly
Pro Gln Ile1 5
1012110PRTArtificialSynthetic polypeptide 121Ala Tyr Leu Met Gln Val Leu
Gln Glu Lys1 5
1012210PRTArtificialSynthetic polypeptide 122Lys Ala Leu Met Gln Val Leu
Gln Glu Lys1 5
1012310PRTArtificialSynthetic polypeptide 123Lys Tyr Ala Met Gln Val Leu
Gln Glu Lys1 5
1012410PRTArtificialSynthetic polypeptide 124Lys Tyr Leu Ala Gln Val Leu
Gln Glu Lys1 5
1012510PRTArtificialSynthetic polypeptide 125Lys Tyr Leu Met Ala Val Leu
Gln Glu Lys1 5
1012610PRTArtificialSynthetic polypeptide 126Lys Tyr Leu Met Gln Ala Leu
Gln Glu Lys1 5
1012710PRTArtificialSynthetic polypeptide 127Lys Tyr Leu Met Gln Val Ala
Gln Glu Lys1 5
1012810PRTArtificialSynthetic polypeptide 128Lys Tyr Leu Met Gln Val Leu
Ala Glu Lys1 5
1012910PRTArtificialSynthetic polypeptide 129Lys Tyr Leu Met Gln Val Leu
Gln Ala Lys1 5
1013010PRTArtificialSynthetic polypeptide 130Lys Tyr Leu Met Gln Val Leu
Gln Glu Ala1 5
1013110PRTArtificialSynthetic polypeptide 131Val Phe Leu His Asp Lys Ile
Lys Ser Leu1 5
1013210PRTArtificialSynthetic polypeptide 132Ala Tyr Ser Thr Ala Ala Ala
Pro Thr Tyr1 5
1013310PRTArtificialSynthetic polypeptide 133Ser Ala Ser Thr Ala Ala Ala
Pro Thr Tyr1 5
1013410PRTArtificialSynthetic polypeptide 134Ser Tyr Ala Thr Ala Ala Ala
Pro Thr Tyr1 5
1013510PRTArtificialSynthetic polypeptide 135Ser Tyr Ser Ala Ala Ala Ala
Pro Thr Tyr1 5
1013610PRTArtificialSynthetic polypeptide 136Ser Tyr Ser Thr Ala Ala Ala
Ala Thr Tyr1 5
1013710PRTArtificialSynthetic polypeptide 137Ser Tyr Ser Thr Ala Ala Ala
Pro Ala Tyr1 5
1013810PRTArtificialSynthetic polypeptide 138Ser Tyr Ser Thr Ala Ala Ala
Pro Thr Ala1 5
1013910PRTArtificialSynthetic polypeptide 139Cys Ile Ser Thr Ser Ala Ala
Pro Thr Tyr1 5
1014010PRTArtificialSynthetic polypeptide 140Ser Tyr Ser Thr Ala Ala Ala
Pro Ala Phe1 5
1014110PRTArtificialSynthetic polypeptide 141Ala Phe Ala Ala Ala Ala Ala
Pro Thr Tyr1 5
1014210PRTArtificialSynthetic polypeptide 142Ser Tyr Ser Thr Ala Ala Ala
Pro Thr Phe1 5
1014310PRTArtificialSynthetic polypeptide 143Ser Thr Ser Ala Ala Pro Thr
Tyr Phe Pro1 5
1014410PRTArtificialSynthetic polypeptide 144Ser Thr Ser Ala Ala Pro Thr
Phe Phe Pro1 5
1014510PRTArtificialSynthetic polypeptide 145Ser Thr Ser Ala Ala Pro Thr
Ala Phe Pro1 5
1014610PRTArtificialSynthetic polypeptide 146Ser Thr Ala Ala Ala Pro Thr
Phe Phe Pro1 5
1014710PRTArtificialSynthetic polypeptide 147Ala Ala Ala Ala Thr Tyr Phe
Pro Pro His1 5
1014810PRTArtificialSynthetic polypeptide 148Ala Ala Ala Pro Ala Tyr Phe
Pro Pro His1 5
1014910PRTArtificialSynthetic polypeptide 149Ala Ala Ala Pro Thr Ala Phe
Pro Pro His1 5
1015010PRTArtificialSynthetic polypeptide 150Ala Ala Ala Pro Thr Tyr Ala
Pro Pro His1 5
1015110PRTArtificialSynthetic polypeptide 151Ala Ala Ala Pro Thr Tyr Phe
Ala Pro His1 5
1015210PRTArtificialSynthetic polypeptide 152Ala Ala Ala Pro Thr Tyr Phe
Pro Ala His1 5
1015310PRTArtificialSynthetic polypeptide 153Ala Ala Ala Pro Thr Tyr Phe
Pro Pro Ala1 5
1015410PRTArtificialSynthetic polypeptide 154Ser Ala Ala Pro Thr Tyr Phe
Pro Ala His1 5
1015510PRTArtificialSynthetic polypeptide 155Ala Ala Ala Pro Ala Phe Phe
Pro Pro His1 5
1015610PRTArtificialSynthetic polypeptide 156Ala Ala Ala Pro Pro Phe Phe
Pro Pro His1 5
1015710PRTArtificialSynthetic polypeptide 157Ala Ala Ala Pro Thr Phe Phe
Pro Pro His1 5
1015810PRTArtificialSynthetic polypeptide 158Ser Ile Ser Val Ala Thr Arg
Leu Ala Gln1 5
1015910PRTArtificialSynthetic polypeptide 159Ala Met Ser Met Leu Thr Lys
Glu Val His1 5
1016010PRTArtificialSynthetic polypeptide 160Pro Ala Phe Ala Ser Ile Arg
Ser Leu Asn1 5
1016110PRTArtificialSynthetic polypeptide 161Pro Asn Phe Asn Ala Gly Ser
Pro Thr Glu1 5
1016210PRTArtificialSynthetic polypeptide 162Lys Met Leu Leu Leu Ser Leu
Gly Thr Gly1 5
1016310PRTArtificialSynthetic polypeptide 163Asn Tyr Leu Ile Ile Ser Val
Gly Thr Gly1 5
1016410PRTArtificialSynthetic polypeptide 164Lys Met Leu Leu Leu Ser Leu
Gly Ala Gly1 5
1016510PRTArtificialSynthetic polypeptide 165Ala Glu Phe Asp Lys Thr Tyr
Thr Ala Lys1 5
1016610PRTArtificialSynthetic polypeptide 166Ser Ala Phe Asp Lys Thr Tyr
Thr Ala Lys1 5
1016710PRTArtificialSynthetic polypeptide 167Ser Glu Ala Asp Lys Thr Tyr
Thr Ala Lys1 5
1016810PRTArtificialSynthetic polypeptide 168Ser Glu Phe Ala Lys Thr Tyr
Thr Ala Lys1 5
1016910PRTArtificialSynthetic polypeptide 169Ser Glu Phe Asp Ala Thr Tyr
Thr Ala Lys1 5
1017010PRTArtificialSynthetic polypeptide 170Ser Glu Phe Asp Lys Ala Tyr
Thr Ala Lys1 5
1017110PRTArtificialSynthetic polypeptide 171Ser Glu Phe Asp Lys Thr Ala
Thr Ala Lys1 5
1017210PRTArtificialSynthetic polypeptide 172Ser Glu Phe Asp Lys Thr Tyr
Ala Ala Lys1 5
1017310PRTArtificialSynthetic polypeptide 173Ser Glu Phe Asp Lys Thr Tyr
Thr Ala Ala1 5
1017410PRTArtificialSynthetic polypeptide 174Lys Gln Ala Glu Lys Tyr Thr
Ala Glu Gln1 5
1017510PRTArtificialSynthetic polypeptide 175Ser Glu Phe Asp Ala Ala Phe
Ala Ala Ala1 5
1017610PRTArtificialSynthetic polypeptide 176Ser Glu Phe Asp Lys Thr Phe
Thr Ala Lys1 5
1017710PRTArtificialSynthetic polypeptide 177Ala Glu Lys Tyr Thr Ala Glu
Gln Cys Ala1 5
1017810PRTArtificialSynthetic polypeptide 178Ala Thr Tyr Thr Ala Lys Glu
Ala Ala Thr1 5
1017910PRTArtificialSynthetic polypeptide 179Lys Ala Tyr Thr Ala Lys Glu
Ala Ala Thr1 5
1018010PRTArtificialSynthetic polypeptide 180Lys Thr Ala Thr Ala Lys Glu
Ala Ala Thr1 5
1018110PRTArtificialSynthetic polypeptide 181Lys Thr Tyr Ala Ala Lys Glu
Ala Ala Thr1 5
1018210PRTArtificialSynthetic polypeptide 182Lys Thr Tyr Thr Ala Ala Glu
Ala Ala Thr1 5
1018310PRTArtificialSynthetic polypeptide 183Lys Thr Tyr Thr Ala Lys Ala
Ala Ala Thr1 5
1018410PRTArtificialSynthetic polypeptide 184Lys Thr Tyr Thr Ala Lys Glu
Ala Ala Ala1 5
1018510PRTArtificialSynthetic polypeptide 185Glu Lys Tyr Thr Ala Glu Gln
Cys Ala Lys1 5
1018610PRTArtificialSynthetic polypeptide 186Ala Ala Phe Ala Ala Ala Glu
Ala Ala Thr1 5
1018710PRTArtificialSynthetic polypeptide 187Lys Thr Phe Thr Ala Lys Glu
Ala Ala Thr1 5
1018810PRTArtificialSynthetic polypeptide 188Gln Ala Leu His Cys Glu Lys
Lys Tyr Leu1 5
1018910PRTArtificialSynthetic polypeptide 189Gln Ala Leu Asp Ser Lys Ala
Ala Tyr Leu1 5
1019010PRTArtificialSynthetic polypeptide 190Gln Ala Leu Asp Ser Lys Asn
Asn Phe Leu1 5
1019110PRTArtificialSynthetic polypeptide 191Gln Ala Leu His Cys Glu Asn
Asn Phe Leu1 5
1019210PRTArtificialSynthetic polypeptide 192Cys Glu Lys Lys Tyr Leu Arg
Ile Gln Asp1 5
1019310PRTArtificialSynthetic polypeptide 193Ser Lys Asn Asn Phe Leu Arg
Val Gln Glu1 5
1019410PRTArtificialSynthetic polypeptide 194Ser Glu Asn Asn Tyr Leu Arg
Val Gln Glu1 5
1019510PRTArtificialSynthetic polypeptide 195Ala Leu Arg Val Gln Glu Asn
Ala Leu Thr1 5
1019610PRTArtificialSynthetic polypeptide 196Tyr Ala Arg Val Gln Glu Asn
Ala Leu Thr1 5
1019710PRTArtificialSynthetic polypeptide 197Tyr Leu Ala Val Gln Glu Asn
Ala Leu Thr1 5
1019810PRTArtificialSynthetic polypeptide 198Tyr Leu Arg Ala Gln Glu Asn
Ala Leu Thr1 5
1019910PRTArtificialSynthetic polypeptide 199Tyr Leu Arg Val Ala Glu Asn
Ala Leu Thr1 5
1020010PRTArtificialSynthetic polypeptide 200Tyr Leu Arg Val Gln Ala Asn
Ala Leu Thr1 5
1020110PRTArtificialSynthetic polypeptide 201Tyr Leu Arg Val Gln Glu Ala
Ala Leu Thr1 5
1020210PRTArtificialSynthetic polypeptide 202Tyr Leu Arg Val Gln Glu Asn
Ala Ala Thr1 5
1020310PRTArtificialSynthetic polypeptide 203Tyr Leu Arg Val Gln Glu Asn
Ala Leu Ala1 5
1020410PRTArtificialSynthetic polypeptide 204Tyr Leu Arg Ile Gln Asp Asp
Thr Leu Thr1 5
1020510PRTArtificialSynthetic polypeptide 205Tyr Leu Thr Val Ala Ala Ala
Ala Leu Thr1 5
1020610PRTArtificialSynthetic polypeptide 206Phe Leu Arg Val Gln Glu Asn
Ala Leu Thr1 5
1020710PRTArtificialSynthetic polypeptide 207Asn Asn Tyr Leu Arg Val Gln
Glu Asn Ala1 5
1020810PRTArtificialSynthetic polypeptide 208Lys Lys Tyr Leu Arg Ile Gln
Asp Asp Thr1 5
1020910PRTArtificialSynthetic polypeptide 209Asn Asn Phe Leu Arg Val Gln
Glu Asn Ala1 5
1021010PRTArtificialSynthetic polypeptide 210Asn Ala Tyr Leu Arg Val Gln
Glu Asn Ala1 5
1021110PRTArtificialSynthetic polypeptide 211Ala Thr Tyr Glu Glu Ala Lys
Leu Arg Phe1 5
1021210PRTArtificialSynthetic polypeptide 212Glu Ala Tyr Glu Glu Ala Leu
Lys Arg Phe1 5
