ENHANCED HETEROLOGOUS PRODUCTION OF LIPOXYGENASES

Abstract

The invention is directed to the enhanced expression and purification of lipoxygenase enzymes. These enzymes are of wide-spread industrial importance, often produced in heterologous microbial systems. Preferably, the lipoxygenase produced by the methods of the invention is a plant-derived enzyme and expressed at high-levels in a microbial system that includes a protease-deficient host and one or more chaperone expression plasmids. The invention is also directed to amino acid and nucleic acid fragments of the lipoxygenase enzyme including fragments in expression constructs encoding all or a portion of one or more lipoxygenase genes. The invention is also directed to methods of manufacturing bread and other food and also non-food products with lipoxygenase manufactured by the methods of the invention.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Non-Provisional application Ser. No. 14/263,105, filed Apr. 28, 2014, which claims priority to U.S. Provisional Application No. 61/817,077, filed Apr. 29, 2013, both of the same title and both of which are incorporated by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 28, 2014, is named 3060.002.US_SL.txt and is 29,528 bytes in size.

BACKGROUND

[0003] 1. Field of the Invention

[0004] The invention is directed to systems, compositions and methods for the expression and purification of lipoxygenases, to amino acid and nucleic acid sequences of all or portions of lipoxygenases, to molecular constructs for the expression of lipoxygenases, and, in particular, to methods for the large scale production and use of lipoxygenases in food products.

[0005] 2. Description of the Background

[0006] Lipoxygenases (LOXs; EC1.13.11_), also known as lipoxydases, are non-heme iron-containing dioxygenases distributed in plants and animals. LOXs catalyze hydroperoxidation of polyunsaturated fatty acids in the first step of fatty acid metabolite synthesis, to produce an unsaturated fatty acid hydroperoxide. A LOX definition according to enzyme classification is linoleate: oxygen oxidoreductase (for plant LOX) and arachidonate: oxygen oxidoreductase (for mammalian LOX). In plants, the most common LOX substrates linoleic acid and linolenic acids are converted into a variety of bioactive mediators involved in plant defense, senescence, seed germination, as well as plant growth and development (Grechkin A. Recent developments in biochemistry of the plant lipoxygenase pathway; Prog Lipid Res. 1998 Nov. 37(5):317-52). Lipoxygenases with different specificities, subcellular location, and tissue-specific expression patterns have been identified as ubiquitously found across kingdoms from bacteria to mammals.

[0007] LOXs are of commercial value in various industries including but not limited to food-related applications in food processing including bread making (bleaching and improved texture), aroma and flavor enhancement as well as for production of perfumes, paint driers (lipoxygenases: potential starting biocatalysts for the synthesis of signaling compounds. Joo Y C, Oh D K. 2012) and pitch control in softwood pulp (Microbial and enzymatic control of pitch in the pulp and paper industry, Ana Gutierrez & Jose C. del Rio & Angel T. Martinez, Appl Microbiol Biotechnol (2009) 82:1005-1018). Lipoxygenase is present in seeds (e.g. soybeans), grains and many other plant tissues. In the presence of oxygen, lipoxygenase oxidizes unsaturated fatty acids and produces lipid hydroperoxides, which improve dough structure through the oxidation of unsaturated fatty acids and subsequently react with specific chemical components of flour. As a consequence, dough stability and rising is increased, which either or together can increase the volume of the final product.

[0008] Regarding the processing of bread, lipoxygenase enzymes offer an advantage over current chemical additives. The flour ingredient industry had long been using chemical bleach, mostly benzoyl peroxide (BPO). Because of potential health concerns over BPO, some Euro countries and China banned the usage of BPO in flour. In the U.S., BPO is still widely used, but the demand keeps shirking although there is currently no safe alternative. Azodiformamide is another chemical alternative, but the dosage is limited to 40 ppm. At this trace dosage, the bleaching effect is quite restrained. In contrast, enzyme additives especially LOXs can replace chemicals to allow for the processing of flour, resulting in the bleaching of bread and its improved texture. In addition, lipid hydroperoxidases decolorize dough and oxidizes carotinoids, converting them into colorless compounds. This blanching of the dough results in lighter colored product, which is highly desired.