1021310PRTArtificialSynthetic polypeptide 213Glu Thr Ala Glu Glu Ala Leu
Lys Arg Phe1 5
1021410PRTArtificialSynthetic polypeptide 214Glu Thr Tyr Ala Glu Ala Leu
Lys Arg Phe1 5
1021510PRTArtificialSynthetic polypeptide 215Glu Thr Tyr Glu Ala Ala Leu
Lys Arg Phe1 5
1021610PRTArtificialSynthetic polypeptide 216Glu Thr Tyr Glu Glu Ala Ala
Lys Arg Phe1 5
1021710PRTArtificialSynthetic polypeptide 217Glu Thr Tyr Glu Glu Ala Leu
Ala Arg Phe1 5
1021810PRTArtificialSynthetic polypeptide 218Glu Thr Tyr Glu Glu Ala Leu
Lys Ala Phe1 5
1021910PRTArtificialSynthetic polypeptide 219Glu Thr Tyr Glu Glu Ala Leu
Lys Arg Ala1 5
1022010PRTArtificialSynthetic polypeptide 220Gly Thr Asn Ala Gln Ser Leu
Ala Asp Phe1 5
1022110PRTArtificialSynthetic polypeptide 221Glu Thr Tyr Glu Ala Ala Leu
Ala Ala Phe1 5
1022210PRTArtificialSynthetic polypeptide 222Glu Thr Phe Glu Glu Ala Leu
Lys Arg Phe1 5
1022310PRTArtificialSynthetic polypeptide 223Tyr Glu Glu Ala Leu Lys Thr
Phe Ala Lys1 5
1022410PRTArtificialSynthetic polypeptide 224Phe Glu Glu Ala Leu Lys Arg
Phe Ala Lys1 5
1022510PRTArtificialSynthetic polypeptide 225Ala Ala Leu Lys Arg Phe Ala
Lys Leu Leu1 5
1022610PRTArtificialSynthetic polypeptide 226Glu Ala Ala Lys Arg Phe Ala
Lys Leu Leu1 5
1022710PRTArtificialSynthetic polypeptide 227Glu Ala Leu Ala Arg Phe Ala
Lys Leu Leu1 5
1022810PRTArtificialSynthetic polypeptide 228Glu Ala Leu Lys Ala Phe Ala
Lys Leu Leu1 5
1022910PRTArtificialSynthetic polypeptide 229Glu Ala Leu Lys Arg Ala Ala
Lys Leu Leu1 5
1023010PRTArtificialSynthetic polypeptide 230Glu Ala Leu Lys Arg Phe Ala
Ala Leu Leu1 5
1023110PRTArtificialSynthetic polypeptide 231Glu Ala Leu Lys Arg Phe Ala
Lys Ala Leu1 5
1023210PRTArtificialSynthetic polypeptide 232Glu Ala Leu Lys Arg Phe Ala
Lys Leu Ala1 5
1023310PRTArtificialSynthetic polypeptide 233Gln Ser Leu Ala Asp Phe Ala
Lys Gln Leu1 5
1023410PRTArtificialSynthetic polypeptide 234Ala Ala Leu Ala Ala Phe Ala
Lys Leu Leu1 5
1023510PRTArtificialSynthetic polypeptide 235Leu Ala Asp Phe Ala Lys Gln
Leu Ser Asp1 5
1023610PRTArtificialSynthetic polypeptide 236Asp Phe Ala Lys Gln Leu Ser
Asp Glu Arg1 5
1023710PRTArtificialSynthetic polypeptide 237Ala Phe Ala Ala Leu Leu Ser
Asp Arg Lys1 5
1023810PRTArtificialSynthetic polypeptide 238Ser Thr Ala Ala Ala Pro Thr
Tyr Phe Pro1 5
1023910PRTArtificialSynthetic polypeptide 239Leu Lys Arg Phe Ala Lys Leu
Leu Ser Asp1 5
1024010PRTArtificialSynthetic polypeptide 240Gln Ala Leu Asp Ser Glu Asn
Asn Phe Leu1 5
1024110PRTArtificialSynthetic polypeptide 241Ser Asp Leu Ala Asp Phe Ala
Lys Gln Leu1 5
1024255DNAArtificialSynthetic construct 242ggagctcgag aaaagagagg
ctgaagcttc attgaattac aaaaaaatgc tgttg
5524342DNAArtificialSynthetic construct 243tcccaactgt cctggtccat
aagaagcttt gtttgctcgg ag
4224436DNAArtificialSynthetic construct 244gcttcttatg gaccaggaca
gttgggagaa atggtg
3624539DNAArtificialSynthetic construct 245ggtctagagg aattctcatt
acctaattga agcaaatgc
392461128DNAArtificialSynthetic construct 246tcgagaaaag agaggctgaa
gcttcattga attacaaaaa aatgctgttg ctctcattag 60gcactggcac tacttcagag
tttgataaaa catatacagc aaaagaggca gctacctgga 120ctgctgtaca ttggatgtta
gttatacaga aaatgactga tgcagcaagt tcttacatga 180ctgattatta cctttctact
gcttttcaag ctcttgattc aaaaaacaat tacctcaggg 240ttcaagaaaa tgcattaaca
ggcacaacta ctgaaatgga tgatgcttct gaggctaata 300tggaattatt agtacaagtt
ggtgaaaact tattgaagaa accagtttcc gaagacaatc 360ctgaaaccta tgaggaagct
ctaaagaggt ttgcaaaatt gctctctgat aggaagaaac 420tccgagcaaa caaagcttct
tatggaccag gacagttggg agaaatggtg actgttctta 480gtattgatgg aggtggaatt
agagggatca ttccggctac cattctcgaa tttcttgaag 540gacaacttca ggaaatggac
aataatgcag atgcaagact tgcagattac tttgatgtaa 600ttggaggaac aagtacagga
ggtttattga ctgctatgat aagtactcca aatgaaaaca 660atcgaccctt tgctgctgcc
aaagaaattg taccttttta cttcgaacat ggccctcaga 720tttttaatcc tagtggtcaa
attttaggcc caaaatatga tggaaaatat cttatgcaag 780ttcttcaaga aaaacttgga
gaaactcgtg tgcatcaagc tttgacagaa gttgtcatct 840caagctttga catcaaaaca
aataagccag taatattcac taagtcaaat ttagcaaact 900ctccagaatt ggatgctaag
atgtatgaca taagttattc cacagcagca gctccaacat 960attttcctcc gcattacttt
gttactaata ctagtaatgg agatgaatat gagttcaatc 1020ttgttgatgg tgctgttgct
actgttgctg atccggcgtt attatccatt agcgttgcaa 1080cgagacttgc acaaaaggat
ccagcatttg cttcaattag gtaatgag
1128247366PRTArtificialSynthetic polypeptide 247Ser Leu Asn Tyr Lys Lys
Met Leu Leu Leu Ser Leu Gly Thr Gly Thr1 5
10 15Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala Lys Glu
Ala Ala Thr Trp 20 25 30Thr
Ala Val His Trp Met Leu Val Ile Gln Lys Met Thr Asp Ala Ala 35
40 45Ser Ser Tyr Met Thr Asp Tyr Tyr Leu
Ser Thr Ala Phe Gln Ala Leu 50 55
60Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr Gly65
70 75 80Thr Thr Thr Glu Met
Asp Asp Ala Ser Glu Ala Asn Met Glu Leu Leu 85
90 95Val Gln Val Gly Glu Asn Leu Leu Lys Lys Pro
Val Ser Glu Asp Asn 100 105
110Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser
115 120 125Asp Arg Lys Lys Leu Arg Ala
Asn Lys Ala Ser Tyr Gly Pro Gly Gln 130 135
140Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly Ile
Arg145 150 155 160Gly Ile
Ile Pro Ala Thr Ile Leu Glu Phe Leu Glu Gly Gln Leu Gln
165 170 175Glu Met Asp Asn Asn Ala Asp
Ala Arg Leu Ala Asp Tyr Phe Asp Val 180 185
190Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala Met Ile
Ser Thr 195 200 205Pro Asn Glu Asn
Asn Arg Pro Phe Ala Ala Ala Lys Glu Ile Val Pro 210
215 220Phe Tyr Phe Glu His Gly Pro Gln Ile Phe Asn Pro
Ser Gly Gln Ile225 230 235
240Leu Gly Pro Lys Tyr Asp Gly Lys Tyr Leu Met Gln Val Leu Gln Glu
245 250 255Lys Leu Gly Glu Thr
Arg Val His Gln Ala Leu Thr Glu Val Val Ile 260
265 270Ser Ser Phe Asp Ile Lys Thr Asn Lys Pro Val Ile
Phe Thr Lys Ser 275 280 285Asn Leu
Ala Asn Ser Pro Glu Leu Asp Ala Lys Met Tyr Asp Ile Ser 290
295 300Tyr Ser Thr Ala Ala Ala Pro Thr Tyr Phe Pro
Pro His Tyr Phe Val305 310 315
320Thr Asn Thr Ser Asn Gly Asp Glu Tyr Glu Phe Asn Leu Val Asp Gly
325 330 335Ala Val Ala Thr
Val Ala Asp Pro Ala Leu Leu Ser Ile Ser Val Ala 340
345 350Thr Arg Leu Ala Gln Lys Asp Pro Ala Phe Ala
Ser Ile Arg 355 360
36524855DNAArtificialSynthetic construct 248ggagctcgag aaaagagagg
ctgaagctaa tactagtaat ggagatgaat atgag
5524939DNAArtificialSynthetic construct 249ggtctagagg aattctcatt
aagtaacaaa gtaatgcgg
392501128DNAArtificialSynthetic construct 250tcgagaaaag agaggctgaa
gctaatacta gtaatggaga tgaatatgag ttcaatcttg 60ttgatggtgc tgttgctact
gttgctgatc cggcgttatt atccattagc gttgcaacga 120gacttgcaca aaaggatcca
gcatttgctt caattaggtc attgaattac aaaaaaatgc 180tgttgctctc attaggcact
ggcactactt cagagtttga taaaacatat acagcaaaag 240aggcagctac ctggactgct
gtacattgga tgttagttat acagaaaatg actgatgcag 300caagttctta catgactgat
tattaccttt ctactgcttt tcaagctctt gattcaaaaa 360acaattacct cagggttcaa
gaaaatgcat taacaggcac aactactgaa atggatgatg 420cttctgaggc taatatggaa
ttattagtac aagttggtga aaacttattg aagaaaccag 480tttccgaaga caatcctgaa
acctatgagg aagctctaaa gaggtttgca aaattgctct 540ctgataggaa gaaactccga
gcaaacaaag cttcttatgg accaggacag ttgggagaaa 600tggtgactgt tcttagtatt
gatggaggtg gaattagagg gatcattccg gctaccattc 660tcgaatttct tgaaggacaa
cttcaggaaa tggacaataa tgcagatgca agacttgcag 720attactttga tgtaattgga
ggaacaagta caggaggttt attgactgct atgataagta 780ctccaaatga aaacaatcga
ccctttgctg ctgccaaaga aattgtacct ttttacttcg 840aacatggccc tcagattttt
aatcctagtg gtcaaatttt aggcccaaaa tatgatggaa 900aatatcttat gcaagttctt
caagaaaaac ttggagaaac tcgtgtgcat caagctttga 960cagaagttgt catctcaagc
tttgacatca aaacaaataa gccagtaata ttcactaagt 1020caaatttagc aaactctcca
gaattggatg ctaagatgta tgacataagt tattccacag 1080cagcagctcc aacatatttt
cctccgcatt actttgttac ttaatgag
1128251366PRTArtificialSynthetic polypeptide 251Asn Thr Ser Asn Gly Asp
Glu Tyr Glu Phe Asn Leu Val Asp Gly Ala1 5
10 15Val Ala Thr Val Ala Asp Pro Ala Leu Leu Ser Ile
Ser Val Ala Thr 20 25 30Arg
Leu Ala Gln Lys Asp Pro Ala Phe Ala Ser Ile Arg Ser Leu Asn 35
40 45Tyr Lys Lys Met Leu Leu Leu Ser Leu
Gly Thr Gly Thr Thr Ser Glu 50 55
60Phe Asp Lys Thr Tyr Thr Ala Lys Glu Ala Ala Thr Trp Thr Ala Val65
70 75 80His Trp Met Leu Val
Ile Gln Lys Met Thr Asp Ala Ala Ser Ser Tyr 85
90 95Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe Gln
Ala Leu Asp Ser Lys 100 105
110Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr Gly Thr Thr Thr
115 120 125Glu Met Asp Asp Ala Ser Glu
Ala Asn Met Glu Leu Leu Val Gln Val 130 135
140Gly Glu Asn Leu Leu Lys Lys Pro Val Ser Glu Asp Asn Pro Glu
Thr145 150 155 160Tyr Glu
Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser Asp Arg Lys
165 170 175Lys Leu Arg Ala Asn Lys Ala
Ser Tyr Gly Pro Gly Gln Leu Gly Glu 180 185
190Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly Ile Arg Gly
Ile Ile 195 200 205Pro Ala Thr Ile
Leu Glu Phe Leu Glu Gly Gln Leu Gln Glu Met Asp 210
215 220Asn Asn Ala Asp Ala Arg Leu Ala Asp Tyr Phe Asp
Val Ile Gly Gly225 230 235
240Thr Ser Thr Gly Gly Leu Leu Thr Ala Met Ile Ser Thr Pro Asn Glu
245 250 255Asn Asn Arg Pro Phe
Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe 260
265 270Glu His Gly Pro Gln Ile Phe Asn Pro Ser Gly Gln
Ile Leu Gly Pro 275 280 285Lys Tyr
Asp Gly Lys Tyr Leu Met Gln Val Leu Gln Glu Lys Leu Gly 290
295 300Glu Thr Arg Val His Gln Ala Leu Thr Glu Val
Val Ile Ser Ser Phe305 310 315
320Asp Ile Lys Thr Asn Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala
325 330 335Asn Ser Pro Glu
Leu Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser Thr 340
345 350Ala Ala Ala Pro Thr Tyr Phe Pro Pro His Tyr
Phe Val Thr 355 360
36525255DNAArtificialSynthetic construct 252ggagctcgag aaaagagagg
ctgaagctag ttattccaca gcagcagctc caaca
5525339DNAArtificialSynthetic construct 253ggtctagagg aattctcatt
atatgtcata catcttagc
392541128DNAArtificialSynthetic construct 254tcgagaaaag agaggctgaa
gctagttatt ccacagcagc agctccaaca tattttcctc 60cgcattactt tgttactaat
actagtaatg gagatgaata tgagttcaat cttgttgatg 120gtgctgttgc tactgttgct
gatccggcgt tattatccat tagcgttgca acgagacttg 180cacaaaagga tccagcattt
gcttcaatta ggtcattgaa ttacaaaaaa atgctgttgc 240tctcattagg cactggcact
acttcagagt ttgataaaac atatacagca aaagaggcag 300ctacctggac tgctgtacat
tggatgttag ttatacagaa aatgactgat gcagcaagtt 360cttacatgac tgattattac
ctttctactg cttttcaagc tcttgattca aaaaacaatt 420acctcagggt tcaagaaaat
gcattaacag gcacaactac tgaaatggat gatgcttctg 480aggctaatat ggaattatta
gtacaagttg gtgaaaactt attgaagaaa ccagtttccg 540aagacaatcc tgaaacctat
gaggaagctc taaagaggtt tgcaaaattg ctctctgata 600ggaagaaact ccgagcaaac
aaagcttctt atggaccagg acagttggga gaaatggtga 660ctgttcttag tattgatgga
ggtggaatta gagggatcat tccggctacc attctcgaat 720ttcttgaagg acaacttcag
gaaatggaca ataatgcaga tgcaagactt gcagattact 780ttgatgtaat tggaggaaca
agtacaggag gtttattgac tgctatgata agtactccaa 840atgaaaacaa tcgacccttt
gctgctgcca aagaaattgt acctttttac ttcgaacatg 900gccctcagat ttttaatcct
agtggtcaaa ttttaggccc aaaatatgat ggaaaatatc 960ttatgcaagt tcttcaagaa
aaacttggag aaactcgtgt gcatcaagct ttgacagaag 1020ttgtcatctc aagctttgac
atcaaaacaa ataagccagt aatattcact aagtcaaatt 1080tagcaaactc tccagaattg
gatgctaaga tgtatgacat ataatgag
1128255366PRTArtificialSynthetic polypeptide 255Ser Tyr Ser Thr Ala Ala
Ala Pro Thr Tyr Phe Pro Pro His Tyr Phe1 5
10 15Val Thr Asn Thr Ser Asn Gly Asp Glu Tyr Glu Phe
Asn Leu Val Asp 20 25 30Gly
Ala Val Ala Thr Val Ala Asp Pro Ala Leu Leu Ser Ile Ser Val 35
40 45Ala Thr Arg Leu Ala Gln Lys Asp Pro
Ala Phe Ala Ser Ile Arg Ser 50 55
60Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly Thr Thr65
70 75 80Ser Glu Phe Asp Lys
Thr Tyr Thr Ala Lys Glu Ala Ala Thr Trp Thr 85
90 95Ala Val His Trp Met Leu Val Ile Gln Lys Met
Thr Asp Ala Ala Ser 100 105
110Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe Gln Ala Leu Asp
115 120 125Ser Lys Asn Asn Tyr Leu Arg
Val Gln Glu Asn Ala Leu Thr Gly Thr 130 135
140Thr Thr Glu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu Leu
Val145 150 155 160Gln Val
Gly Glu Asn Leu Leu Lys Lys Pro Val Ser Glu Asp Asn Pro
165 170 175Glu Thr Tyr Glu Glu Ala Leu
Lys Arg Phe Ala Lys Leu Leu Ser Asp 180 185
190Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr Gly Pro Gly
Gln Leu 195 200 205Gly Glu Met Val
Thr Val Leu Ser Ile Asp Gly Gly Gly Ile Arg Gly 210
215 220Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu Glu Gly
Gln Leu Gln Glu225 230 235
240Met Asp Asn Asn Ala Asp Ala Arg Leu Ala Asp Tyr Phe Asp Val Ile
245 250 255Gly Gly Thr Ser Thr
Gly Gly Leu Leu Thr Ala Met Ile Ser Thr Pro 260
265 270Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala Lys Glu
Ile Val Pro Phe 275 280 285Tyr Phe
Glu His Gly Pro Gln Ile Phe Asn Pro Ser Gly Gln Ile Leu 290
295 300Gly Pro Lys Tyr Asp Gly Lys Tyr Leu Met Gln
Val Leu Gln Glu Lys305 310 315
320Leu Gly Glu Thr Arg Val His Gln Ala Leu Thr Glu Val Val Ile Ser
325 330 335Ser Phe Asp Ile
Lys Thr Asn Lys Pro Val Ile Phe Thr Lys Ser Asn 340
345 350Leu Ala Asn Ser Pro Glu Leu Asp Ala Lys Met
Tyr Asp Ile 355 360
36525655DNAArtificialSynthetic construct 256ggagctcgag aaaagagagg
ctgaagctac atatacagca aaagaggcag ctacc
5525739DNAArtificialSynthetic construct 257ggtctagagg aattctcatt
atttatcaaa ctctgaagt
392581128DNAArtificialSynthetic construct 258tcgagaaaag agaggctgaa
gctacatata cagcaaaaga ggcagctacc tggactgctg 60tacattggat gttagttata
cagaaaatga ctgatgcagc aagttcttac atgactgatt 120attacctttc tactgctttt
caagctcttg attcaaaaaa caattacctc agggttcaag 180aaaatgcatt aacaggcaca
actactgaaa tggatgatgc ttctgaggct aatatggaat 240tattagtaca agttggtgaa
aacttattga agaaaccagt ttccgaagac aatcctgaaa 300cctatgagga agctctaaag
aggtttgcaa aattgctctc tgataggaag aaactccgag 360caaacaaagc ttcttatgga
ccaggacagt tgggagaaat ggtgactgtt cttagtattg 420atggaggtgg aattagaggg
atcattccgg ctaccattct cgaatttctt gaaggacaac 480ttcaggaaat ggacaataat
gcagatgcaa gacttgcaga ttactttgat gtaattggag 540gaacaagtac aggaggttta
ttgactgcta tgataagtac tccaaatgaa aacaatcgac 600cctttgctgc tgccaaagaa
attgtacctt tttacttcga acatggccct cagattttta 660atcctagtgg tcaaatttta
ggcccaaaat atgatggaaa atatcttatg caagttcttc 720aagaaaaact tggagaaact
cgtgtgcatc aagctttgac agaagttgtc atctcaagct 780ttgacatcaa aacaaataag
ccagtaatat tcactaagtc aaatttagca aactctccag 840aattggatgc taagatgtat
gacataagtt attccacagc agcagctcca acatattttc 900ctccgcatta ctttgttact
aatactagta atggagatga atatgagttc aatcttgttg 960atggtgctgt tgctactgtt
gctgatccgg cgttattatc cattagcgtt gcaacgagac 1020ttgcacaaaa ggatccagca
tttgcttcaa ttaggtcatt gaattacaaa aaaatgctgt 1080tgctctcatt aggcactggc
actacttcag agtttgataa ataatgag
1128259366PRTArtificialSynthetic polypeptide 259Thr Tyr Thr Ala Lys Glu
Ala Ala Thr Trp Thr Ala Val His Trp Met1 5
10 15Leu Val Ile Gln Lys Met Thr Asp Ala Ala Ser Ser
Tyr Met Thr Asp 20 25 30Tyr
Tyr Leu Ser Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn Tyr 35
40 45Leu Arg Val Gln Glu Asn Ala Leu Thr
Gly Thr Thr Thr Glu Met Asp 50 55
60Asp Ala Ser Glu Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn65
70 75 80Leu Leu Lys Lys Pro
Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu 85
90 95Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser Asp
Arg Lys Lys Leu Arg 100 105
110Ala Asn Lys Ala Ser Tyr Gly Pro Gly Gln Leu Gly Glu Met Val Thr
115 120 125Val Leu Ser Ile Asp Gly Gly
Gly Ile Arg Gly Ile Ile Pro Ala Thr 130 135
140Ile Leu Glu Phe Leu Glu Gly Gln Leu Gln Glu Met Asp Asn Asn
Ala145 150 155 160Asp Ala
Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr
165 170 175Gly Gly Leu Leu Thr Ala Met
Ile Ser Thr Pro Asn Glu Asn Asn Arg 180 185
190Pro Phe Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu
His Gly 195 200 205Pro Gln Ile Phe
Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp 210
215 220Gly Lys Tyr Leu Met Gln Val Leu Gln Glu Lys Leu
Gly Glu Thr Arg225 230 235
240Val His Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys
245 250 255Thr Asn Lys Pro Val
Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro 260
265 270Glu Leu Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser
Thr Ala Ala Ala 275 280 285Pro Thr
Tyr Phe Pro Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly 290
295 300Asp Glu Tyr Glu Phe Asn Leu Val Asp Gly Ala
Val Ala Thr Val Ala305 310 315
320Asp Pro Ala Leu Leu Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys
325 330 335Asp Pro Ala Phe
Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu 340
345 350Leu Leu Ser Leu Gly Thr Gly Thr Thr Ser Glu
Phe Asp Lys 355 360
36526055DNAArtificialSynthetic construct 260ggagctcgag aaaagagagg
ctgaagctaa tgcattaaca ggcacaacta ctgaa
5526139DNAArtificialSynthetic construct 261ggtctagagg aattctcatt