[0009] With regard to enzymes employed in the food industry, regulations frequently require enzymes to be recognized or proven as safe for use. In the case of lipoxygenases, considering that they are ubiquitously found in plants and consumed by humans and animals alike, plant lipoxygenases are considered safe for use, and therefore, of major value to the industry. Although soy extracts containing high levels of lipoxygenases have been used as an additive for bread manufacturing, soy produces an undesirable taste and smell and, accordingly, not often a useful option.

[0010] Because of plant LOX value, many attempts at high-level expression of recombinant plant derived LOX from soy, rice, potato and other sources by heterologous expression in microbial hosts including, but not limited to bacteria such as E. coli (BL21 strain), Bacilli, and in yeast has been attempted, though production was limited [3-8]. The best of these, although still a poor expression from E. coli, was observed at very cold temperatures [8]. Only one lipoxygenase was produced in Bacilli at high-levels (.about.160 mg/L), but the lipoxygenase was from a bacterial enzyme, not a plant and consequently not approved for use in the human food industry [9, 10]. In addition, yields still could not achieve desired levels. Accordingly, a need exists for high level expression of plant lipoxygenases that is generally recognized as safe for use in foods, and easily produced in large quantities.

SUMMARY OF THE INVENTION

[0011] The present invention overcomes the problems and disadvantages associated with current strategies and designs, and provides new methods and compositions involving the heterologous expression, purification and use of lipoxygenases.

[0012] One embodiment of the invention is directed to the heterologous expression of lipoxygenases in microbes.

[0013] Another embodiment of the invention is directed to methods for the purification of lipoxygenases, preferably from heterologous expression systems according to the invention.

[0014] Another embodiment of the invention is directed to lipoxygenase polypeptide and nucleic acids sequences and molecular constructs of lipoxygenase coding sequences, preferably for the high level expression of lipoxygenase as compared to expression in wild-type host cells. Preferably wild-type host cells are cells that do not contain a protease deficiency and/or cells that do not contain one or more chaperones.

[0015] Another embodiment of the invention is directed to methods for the manufacture of bread products comprising adding lipoxygenases of the invention to a dough containing unsaturated fatty acids and/or carotinoids. Preferably the lipoxygenase reacts with components of the flour forming lipid hydroperoxides increasing the stability of the dough and enhancing the volume of the baked goods.

[0016] Another embodiment of the invention is directed to purified lipoxygenase enzyme made by the methods of the invention. When the purified enzyme is added to dough, another embodiment of the invention comprises products made with the purified enzyme added to dough such as, preferably, bread products. The manufacture of bread products of the invention preferably comprises adding lipoxygenases to a dough containing unsaturated fatty acids and/or carotinoids. Preferably the lipoxygenase reacts with components of the flour forming lipid hydroperoxides increasing the stability of the dough and enhancing the volume of the baked goods.

[0017] Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the invention.

DESCRIPTION OF THE FIGURES

[0018] FIG. 1 Western analysis of SLP1 indicates varying profiles of degradation. M=marker. Different K12 cells with different genotypes are presented in sets of "U" (uninduced) and "I" (induced).

[0019] FIG. 2 Co-expression of the GroEL-GroES chaperone enhances SLP1 production. Four chaperones A-D were co-expressed in E. coli with SLP1. Left Panel: SLP1 is directed detected as a weak band in SDS-PAGE as a result of Co-expression with Chaperone B. Right Lane: Enhanced expression of SLP1 with co-expressed GroEL-GroES is confirmed by standard Western analysis.

[0020] FIG. 3 Single step purification of SLP1 from the 424 vector (native protein sequence, no polyhistidine tag). All lanes: SLP1 lacking a his tag was eluted from Zinc-NTA (IMAC) columns and run in SDS PAGE followed by protein staining: Lanes 1--crude lysate; Lane 2--column flow-through; Lane 3--column wash; Lanes 4-7--Zinc-NTA (IMAC) elution with SLP1 loaded in the presence of 0, 2, 5 and 10 mM imidazole, each eluted with 80 mM imidazole.

[0021] FIG. 4 E. coli cell lines used to verify experiments.

[0022] FIG. 5 SLP1 expression in E. coli; SDS PAGE protein gel of whole cell K12 E. coli lysate expressing SLP1.