attcttgaac cctgaggta
392621128DNAArtificialSynthetic construct 262tcgagaaaag agaggctgaa
gctaatgcat taacaggcac aactactgaa atggatgatg 60cttctgaggc taatatggaa
ttattagtac aagttggtga aaacttattg aagaaaccag 120tttccgaaga caatcctgaa
acctatgagg aagctctaaa gaggtttgca aaattgctct 180ctgataggaa gaaactccga
gcaaacaaag cttcttatgg accaggacag ttgggagaaa 240tggtgactgt tcttagtatt
gatggaggtg gaattagagg gatcattccg gctaccattc 300tcgaatttct tgaaggacaa
cttcaggaaa tggacaataa tgcagatgca agacttgcag 360attactttga tgtaattgga
ggaacaagta caggaggttt attgactgct atgataagta 420ctccaaatga aaacaatcga
ccctttgctg ctgccaaaga aattgtacct ttttacttcg 480aacatggccc tcagattttt
aatcctagtg gtcaaatttt aggcccaaaa tatgatggaa 540aatatcttat gcaagttctt
caagaaaaac ttggagaaac tcgtgtgcat caagctttga 600cagaagttgt catctcaagc
tttgacatca aaacaaataa gccagtaata ttcactaagt 660caaatttagc aaactctcca
gaattggatg ctaagatgta tgacataagt tattccacag 720cagcagctcc aacatatttt
cctccgcatt actttgttac taatactagt aatggagatg 780aatatgagtt caatcttgtt
gatggtgctg ttgctactgt tgctgatccg gcgttattat 840ccattagcgt tgcaacgaga
cttgcacaaa aggatccagc atttgcttca attaggtcat 900tgaattacaa aaaaatgctg
ttgctctcat taggcactgg cactacttca gagtttgata 960aaacatatac agcaaaagag
gcagctacct ggactgctgt acattggatg ttagttatac 1020agaaaatgac tgatgcagca
agttcttaca tgactgatta ttacctttct actgcttttc 1080aagctcttga ttcaaaaaac
aattacctca gggttcaaga ataatgag
1128263366PRTArtificialSynthetic polypeptide 263Asn Ala Leu Thr Gly Thr
Thr Thr Glu Met Asp Asp Ala Ser Glu Ala1 5
10 15Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn Leu
Leu Lys Lys Pro 20 25 30Val
Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe 35
40 45Ala Lys Leu Leu Ser Asp Arg Lys Lys
Leu Arg Ala Asn Lys Ala Ser 50 55
60Tyr Gly Pro Gly Gln Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp65
70 75 80Gly Gly Gly Ile Arg
Gly Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu 85
90 95Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala
Asp Ala Arg Leu Ala 100 105
110Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr
115 120 125Ala Met Ile Ser Thr Pro Asn
Glu Asn Asn Arg Pro Phe Ala Ala Ala 130 135
140Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly Pro Gln Ile Phe
Asn145 150 155 160Pro Ser
Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys Tyr Leu Met
165 170 175Gln Val Leu Gln Glu Lys Leu
Gly Glu Thr Arg Val His Gln Ala Leu 180 185
190Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys Thr Asn Lys
Pro Val 195 200 205Ile Phe Thr Lys
Ser Asn Leu Ala Asn Ser Pro Glu Leu Asp Ala Lys 210
215 220Met Tyr Asp Ile Ser Tyr Ser Thr Ala Ala Ala Pro
Thr Tyr Phe Pro225 230 235
240Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly Asp Glu Tyr Glu Phe
245 250 255Asn Leu Val Asp Gly
Ala Val Ala Thr Val Ala Asp Pro Ala Leu Leu 260
265 270Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys Asp
Pro Ala Phe Ala 275 280 285Ser Ile
Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly 290
295 300Thr Gly Thr Thr Ser Glu Phe Asp Lys Thr Tyr
Thr Ala Lys Glu Ala305 310 315
320Ala Thr Trp Thr Ala Val His Trp Met Leu Val Ile Gln Lys Met Thr
325 330 335Asp Ala Ala Ser
Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe 340
345 350Gln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg
Val Gln Glu 355 360
3652641158DNAArtificialSynthetic construct 264atggccacca ccaagagctt
cctcatcctg atcttcatga tcctggccac caccagcagc 60accttcgccc agctcggcga
gatggtgacc gtgctctcca tcgacggcgg tggcatcagg 120ggcatcatcc cggccaccat
cctggagttc ctggagggcc aactccagga gatggacaac 180aacgccgacg cccgcctggc
cgactacttc gacgtgatcg gtggcaccag caccggcggt 240ctcctgaccg ccatgatctc
cactccgaac gagaacaacc gccccttcgc cgctgcgaag 300gagatcgtcc cgttctactt
cgaacacggc cctcagattt tcaacccctc gggtcaaatc 360ctgggcccca agtacgacgg
caagtacctt atgcaagtgc ttcaggagaa gctgggcgag 420actagggtgc accaggcgct
gaccgaggtc gtcatctcca gcttcgacat caagaccaac 480aagccagtca tcttcaccaa
gtccaacctg gccaacagcc cggagctgga cgctaagatg 540tacgacatct cctactccac
tgctgccgct cccacgtact tccctccgca ctacttcgtc 600accaacacca gcaacggcga
cgagtacgag ttcaaccttg ttgacggtgc ggtggctacg 660gtggcggacc cggcgctcct
gtccatcagc gtcgccacgc gcctggccca gaaggatcca 720gccttcgcta gcattaggag
cctcaactac aagaagatgc tgctgctcag cctgggcact 780ggcacgacct ccgagttcga
caagacctac actgccaagg aggccgctac ctggaccgcc 840gtccattgga tgctggtcat
ccagaagatg acggacgccg cttccagcta catgaccgac 900tactacctct ccactgcgtt
ccaggcgctt gactccaaga acaactacct ccgtgttcag 960gagaatgccc tcactggcac
cacgaccgag atggacgatg cctccgaggc caacatggag 1020ctgctcgtcc aggtgggtga
gaacctcctg aagaagcccg tctccgaaga caatcccgag 1080acctatgagg aagcgctcaa
gcgctttgcc aagctgctct ctgataggaa gaaactccgc 1140gctaacaagg ccagctac
1158265386PRTArtificialSynthetic polypeptide 265Met Ala Thr Thr Lys Ser
Phe Leu Ile Leu Ile Phe Met Ile Leu Ala1 5
10 15Thr Thr Ser Ser Thr Phe Ala Gln Leu Gly Glu Met
Val Thr Val Leu 20 25 30Ser
Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu 35
40 45Glu Phe Leu Glu Gly Gln Leu Gln Glu
Met Asp Asn Asn Ala Asp Ala 50 55
60Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly65
70 75 80Leu Leu Thr Ala Met
Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe 85
90 95Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe
Glu His Gly Pro Gln 100 105
110Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys
115 120 125Tyr Leu Met Gln Val Leu Gln
Glu Lys Leu Gly Glu Thr Arg Val His 130 135
140Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys Thr
Asn145 150 155 160Lys Pro
Val Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro Glu Leu
165 170 175Asp Ala Lys Met Tyr Asp Ile
Ser Tyr Ser Thr Ala Ala Ala Pro Thr 180 185
190Tyr Phe Pro Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly
Asp Glu 195 200 205Tyr Glu Phe Asn
Leu Val Asp Gly Ala Val Ala Thr Val Ala Asp Pro 210
215 220Ala Leu Leu Ser Ile Ser Val Ala Thr Arg Leu Ala
Gln Lys Asp Pro225 230 235
240Ala Phe Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu
245 250 255Ser Leu Gly Thr Gly
Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260
265 270Lys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met
Leu Val Ile Gln 275 280 285Lys Met
Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser 290
295 300Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn
Tyr Leu Arg Val Gln305 310 315
320Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu
325 330 335Ala Asn Met Glu
Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys 340
345 350Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu
Glu Ala Leu Lys Arg 355 360 365Phe
Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370
375 380Ser Tyr38526655DNAArtificialSynthetic
construct 266ggagctcgag aaaagagagg ctgaagctag cctcaactac aagaagatgc tgctg
5526742DNAArtificialSynthetic construct 267gccgagctgt cctggtccgt
agctggcctt gttagcgcgg ag
4226836DNAArtificialSynthetic construct 268gccagctacg gaccaggaca
gctcggcgag atggtg
3626939DNAArtificialSynthetic construct 269ggtctagagg aattctcatt
acctaatgct agcgaaggc
392701167DNAArtificialSynthetic construct 270atggccacca ccaagagctt
cctcatcctg atcttcatga tcctggccac caccagcagc 60accttcgcca gcctcaacta
caagaagatg ctgctgctca gcctgggcac tggcacgacc 120tccgagttcg acaagaccta
cactgccaag gaggccgcta cctggaccgc cgtccattgg 180atgctggtca tccagaagat
gacggacgcc gcttccagct acatgaccga ctactacctc 240tccactgcgt tccaggcgct
tgactccaag aacaactacc tccgtgttca ggagaatgcc 300ctcactggca ccacgaccga
gatggacgat gcctccgagg ccaacatgga gctgctcgtc 360caggtgggtg agaacctcct
gaagaagccc gtctccgaag acaatcccga gacctatgag 420gaagcgctca agcgctttgc
caagctgctc tctgatagga agaaactccg cgctaacaag 480gccagctacg gaccaggaca
gctcggcgag atggtgaccg tgctctccat cgacggcggt 540ggcatcaggg gcatcatccc
ggccaccatc ctggagttcc tggagggcca actccaggag 600atggacaaca acgccgacgc
ccgcctggcc gactacttcg acgtgatcgg tggcaccagc 660accggcggtc tcctgaccgc
catgatctcc actccgaacg agaacaaccg ccccttcgcc 720gctgcgaagg agatcgtccc
gttctacttc gaacacggcc ctcagatttt caacccctcg 780ggtcaaatcc tgggccccaa
gtacgacggc aagtacctta tgcaagtgct tcaggagaag 840ctgggcgaga ctagggtgca
ccaggcgctg accgaggtcg tcatctccag cttcgacatc 900aagaccaaca agccagtcat
cttcaccaag tccaacctgg ccaacagccc ggagctggac 960gctaagatgt acgacatctc
ctactccact gctgccgctc ccacgtactt ccctccgcac 1020tacttcgtca ccaacaccag