DESCRIPTION OF THE INVENTION

[0023] Lipoxygenase enzymes (also referred to herein as LOX) are widely used in commercial processing of food products, the manufacture of perfumes and painting products, and in the processing of wood pulp. Although all lipoxygenase catalyze the same basic function, only plant lipoxygenases have been approved by the United States Food and Drug Administration for use in foods and food products. Despite their broad uses, lipoxygenase enzymes are only expressed at low levels and, consequently, commercial quantities are both expensive and difficult to produce.

[0024] Despite previous failures in achieving high level LOX expression, it has been surprisingly discovered that considerable enhancement of plant lipoxygenase expression can be achieved. At least part of this high-level of expression is attributed to the selection of sequences being expressed, expression of the sequences in a protease deficient host, and/or the co-expression with one or more chaperone plasmid sequences. Preferable, the increased expression achieved is at a higher level than expression in host cells that do not contain a protease deficiency and/or cells that do not contain one or more chaperone plasmids. Preferably the expression of the one or more proteases is eliminated, reduced to an undetectable level using conventional detection or reduced by at least 90%, all as compared to wild-type expression levels.

[0025] One embodiment of the invention comprises a system containing a bacterial cell host, preferably with a deficiency or one or more proteases, containing a coding sequence for lipoxygenase enzyme and preferably a chaperone system comprising one or more chaperone molecules. The system is preferably inducible and also preferably maintained from about 10.degree. C. to about 37.degree. C. for a period of time for maximal expression of enzyme product. The period of time is preferably from minutes to hours to days, and more preferably from about 1 to about 24 hours, more preferably from 2 to 12 hours and more preferably from about 2 to about 4 hours. The cells are preferably maintained at temperatures from about 15.degree. C. to about 25.degree. C. during this period.

[0026] The lipoxygenase enzyme may be derived from animal or bacterial cells, and is preferably derived from plant cells. Expression constructs may contain all or a portion of the lipoxygenase gene or coding region. Preferably constructs contain a portion of the coding region sufficient to create functional lipoxygenase activity. Preferably the constructs of the invention encode the sequences of SEQ ID NOs 1-3, or contain the nucleic acid sequences of SEQ ID NOs 4-6. Also preferably the sequence is a functional sequences that generates functional lipoxygenase activity.

[0027] Preferably the host cell is a microorganism that rapidly and economically proliferates in vitro such as, for example, one or more of the bacterial cell strains of K12 cells, E. coli cells, Bacillus cells, Lactococci or yeast cells. Also preferably, the host cells contain one or more protease deficiencies as compared to wild-type cells. For E. coli host cells, the deficiency is preferably of one or more of the proteases Lon, OMPT, and/or Lon/ClpP.

[0028] Preferably the host cells further contain one or more chaperone plasmid expression vectors. Chaperones function in assisting protein folding, benefiting the co-expressed molecules.

[0029] Expression of lipoxygenase in the systems of the invention typically involves inducing expression of the lipoxygenase sequence and also preferably the chaperone sequences before, during or after expression of the lipoxygenase, and preferably simultaneously or nearly simultaneously to allow for maximal expression of the enzyme.

[0030] Lipoxygenase produced according to methods of the invention can be further isolated and purified. Preferably, purification of lipoxygenase produced according to the methods of the invention involves contact the with immobilized-metal affinity chromatography media. The enzyme remains bound and can be washed with wash buffer and subsequently eluted with elution buffer.

[0031] Preferably the increased lipoxygenase expression of the invention is 5 fold greater as compared to expression in wild-type cells (e.g., cells that are not protease deficient and/or cells without one or more expression chaperones), more preferably 10 fold greater, more preferably 50 fold greater, more preferably 100 fold greater, more preferably 200 fold greater, more preferably 300 fold greater, more preferably 400 fold greater, and more preferably 500 fold greater or more.

[0032] Lipoxygenase made according to the invention is preferably useful in the manufacture of food products such as bread products (for either, or both bleaching and improving texture), the manufacture of paints thinners, perfumes, aroma and flavor enhancers, as signaling compounds, and for pitch control in softwood pulp in paper industry.

[0033] The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.

EXAMPLES

Example 1

LOXs Employed For Protein Production

[0034] SLP1 (seed linoleate 13S-lipoxygenase-1 [Glycine max] NCBI Reference Sequence: NP_001236153.1, length 839 amino acids) and SLP3 seed linoleate 9S-lipoxygenase-3 [Glycine max] NCBI Reference Sequence: NP_001235383.1) were employed as LOXs for production in microbes. In addition, a shortened version of SLP1 (herein minilox) from amino acid Serine 278 containing an additional methionine before the Serine 278 were cloned and expressed in microbes.