caacggcgac gagtacgagt tcaaccttgt tgacggtgcg 1080gtggctacgg tggcggaccc
ggcgctcctg tccatcagcg tcgccacgcg cctggcccag 1140aaggatccag ccttcgctag
cattagg
1167271389PRTArtificialSynthetic polypeptide 271Met Ala Thr Thr Lys Ser
Phe Leu Ile Leu Ile Phe Met Ile Leu Ala1 5
10 15Thr Thr Ser Ser Thr Phe Ala Ser Leu Asn Tyr Lys
Lys Met Leu Leu 20 25 30Leu
Ser Leu Gly Thr Gly Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr 35
40 45Ala Lys Glu Ala Ala Thr Trp Thr Ala
Val His Trp Met Leu Val Ile 50 55
60Gln Lys Met Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu65
70 75 80Ser Thr Ala Phe Gln
Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val 85
90 95Gln Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu
Met Asp Asp Ala Ser 100 105
110Glu Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys
115 120 125Lys Pro Val Ser Glu Asp Asn
Pro Glu Thr Tyr Glu Glu Ala Leu Lys 130 135
140Arg Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn
Lys145 150 155 160Ala Ser
Tyr Gly Pro Gly Gln Leu Gly Glu Met Val Thr Val Leu Ser
165 170 175Ile Asp Gly Gly Gly Ile Arg
Gly Ile Ile Pro Ala Thr Ile Leu Glu 180 185
190Phe Leu Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala Asp
Ala Arg 195 200 205Leu Ala Asp Tyr
Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly Leu 210
215 220Leu Thr Ala Met Ile Ser Thr Pro Asn Glu Asn Asn
Arg Pro Phe Ala225 230 235
240Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly Pro Gln Ile
245 250 255Phe Asn Pro Ser Gly
Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys Tyr 260
265 270Leu Met Gln Val Leu Gln Glu Lys Leu Gly Glu Thr
Arg Val His Gln 275 280 285Ala Leu
Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys Thr Asn Lys 290
295 300Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Asn
Ser Pro Glu Leu Asp305 310 315
320Ala Lys Met Tyr Asp Ile Ser Tyr Ser Thr Ala Ala Ala Pro Thr Tyr
325 330 335Phe Pro Pro His
Tyr Phe Val Thr Asn Thr Ser Asn Gly Asp Glu Tyr 340
345 350Glu Phe Asn Leu Val Asp Gly Ala Val Ala Thr
Val Ala Asp Pro Ala 355 360 365Leu
Leu Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys Asp Pro Ala 370
375 380Phe Ala Ser Ile
Arg38527255DNAArtificialSynthetic construct 272ggagctcgag aaaagagagg
ctgaagctac tgccaaggag gccgctacct ggacc
5527339DNAArtificialSynthetic construct 273ggtctagagg aattctcatt
acttgtcgaa ctcggaggt
392741167DNAArtificialSynthetic construct 274atggccacca ccaagagctt
cctcatcctg atcttcatga tcctggccac caccagcagc 60accttcgcca cctacactgc
caaggaggcc gctacctgga ccgccgtcca ttggatgctg 120gtcatccaga agatgacgga
cgccgcttcc agctacatga ccgactacta cctctccact 180gcgttccagg cgcttgactc
caagaacaac tacctccgtg ttcaggagaa tgccctcact 240ggcaccacga ccgagatgga
cgatgcctcc gaggccaaca tggagctgct cgtccaggtg 300ggtgagaacc tcctgaagaa
gcccgtctcc gaagacaatc ccgagaccta tgaggaagcg 360ctcaagcgct ttgccaagct
gctctctgat aggaagaaac tccgcgctaa caaggccagc 420tacggaccag gacagctcgg
cgagatggtg accgtgctct ccatcgacgg cggtggcatc 480aggggcatca tcccggccac
catcctggag ttcctggagg gccaactcca ggagatggac 540aacaacgccg acgcccgcct
ggccgactac ttcgacgtga tcggtggcac cagcaccggc 600ggtctcctga ccgccatgat
ctccactccg aacgagaaca accgcccctt cgccgctgcg 660aaggagatcg tcccgttcta
cttcgaacac ggccctcaga ttttcaaccc ctcgggtcaa 720atcctgggcc ccaagtacga
cggcaagtac cttatgcaag tgcttcagga gaagctgggc 780gagactaggg tgcaccaggc
gctgaccgag gtcgtcatct ccagcttcga catcaagacc 840aacaagccag tcatcttcac
caagtccaac ctggccaaca gcccggagct ggacgctaag 900atgtacgaca tctcctactc
cactgctgcc gctcccacgt acttccctcc gcactacttc 960gtcaccaaca ccagcaacgg
cgacgagtac gagttcaacc ttgttgacgg tgcggtggct 1020acggtggcgg acccggcgct
cctgtccatc agcgtcgcca cgcgcctggc ccagaaggat 1080ccagccttcg ctagcattag
gagcctcaac tacaagaaga tgctgctgct cagcctgggc 1140actggcacga cctccgagtt
cgacaag
1167275389PRTArtificialSynthetic polypeptide 275Met Ala Thr Thr Lys Ser
Phe Leu Ile Leu Ile Phe Met Ile Leu Ala1 5
10 15Thr Thr Ser Ser Thr Phe Ala Thr Tyr Thr Ala Lys
Glu Ala Ala Thr 20 25 30Trp
Thr Ala Val His Trp Met Leu Val Ile Gln Lys Met Thr Asp Ala 35
40 45Ala Ser Ser Tyr Met Thr Asp Tyr Tyr
Leu Ser Thr Ala Phe Gln Ala 50 55
60Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr65
70 75 80Gly Thr Thr Thr Glu
Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu 85
90 95Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys
Pro Val Ser Glu Asp 100 105
110Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu
115 120 125Ser Asp Arg Lys Lys Leu Arg
Ala Asn Lys Ala Ser Tyr Gly Pro Gly 130 135
140Gln Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly
Ile145 150 155 160Arg Gly
Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu Glu Gly Gln Leu
165 170 175Gln Glu Met Asp Asn Asn Ala
Asp Ala Arg Leu Ala Asp Tyr Phe Asp 180 185
190Val Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala Met
Ile Ser 195 200 205Thr Pro Asn Glu
Asn Asn Arg Pro Phe Ala Ala Ala Lys Glu Ile Val 210
215 220Pro Phe Tyr Phe Glu His Gly Pro Gln Ile Phe Asn
Pro Ser Gly Gln225 230 235
240Ile Leu Gly Pro Lys Tyr Asp Gly Lys Tyr Leu Met Gln Val Leu Gln
245 250 255Glu Lys Leu Gly Glu
Thr Arg Val His Gln Ala Leu Thr Glu Val Val 260
265 270Ile Ser Ser Phe Asp Ile Lys Thr Asn Lys Pro Val
Ile Phe Thr Lys 275 280 285Ser Asn
Leu Ala Asn Ser Pro Glu Leu Asp Ala Lys Met Tyr Asp Ile 290
295 300Ser Tyr Ser Thr Ala Ala Ala Pro Thr Tyr Phe
Pro Pro His Tyr Phe305 310 315
320Val Thr Asn Thr Ser Asn Gly Asp Glu Tyr Glu Phe Asn Leu Val Asp
325 330 335Gly Ala Val Ala
Thr Val Ala Asp Pro Ala Leu Leu Ser Ile Ser Val 340
345 350Ala Thr Arg Leu Ala Gln Lys Asp Pro Ala Phe
Ala Ser Ile Arg Ser 355 360 365Leu
Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly Thr Thr 370
375 380Ser Glu Phe Asp
Lys3852767PRTArtificialSynthetic polypeptide 276Gly Gly Gly Ser Gly Gly
Gly1 52773PRTArtificialSynthetic polypeptide 277Gly Pro
Gly1278386PRTSolanum tuberosum 278Met Ala Thr Thr Lys Ser Phe Leu Ile Leu
Phe Phe Met Ile Leu Ala1 5 10
15Thr Thr Ser Ser Thr Cys Ala Lys Leu Glu Glu Met Val Thr Val Leu
20 25 30Ser Ile Asp Gly Gly Gly
Ile Lys Gly Ile Ile Pro Ala Ile Ile Leu 35 40
45Glu Phe Leu Glu Gly Gln Leu Gln Glu Val Asp Asn Asn Lys
Asp Ala 50 55 60Arg Leu Ala Asp Tyr
Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly65 70
75 80Leu Leu Thr Ala Met Ile Thr Thr Pro Asn
Glu Asn Asn Arg Pro Phe 85 90
95Ala Ala Ala Lys Asp Ile Val Pro Phe Tyr Phe Glu His Gly Pro His
100 105 110Ile Phe Asn Tyr Ser
Gly Ser Ile Ile Gly Pro Met Tyr Asp Gly Lys 115
120 125Tyr Leu Leu Gln Val Leu Gln Glu Lys Leu Gly Glu
Thr Arg Val His 130 135 140Gln Ala Leu
Thr Glu Val Ala Ile Ser Ser Phe Asp Ile Lys Thr Asn145
150 155 160Lys Pro Val Ile Phe Thr Lys
Ser Asn Leu Ala Lys Ser Pro Glu Leu 165
170 175Asp Ala Lys Met Tyr Asp Ile Cys Tyr Ser Thr Ala
Ala Ala Pro Ile 180 185 190Tyr
Phe Pro Pro His Tyr Phe Ile Thr His Thr Ser Asn Gly Asp Ile 195
200 205Tyr Glu Phe Asn Leu Val Asp Gly Gly
Val Ala Thr Val Gly Asp Pro 210 215
220Ala Leu Leu Ser Leu Ser Val Ala Thr Arg Leu Ala Gln Glu Asp Pro225
230 235 240Ala Phe Ser Ser
Ile Lys Ser Leu Asp Tyr Lys Gln Met Leu Leu Leu 245
250 255Ser Leu Gly Thr Gly Thr Asn Ser Glu Phe
Asp Lys Thr Tyr Thr Ala 260 265
270Gln Glu Ala Ala Lys Trp Gly Pro Leu Arg Trp Met Leu Ala Ile Gln
275 280 285Gln Met Thr Asn Ala Ala Ser
Ser Tyr Met Thr Asp Tyr Tyr Ile Ser 290 295
300Thr Val Phe Gln Ala Arg His Ser Gln Asn Asn Tyr Leu Arg Val
Gln305 310 315 320Glu Asn
Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu
325 330 335Ala Asn Met Glu Leu Leu Val
Gln Val Gly Glu Thr Leu Leu Lys Lys 340 345
350Pro Val Ser Lys Asp Ser Pro Glu Thr Tyr Glu Glu Ala Leu
Lys Arg 355 360 365Phe Ala Lys Leu
Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370
375 380Ser Tyr385279386PRTSolanum tuberosum 279Met Ala
Thr Thr Lys Ser Val Leu Val Leu Phe Phe Met Ile Leu Ala1 5
10 15Thr Thr Ser Ser Thr Cys Ala Thr
Leu Gly Glu Met Val Thr Val Leu 20 25
30Ser Ile Asp Gly Gly Gly Ile Lys Gly Ile Ile Pro Ala Thr Ile
Leu 35 40 45Glu Phe Leu Glu Gly
Gln Leu Gln Glu Val Asp Asn Asn Lys Asp Ala 50 55
60Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr
Gly Gly65 70 75 80Leu
Leu Thr Ala Met Ile Thr Thr Pro Asn Glu Asn Asn Arg Pro Phe
85 90 95Ala Ala Ala Lys Asp Ile Val
Pro Phe Tyr Phe Glu His Gly Pro His 100 105
110Ile Phe Asn Ser Ser Gly Ser Ile Phe Gly Pro Met Tyr Asp
Gly Lys 115 120 125Tyr Phe Leu Gln
Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His 130
135 140Gln Ala Leu Thr Glu Val Ala Ile Ser Ser Phe Asp
Ile Lys Thr Asn145 150 155
160Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu
165 170 175Asp Ala Lys Met Asn
Asp Ile Cys Tyr Ser Thr Ala Ala Ala Pro Thr 180
185 190Tyr Phe Pro Pro His Tyr Phe Val Thr His Thr Ser
Asn Gly Asp Lys 195 200 205Tyr Glu
Phe Asn Leu Val Asp Gly Ala Val Ala Thr Val Gly Asp Pro 210
215 220Ala Leu Leu Ser Leu Ser Val Arg Thr Lys Leu
Ala Gln Val Asp Pro225 230 235
240Lys Phe Ala Ser Ile Lys Ser Leu Asn Tyr Asn Glu Met Leu Leu Leu
245 250 255Ser Leu Gly Thr
Gly Thr Asn Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260
265 270Glu Glu Ala Ala Lys Trp Gly Pro Leu Arg Trp
Ile Leu Ala Ile Gln 275 280 285Gln
Met Thr Asn Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser 290
295 300Thr Val Phe Gln Ala Arg His Ser Gln Asn
Asn Tyr Leu Arg Val Gln305 310 315
320Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser
Glu 325 330 335Ala Asn Met
Glu Leu Leu Val Gln Val Gly Glu Lys Leu Leu Lys Lys 340
345 350Pro Val Ser Lys Asp Ser Pro Glu Thr Tyr
Glu Glu Ala Leu Lys Arg 355 360
365Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370
375 380Ser Tyr385280365PRTSolanum
tuberosum 280Met Ala Leu Glu Glu Met Val Ala Val Leu Ser Ile Asp Gly Gly
Gly1 5 10 15Ile Lys Gly
Ile Ile Pro Gly Thr Ile Leu Glu Phe Leu Glu Gly Gln 20
25 30Leu Gln Lys Met Asp Asn Asn Ala Asp Ala
Arg Leu Ala Asp Tyr Phe 35 40
45Asp Val Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala Met Ile 50
55 60Thr Thr Pro Asn Glu Asn Asn Arg Pro
Phe Ala Ala Ala Asn Glu Ile65 70 75
80Val Pro Phe Tyr Phe Glu His Gly Pro His Ile Phe Asn Ser
Arg Tyr 85 90 95Trp Pro
Ile Phe Trp Pro Lys Tyr Asp Gly Lys Tyr Leu Met Gln Val 100
105 110Leu Gln Glu Lys Leu Gly Glu Thr Arg
Val His Gln Ala Leu Thr Glu 115 120
125Val Ala Ile Ser Ser Phe Asp Ile Lys Thr Asn Lys Pro Val Ile Phe
130 135 140Thr Lys Ser Asn Leu Ala Lys
Ser Pro Glu Leu Asp Ala Lys Thr Tyr145 150
155 160Asp Ile Cys Tyr Ser Thr Ala Ala Ala Pro Thr Tyr
Phe Pro Pro His 165 170
175Tyr Phe Ala Thr Asn Thr Ile Asn Gly Asp Lys Tyr Glu Phe Asn Leu
180 185 190Val Asp Gly Ala Val Ala
Thr Val Ala Asp Pro Ala Leu Leu Ser Val 195 200
205Ser Val Ala Thr Arg Arg Ala Gln Glu Asp Pro Ala Phe Ala
Ser Ile 210 215 220Arg Ser Leu Asn Tyr
Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly225 230
235 240Thr Thr Ser Glu Phe Asp Lys Thr His Thr
Ala Glu Glu Thr Ala Lys 245 250
255Trp Gly Ala Leu Gln Trp Met Leu Val Ile Gln Gln Met Thr Glu Ala
260 265 270Ala Ser Ser Tyr Met
Thr Asp Tyr Tyr Leu Ser Thr Val Phe Gln Asp 275
280 285Leu His Ser Gln Asn Asn Tyr Leu Arg Val Gln Glu
Asn Ala Leu Thr 290 295 300Gly Thr Thr
Thr Lys Ala Asp Asp Ala Ser Glu Ala Asn Met Glu Leu305
310 315 320Leu Ala Gln Val Gly Glu Asn
Leu Leu Lys Lys Pro Val Ser Lys Asp 325
330 335Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe
Ala Lys Leu Leu 340 345 350Ser
Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr 355
360 365281364PRTSolanum tuberosum 281Pro Trp Leu Glu Glu
Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly1 5
10 15Ile Lys Gly Ile Ile Pro Ala Ile Ile Leu Glu
Phe Leu Glu Gly Gln 20 25
30Leu Gln Glu Val Asp Asn Asn Lys Asp Ala Arg Leu Ala Asp Tyr Phe
35 40 45Asp Val Ile Gly Gly Thr Ser Thr
Gly Gly Leu Leu Thr Ala Met Ile 50 55
60Thr Thr Pro Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala Lys Asp Ile65
70 75 80Val Pro Phe Tyr Phe
Glu His Gly Pro His Ile Phe Asn Tyr Ser Gly 85
90 95Ser Ile Leu Gly Pro Met Tyr Asp Gly Lys Tyr
Leu Leu Gln Val Leu 100 105
110Gln Glu Lys Leu Gly Glu Thr Arg Val His Gln Ala Leu Thr Glu Val
115 120 125Ala Ile Ser Ser Phe Asp Ile
Lys Thr Asn Lys Pro Val Ile Phe Thr 130 135
140Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu Asp Ala Lys Met Tyr
Asp145 150 155 160Ile Cys
Tyr Ser Thr Ala Ala Ala Pro Ile Tyr Phe Pro Pro His His
165 170 175Phe Val Thr His Thr Ser Asn
Gly Ala Arg Tyr Glu Phe Asn Leu Val 180 185
190Asp Gly Ala Val Ala Thr Val Gly Asp Pro Ala Leu Leu Ser
Leu Ser 195 200 205Val Ala Thr Arg
Leu Ala Gln Glu Asp Pro Ala Phe Ser Ser Ile Lys 210
215 220Ser Leu Asp Tyr Lys Gln Met Leu Leu Leu Ser Leu
Gly Thr Gly Thr225 230 235
240Asn Ser Glu Phe Asp Lys Thr Tyr Thr Ala Glu Glu Ala Ala Lys Trp
245 250 255Gly Pro Leu Arg Trp
Met Leu Ala Ile Gln Gln Met Thr Asn Ala Ala 260
265 270Ser Phe Tyr Met Thr Asp Tyr Tyr Ile Ser Thr Val
Phe Gln Ala Arg 275 280 285His Ser
Gln Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Asn Gly 290
295 300Thr Thr Thr Glu Met Asp Asp Ala Ser Glu Ala
Asn Met Glu Leu Leu305 310 315
320Val Gln Val Gly Glu Thr Leu Leu Lys Lys Pro Val Ser Arg Asp Ser
325 330 335Pro Glu Thr Tyr
Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser 340
345 350Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser
Tyr 355 360282386PRTSolanum tuberosum 282Met Ala
Thr Thr Lys Ser Phe Leu Ile Leu Phe Phe Met Ile Leu Ala1 5
10 15Thr Thr Ser Ser Thr Cys Ala Lys
Leu Glu Glu Met Val Thr Val Leu 20 25
30Ser Ile Asp Gly Gly Gly Ile Lys Gly Ile Ile Pro Ala Ile Ile
Leu 35 40 45Glu Phe Leu Glu Gly
Gln Leu Gln Glu Val Asp Asn Asn Lys Asp Ala 50 55
60Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr
Gly Gly65 70 75 80Leu
Leu Thr Ala Met Ile Thr Thr Pro Asn Glu Asn Asn Arg Pro Phe
85 90 95Ala Ala Ala Lys Asp Ile Val
Pro Phe Tyr Phe Glu His Gly Pro His 100 105
110Ile Phe Asn Tyr Ser Gly Ser Ile Leu Gly Pro Met Tyr Asp
Gly Lys 115 120 125Tyr Leu Leu Gln
Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His 130
135 140Gln Ala Leu Thr Glu Val Ala Ile Ser Ser Phe Asp
Ile Lys Thr Asn145 150 155
160Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu
165 170 175Asp Ala Lys Met Tyr
Asp Ile Cys Tyr Ser Thr Ala Ala Ala Pro Ile 180
185 190Tyr Phe Pro Pro His His Phe Val Thr His Thr Ser
Asn Gly Ala Arg 195 200 205Tyr Glu
Phe Asn Leu Val Asp Gly Ala Val Ala Thr Val Gly Asp Pro 210
215 220Ala Leu Leu Ser Leu Ser Val Ala Thr Arg Leu
Ala Gln Glu Asp Pro225 230 235
240Ala Phe Ser Ser Ile Lys Ser Leu Asp Tyr Lys Gln Met Leu Leu Leu
245 250 255Ser Leu Gly Thr
Gly Thr Asn Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260
265 270Glu Glu Ala Ala Lys Trp Gly Pro Leu Arg Trp
Met Leu Ala Ile Gln 275 280 285Gln
Met Thr Asn Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Ile Ser 290
295 300Thr Val Phe Gln Ala Arg His Ser Gln Asn
Asn Tyr Leu Arg Val Gln305 310 315
320Glu Asn Ala Leu Asn Gly Thr Thr Thr Glu Met Asp Asp Ala Ser
Glu 325 330 335Ala Asn Met
Glu Leu Leu Val Gln Val Gly Ala Thr Leu Leu Lys Lys 340
345 350Pro Val Ser Lys Asp Ser Pro Glu Thr Tyr
Glu Glu Ala Leu Lys Arg 355 360
365Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370
375 380Ser
Tyr38528310PRTArtificialSynthetic polypeptide 283Ala Phe Phe Asp Lys Thr
Tyr Thr Ala Lys1 5
1028410PRTArtificialSynthetic polypeptide 284Cys Ile Phe Asp Ser Thr Tyr
Thr Ala Lys1 5 102851161DNASolanum
tuberosum 285atggcaacta ctaaatcttt tttaatttta atatttatga tattagcaac
tactagttca 60acatttgctc agttgggaga aatggtgact gttcttagta ttgatggagg
tggaattaga 120gggatcattc cggctaccat tctcgaattt cttgaaggac aacttcagga
aatggacaat 180aatgcagatg caagacttgc agattacttt gatgtaattg gaggaacaag
tacaggaggt 240ttattgactg ctatgataag tactccaaat gaaaacaatc gaccctttgc
tgctgccaaa 300gaaattgtac ctttttactt cgaacatggc cctcagattt ttaatcctag
tggtcaaatt 360ttaggcccaa aatatgatgg aaaatatctt atgcaagttc ttcaagaaaa
acttggagaa 420actcgtgtgc atcaagcttt gacagaagtt gtcatctcaa gctttgacat
caaaacaaat 480aagccagtaa tattcactaa gtcaaattta gcaaactctc cagaattgga
tgctaagatg 540tatgacataa gttattccac agcagcagct ccaacatatt ttcctccgca
ttactttgtt 600actaatacta gtaatggaga tgaatatgag ttcaatcttg ttgatggtgc
tgttgctact 660gttgctgatc cggcgttatt atccattagc gttgcaacga gacttgcaca
aaaggatcca 720gcatttgctt caattaggtc attgaattac aaaaaaatgc tgttgctctc
attaggcact 780ggcactactt cagagtttga taaaacatat acagcaaaag aggcagctac
ctggactgct 840gtacattgga tgttagttat acagaaaatg actgatgcag caagttctta
catgactgat 900tattaccttt ctactgcttt tcaagctctt gattcaaaaa acaattacct
cagggttcaa 960gaaaatgcat taacaggcac aactactgaa atggatgatg cttctgaggc
taatatggaa 1020ttattagtac aagttggtga aaacttattg aagaaaccag tttccgaaga
caatcctgaa 1080acctatgagg aagctctaaa gaggtttgca aaattgctct ctgataggaa
gaaactccga 1140gcaaacaaag cttcttatta a
1161286386PRTSolanum tuberosum 286Met Ala Thr Thr Lys Ser Phe
Leu Ile Leu Ile Phe Met Ile Leu Ala1 5 10
15Thr Thr Ser Ser Thr Phe Ala Gln Leu Gly Glu Met Val
Thr Val Leu 20 25 30Ser Ile
Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu 35
40 45Glu Phe Leu Glu Gly Gln Leu Gln Glu Met
Asp Asn Asn Ala Asp Ala 50 55 60Arg
Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly65
70 75 80Leu Leu Thr Ala Met Ile
Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe 85
90 95Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu
His Gly Pro Gln 100 105 110Ile
Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys 115
120 125Tyr Leu Met Gln Val Leu Gln Glu Lys
Leu Gly Glu Thr Arg Val His 130 135
140Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys Thr Asn145
150 155 160Lys Pro Val Ile
Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro Glu Leu 165
170 175Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser
Thr Ala Ala Ala Pro Thr 180 185
190Tyr Phe Pro Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly Asp Glu
195 200 205Tyr Glu Phe Asn Leu Val Asp
Gly Ala Val Ala Thr Val Ala Asp Pro 210 215
220Ala Leu Leu Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys Asp
Pro225 230 235 240Ala Phe
Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu
245 250 255Ser Leu Gly Thr Gly Thr Thr
Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260 265
270Lys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met Leu Val
Ile Gln 275 280 285Lys Met Thr Asp
Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser 290
295 300Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn Tyr
Leu Arg Val Gln305 310 315
320Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu
325 330 335Ala Asn Met Glu Leu
Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys 340
345 350Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu
Ala Leu Lys Arg 355 360 365Phe Ala
Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370
375 380Ser Tyr385287408PRTArtificialSynthetic
polypeptide 287Met Lys Ser Lys Met Ala Met Leu Leu Leu Leu Phe Cys Val
Leu Ser1 5 10 15Asn Gln
Leu Val Ala Ala Phe Ser Thr Gln Ala Lys Ala Ser Lys Asp 20
25 30Gly Asn Leu Val Thr Val Leu Ala Ile
Asp Gly Gly Gly Ile Arg Gly 35 40
45Ile Ile Pro Gly Val Ile Leu Lys Gln Leu Glu Ala Thr Leu Gln Arg 50
55 60Trp Asp Ser Ser Ala Arg Leu Ala Glu
Tyr Phe Asp Val Val Ala Gly65 70 75
80Thr Ser Thr Gly Gly Ile Ile Thr Ala Ile Leu Thr Ala Pro
Asp Pro 85 90 95Gln Asn
Lys Asp Arg Pro Leu Tyr Ala Ala Glu Glu Ile Ile Asp Phe 100
105 110Tyr Ile Glu His Gly Pro Ser Ile Phe
Asn Lys Ser Thr Ala Cys Ser 115 120
125Leu Pro Gly Ile Phe Cys Pro Lys Tyr Asp Gly Lys Tyr Leu Gln Glu
130 135 140Ile Ile Ser Gln Lys Leu Asn
Glu Thr Leu Leu Asp Gln Thr Thr Thr145 150
155 160Asn Val Val Ile Pro Ser Phe Asp Ile Lys Leu Leu
Arg Pro Thr Ile 165 170
175Phe Ser Thr Phe Lys Leu Glu Glu Val Pro Glu Leu Asn Val Lys Leu
180 185 190Ser Asp Val Cys Met Gly
Thr Ser Ala Ala Pro Ile Val Phe Pro Pro 195 200
205Tyr Tyr Phe Lys His Gly Asp Thr Glu Phe Asn Leu Val Asp
Gly Ala 210 215 220Ile Ile Ala Asp Ile
Pro Ala Pro Val Ala Leu Ser Glu Val Leu Gln225 230
235 240Gln Glu Lys Tyr Lys Asn Lys Glu Ile Leu
Leu Leu Ser Ile Gly Thr 245 250
255Gly Val Val Lys Pro Gly Glu Gly Tyr Ser Ala Asn Arg Thr Trp Thr
260 265 270Ile Phe Asp Trp Ser
Ser Glu Thr Leu Ile Gly Leu Met Gly His Gly 275
280 285Thr Arg Ala Met Ser Asp Tyr Tyr Val Gly Ser His
Phe Lys Ala Leu 290 295 300Gln Pro Gln
Asn Asn Tyr Leu Arg Ile Gln Glu Tyr Asp Leu Asp Pro305
310 315 320Ala Leu Glu Ser Ile Asp Asp
Ala Ser Thr Glu Asn Met Glu Asn Leu 325
330 335Glu Lys Val Gly Gln Ser Leu Leu Asn Glu Pro Val
Lys Arg Met Asn 340 345 350Leu
Asn Thr Phe Val Val Glu Glu Thr Gly Glu Gly Thr Asn Ala Glu 355
360 365Ala Leu Asp Arg Leu Ala Gln Ile Leu
Tyr Glu Glu Lys Ile Thr Arg 370 375
380Gly Leu Gly Lys Ile Ser Leu Glu Val Asp Asn Ile Asp Pro Tyr Thr385
390 395 400Glu Arg Val Arg
Lys Leu Leu Phe 405288410PRTZea mays 288Met Gly Ser Ile
Gly Arg Gly Thr Ala Asn Cys Ala Thr Val Pro Gln1 5
10 15Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr
Ile Leu Ser Ile Asp Gly 20 25
30Gly Gly Ile Arg Gly Leu Ile Pro Ala Thr Ile Ile Ala Tyr Leu Glu
35 40 45Ala Lys Leu Gln Glu Leu Asp Gly
Pro Asp Ala Arg Ile Ala Asp Tyr 50 55
60Phe Asp Val Ile Ala Gly Thr Ser Thr Gly Ala Leu Leu Ala Ser Met65
70 75 80Leu Ala Ala Pro Asp
Glu Asn Asn Arg Pro Leu Phe Ala Ala Lys Asp 85
90 95Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys
Ile Phe Pro Gln Lys 100 105
110Lys Ala Gly Leu Leu Thr Pro Leu Arg Asn Leu Leu Gly Leu Val Arg
115 120 125Gly Pro Lys Tyr Asp Gly Val
Phe Leu His Asp Lys Ile Lys Ser Leu 130 135
140Thr His Asp Val Arg Val Ala Asp Thr Val Thr Asn Val Ile Val
Pro145 150 155 160Ala Phe
Asp Val Lys Tyr Leu Gln Pro Ile Ile Phe Ser Thr Tyr Glu
165 170 175Ala Lys Thr Asp Thr Leu Lys
Asn Ala His Leu Ser Asp Ile Cys Ile 180 185
190Ser Thr Ser Ala Ala Pro Thr Tyr Phe Pro Ala His Phe Phe
Lys Thr 195 200 205Glu Ala Thr Asp
Gly Arg Pro Pro Arg Glu Tyr His Leu Val Asp Gly 210
215 220Gly Val Ala Ala Asn Asn Pro Thr Met Val Ala Met
Ser Met Leu Thr225 230 235
240Lys Glu Val His Arg Arg Asn Pro Asn Phe Asn Ala Gly Ser Pro Thr
245 250 255Glu Tyr Thr Asn Tyr
Leu Ile Ile Ser Val Gly Thr Gly Ser Ala Lys 260
265 270Gln Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys
Trp Gly Leu Ile 275 280 285Gln Trp
Leu Tyr Asn Gly Gly Phe Thr Pro Ile Ile Asp Ile Phe Ser 290
295 300His Ala Ser Ser Asp Met Val Asp Ile His Ala
Ser Ile Leu Phe Gln305 310 315
320Ala Leu His Cys Glu Lys Lys Tyr Leu Arg Ile Gln Asp Asp Thr Leu
325 330 335Thr Gly Asn Ala
Ser Ser Val Asp Ile Ala Thr Lys Glu Asn Met Glu 340
345 350Ser Leu Ile Ser Ile Gly Gln Glu Leu Leu Lys
Lys Pro Val Ala Arg 355 360 365Val
Asn Ile Asp Thr Gly Val Tyr Glu Ser Cys Asp Gly Glu Gly Thr 370
375 380Asn Ala Gln Ser Leu Ala Asp Phe Ala Lys
Gln Leu Ser Asp Glu Arg385 390 395
400Lys Leu Arg Lys Ser Asn Leu Asn Ser Asn 405
410289508PRTZea mays 289Arg Pro Thr Arg Pro Arg His Pro Arg
Asn Thr Gln Lys Arg Gly Ala1 5 10
15Leu Leu Val Gly Trp Ile Leu Phe Ser Leu Ala Ala Ser Pro Val
Lys 20 25 30Phe Gln Thr His
Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala 35
40 45Thr Val Pro Gln Pro Pro Pro Ser Thr Gly Lys Leu
Ile Thr Ile Leu 50 55 60Ser Ile Asp
Gly Gly Gly Ile Arg Gly Leu Ile Pro Ala Thr Ile Ile65 70
75 80Ala Tyr Leu Glu Ala Lys Leu Gln
Glu Leu Asp Gly Pro Asp Ala Arg 85 90
95Ile Ala Asp Tyr Phe Asp Val Ile Ala Gly Thr Ser Thr Gly
Ala Leu 100 105 110Leu Ala Ser
Met Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu Phe 115
120 125Ala Ala Lys Asp Leu Thr Thr Phe Tyr Leu Glu
Asn Gly Pro Lys Ile 130 135 140Phe Pro
Gln Lys Lys Ala Gly Leu Leu Thr Pro Leu Arg Asn Leu Leu145
150 155 160Gly Leu Val Arg Gly Pro Lys
Tyr Asp Gly Val Phe Leu His Asp Lys 165
170 175Ile Lys Ser Leu Thr His Asp Val Arg Val Ala Asp
Thr Val Thr Asn 180 185 190Val
Ile Val Pro Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile Ile Phe 195
200 205Ser Thr Tyr Glu Ala Lys Thr Asp Ala
Leu Lys Asn Ala His Leu Ser 210 215
220Asp Ile Cys Ile Ser Thr Ser Ala Ala Pro Thr Tyr Phe Pro Ala His225
230 235 240Phe Phe Lys Thr
Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His 245
250 255Leu Val Asp Gly Gly Val Ala Ala Asn Asn
Pro Thr Met Val Ala Met 260 265
270Ser Met Leu Thr Lys Glu Val His Arg Arg Asn Pro Asn Phe Asn Ala
275 280 285Gly Ser Pro Thr Glu Tyr Thr
Asn Tyr Leu Ile Ile Ser Val Gly Thr 290 295
300Gly Ser Ala Lys Gln Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala
Lys305 310 315 320Trp Gly
Leu Ile Gln Trp Leu Tyr Asn Gly Gly Phe Thr Pro Ile Ile
325 330 335Asp Ile Phe Ser His Ala Ser
Ser Asp Met Val Asp Ile His Ala Ser 340 345
350Ile Leu Phe Gln Ala Leu His Cys Glu Lys Lys Tyr Leu Arg
Ile Gln 355 360 365Leu Tyr Tyr Ala
Gly Tyr Phe Asp Trp Glu Arg Ile Val Arg Gly His 370
375 380Arg His Gln Gly Glu His Gly Val Ser Asp Ile Asp
Arg Pro Gly Ala385 390 395
400Ala Gln Glu Ala Ser Gly Glu Ser Glu His Arg His Arg Ala Val Arg
405 410 415Val Leu Arg Arg Gly
His Lys Cys Thr Val Ala Ser Leu Arg Gln Ala 420
425 430Thr Leu Arg Ala Gln Ala Thr Gln Glu Gln Ser Gln
Leu Gln Leu Ile 435 440 445Asn Thr
Ser Leu Ser His Ser Met Cys Ser Phe Arg Arg Phe Thr Val 450
455 460Ser Tyr Phe Phe Asn Phe Asn Ser Val Cys Val
Leu Cys Val Leu Cys465 470 475
480Val Tyr Gln Thr Phe Lys Phe Asn Gln Lys Lys Lys Lys Lys Lys Lys
485 490 495Lys Lys Lys Lys
Lys Lys Lys Lys Lys Arg Ala Ala 500
505290410PRTZea mays 290Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala
Thr Val Pro Gln1 5 10
15Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly
20 25 30Gly Gly Ile Arg Gly Leu Ile
Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 40
45Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg Ile Ala Asp
Tyr 50 55 60Phe Asp Val Ile Ala Gly
Thr Ser Thr Gly Ala Leu Leu Ala Ser Met65 70
75 80Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu
Phe Ala Ala Lys Asp 85 90
95Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys
100 105 110Lys Ala Gly Leu Leu Thr
Pro Leu Arg Asn Leu Leu Gly Leu Val Arg 115 120
125Gly Pro Lys Tyr Asp Gly Val Phe Leu His Asp Lys Ile Lys
Ser Leu 130 135 140Thr His Asp Val Arg
Val Ala Asp Thr Val Thr Asn Val Ile Val Pro145 150
155 160Ala Phe Asp Val Lys Ser Leu Gln Pro Ile
Ile Phe Ser Thr Tyr Glu 165 170
175Ala Lys Thr Asp Thr Leu Lys Asn Ala His Leu Ser Asp Ile Cys Ile
180 185 190Ser Thr Ser Ala Ala
Pro Thr Tyr Phe Pro Ala His Phe Phe Lys Thr 195
200 205Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His
Leu Val Asp Gly 210 215 220Gly Val Ala
Ala Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr225
230 235 240Lys Glu Val His Arg Arg Asn
Pro Asn Phe Asn Ala Gly Ser Pro Thr 245
250 255Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr
Gly Ser Ala Lys 260 265 270Gln
Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile 275
280 285Gln Trp Leu Tyr Asn Gly Gly Phe Thr
Pro Ile Ile Asp Ile Phe Ser 290 295
300His Ala Ser Ser Asp Met Val Asp Ile His Ala Ser Ile Leu Phe Gln305
310 315 320Ala Leu His Cys
Glu Lys Lys Tyr Leu Arg Ile Gln Asp Asp Thr Leu 325
330 335Thr Gly Asn Ala Ser Ser Val Asp Ile Ala
Thr Lys Glu Asn Met Glu 340 345
350Ser Leu Ile Ser Ile Gly Gln Glu Leu Leu Asn Lys Pro Val Ala Arg
355 360 365Val Asn Ile Asp Thr Gly Leu
Tyr Glu Ser Cys Glu Gly Glu Gly Thr 370 375
380Asn Ala Gln Ser Leu Ala Asp Phe Ala Lys Gln Leu Ser Asp Glu
Arg385 390 395 400Lys Leu
Arg Lys Ser Asn Leu Asn Ser Asn 405
410291410PRTZea mays 291Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala
Thr Val Pro Gln1 5 10
15Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly
20 25 30Gly Gly Ile Arg Gly Leu Ile
Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 40
45Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg Ile Ala Asp
Tyr 50 55 60Phe Asp Val Ile Ala Gly
Thr Ser Thr Gly Ala Leu Leu Ala Ser Met65 70
75 80Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu
Phe Ala Ala Lys Asp 85 90
95Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys
100 105 110Lys Ala Gly Leu Leu Thr
Pro Leu Arg Asn Leu Leu Gly Leu Val Arg 115 120
125Gly Pro Lys Tyr Asp Gly Val Phe Leu His Asp Lys Ile Lys
Ser Leu 130 135 140Thr His Asp Val Arg
Val Ala Asp Thr Val Thr Asn Val Ile Val Pro145 150
155 160Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile
Ile Phe Ser Thr Tyr Glu 165 170
175Ala Lys Thr Asp Ala Leu Lys Asn Ala His Leu Ser Asp Ile Cys Ile
180 185 190Ser Thr Ser Ala Ala
Pro Thr Tyr Phe Pro Ala His Phe Phe Lys Thr 195
200 205Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His
Leu Val Asp Gly 210 215 220Gly Val Ala
Ala Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr225
230 235 240Lys Glu Val His Arg Arg Asn
Pro Asn Phe Asn Ala Gly Ser Pro Thr 245
250 255Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr
Gly Ser Ala Lys 260 265 270Gln
Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile 275
280 285Gln Trp Leu Tyr Asn Gly Gly Phe Thr
Pro Ile Ile Asp Ile Phe Ser 290 295
300His Ala Ser Ser Asp Met Val Asp Ile His Ala Ser Ile Leu Phe Gln305
310 315 320Ala Leu His Cys
Glu Lys Lys Tyr Leu Arg Ile Gln Asp Asp Thr Leu 325
330 335Thr Gly Asn Ala Ser Ser Val Asp Ile Ala
Thr Lys Glu Asn Met Glu 340 345
350Ser Leu Ile Ser Ile Gly Gln Glu Leu Leu Lys Lys Pro Val Ala Arg
355 360 365Val Asn Ile Asp Thr Gly Leu
Tyr Glu Ser Cys Asp Gly Glu Gly Thr 370 375
380Asn Ala Gln Ser Leu Ala Asp Phe Ala Lys Gln Leu Ser Asp Glu
Arg385 390 395 400Lys Leu
Arg Lys Ser Asn Leu Asn Ser Asn 405
410292410PRTZea mays 292Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala
Thr Val Pro Gln1 5 10
15Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly
20 25 30Gly Gly Ile Arg Gly Leu Ile
Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 40
45Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg Ile Ala Asp
Tyr 50 55 60Phe Asp Val Ile Ala Gly
Thr Ser Thr Gly Ala Leu Leu Ala Ser Met65 70
75 80Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu
Phe Ala Ala Lys Asp 85 90
95Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys
100 105 110Lys Ala Gly Leu Leu Thr
Pro Leu Arg Asn Leu Leu Gly Leu Val Arg 115 120
125Gly Pro Lys Tyr Asp Gly Val Phe Leu His Asp Lys Ile Lys
Ser Leu 130 135 140Thr His Asp Val Arg
Val Ala Asp Thr Val Thr Asn Val Ile Val Pro145 150
155 160Ala Phe Asp Val Lys Ser Leu Gln Pro Ile
Ile Phe Ser Thr Tyr Glu 165 170
175Ala Lys Thr Asp Thr Leu Lys Asn Ala His Leu Ser Asp Ile Cys Ile
180 185 190Ser Thr Ser Ala Ala
Pro Thr Tyr Phe Pro Ala His Phe Phe Lys Ile 195
200 205Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His
Leu Val Asp Gly 210 215 220Gly Val Ala
Ala Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr225
230 235 240Lys Glu Val His Arg Arg Asn
Pro Asn Phe Asn Ala Gly Ser Pro Thr 245
250 255Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr
Gly Ser Ala Lys 260 265 270Gln
Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile 275
280 285Gln Trp Leu Tyr Asn Gly Gly Phe Thr
Pro Ile Ile Asp Ile Phe Ser 290 295
300His Ala Ser Ser Asp Met Val Asp Ile His Ala Ser Ile Leu Phe Gln305
310 315 320Ala Leu His Cys
Glu Lys Lys Tyr Leu Arg Ile Gln Asp Asp Thr Leu 325
330 335Thr Gly Asn Ala Ser Ser Val Asp Ile Ala
Thr Lys Glu Asn Met Glu 340 345
350Ser Leu Ile Ser Ile Gly Gln Glu Leu Leu Asn Lys Pro Val Ala Arg
355 360 365Val Asn Ile Asp Thr Gly Leu
Tyr Glu Ser Cys Glu Gly Glu Gly Thr 370 375
380Asn Ala Gln Ser Leu Ala Asp Phe Ala Lys Gln Leu Ser Asp Glu
Arg385 390 395 400Lys Leu
Arg Lys Ser Asn Leu Asn Ser Asn 405
410293337PRTZea mays 293Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala
Thr Val Pro Gln1 5 10
15Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly
20 25 30Gly Gly Ile Arg Gly Leu Ile
Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 40
45Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg Ile Ala Asp
Tyr 50 55 60Phe Asp Val Ile Ala Gly
Thr Ser Thr Gly Ala Leu Leu Ala Ser Met65 70
75 80Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu
Phe Ala Ala Lys Asp 85 90
95Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys
100 105 110Lys Ala Gly Leu Leu Thr
Pro Leu Arg Asn Leu Leu Gly Leu Val Arg 115 120
125Gly Pro Lys Tyr Asp Gly Val Phe Leu His Asp Lys Ile Lys
Ser Leu 130 135 140Thr His Asp Val Arg
Val Ala Asp Thr Val Thr Asn Val Ile Val Pro145 150
155 160Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile
Ile Phe Ser Thr Tyr Glu 165 170
175Ala Lys Thr Asp Ala Leu Lys Asn Ala His Leu Ser Asp Ile Cys Ile
180 185 190Ser Thr Ser Ala Ala
Pro Thr Tyr Phe Pro Ala His Phe Phe Lys Thr 195
200 205Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His
Leu Val Asp Gly 210 215 220Gly Val Ala
Ala Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr225
230 235 240Lys Glu Val His Arg Arg Asn
Pro Asn Phe Asn Ala Gly Ser Pro Thr 245
250 255Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr
Gly Ser Ala Lys 260 265 270Gln
Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile 275
280 285Gln Trp Leu Tyr Asn Gly Gly Phe Thr
Pro Ile Ile Asp Ile Phe Ser 290 295
300His Ala Ser Ser Asp Met Val Asp Ile His Ala Ser Ile Leu Phe Gln305
310 315 320Ala Leu His Cys
Glu Lys Lys Tyr Leu Arg Ile Gln Leu Tyr Tyr Ala 325
330 335Gly29429DNAArtificialSynthetic construct
294gggccatggc gcagttggga gaaatggtg
2929537DNAArtificialSynthetic construct 295aacaaagctt cttattgagg
tgcggccgct tgcatgc 37
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