Example 2

Synthesis of DNA Encoding Protein Sequences for SLP1, SLP3 and Minilox Optimized For Expression

[0035] Optimal gene codon usage in plants and bacteria differ. New DNA encoding sequences for SLP1, SLP3 and minilox were determined and synthetically generated according to instructions (Genscript USA Inc.). The sequence for minilox was identical as that of SLP1 with the exception of having an ATG encoding for an initiator methionine prior to nucleotide bases encoding for SLP1 Serine 278. Optimized sequences with desired cloning sites were created.

Example 3

Cloning of Soybean Lipoxygenase 1 (SLP1) and 3 (SLP3) and MiniLox

[0036] Initially, SLP1 and SLP3 were cloned into the pET 47b vector (Novogen) using SmaI-XhoI restriction sites, so that each contained the pET47b initiating methionine and a 6X histidine tag (SEQ ID NO 7). The SLP1 and minilox encoding DNA were then transferred to the DNA2.0 expression vector 424 purple, a low copy number plasmid without the histidine tags using NdeI-Xhol sites, so that expressed proteins would not contain the histidine tags. Similarly, the SLP1 encoding sequence was cloned into the 424-purple vector (herein 424 vector) with the exception of using NdeI-EcoRI cloning sites. The SLP1 encoding sequences were then transferred to the DNA2.0 purple-444 vector (herein 444 vector), a high copy number plasmid using restriction sites Ndel-XhoI. The vectors contained promoters for expression of the insert DNA with the pET47 containing a T7-promotor, and the DNA2.0 vectors contained a T5-promotor.

Example 4

Expression of SLP1, Minilox and SLP3

[0037] Vectors were transfected into cell lines. Initially, expression of SLP1 was performed with the 6 histidine (SEQ ID NO 7) tagged SLP1 vector in E. coli BL21 cells, an E. coli B cell line suitable for the expression of the pET47b vectors. Thereafter, all expression was performed in E. coli K12 strains. Expression was tested in LB media, with 50-100 .mu.g/m1 ampicillin, and induction of expression for all vectors was with 0.5-1 mM IPTG.

Example 5

Activity Assay

[0038] The activity assay utilized linoleic acid as a substrate and colorimetric detection of product. Detected values for the assay varied depending on the substrate preparation, age of substrate, and substrate batch, which may be subject to variation due to oxidation from the environment. As such, approximate expression levels of SLP1 in BL21 are presented as 1 unit/cell OD550 SLP1-LB culture and relative and approximate values for expression in other strains is relative to the BL21 expression. Cell OD550 is defined as a cell density at OD550.

Example 6

Improvement of SLP1 Production

[0039] SLP1 expressed with or without a histidine tag using the pET47b vector and BL21 cells was very poor when induced at room temperature. The standard level of activity, 1 unit/OD cells was established for induction at 15.degree. C. with and overnight expression. Dramatically improved activity was observed using the purple-424 vector (herein 424 vector), in the K12 HMS 174 cell line (4 units/Cell OD550). Unlike BL21 cells, activity was also observed when induced at 20.degree. C.-25.degree. C. with overnight expression. Slp1 activity could be further enhanced by growing cells at 15.degree. C. for up to several days. In all E. coli strains tested, growth at 37C of was found to generate little or no SLP1 activity, and protein degradation products were observed upon western analysis (FIG. 2).

[0040] An additional increase in activity was discovered using protease deficient E. coli K12 strains with the 424 vector. Lon, OMPT, or Lon/ClpP mutants all showed a further minimum two-fold increase in activity with (.about.10 units/cell OD550). The specific E. coli cell lines with specific protease deficiencies also showed some similar characteristics of protein degradation (FIG. 3) yet some cell lines had less degradation than others, signifying that proteases play a role in the limited production of lipoxygenases.

[0041] An additional enhancement of activity was observed when using the 444 high plasmid copy vector in the K12 Pam155 (lon protease deficient) E. coli cell line and with chaperones. Chaperone plasmid sets consisting of five different plasmids from Takara Bio Inc. each designed to express a single or multiple molecular chaperone sets can enable optimal protein expression and folding and reduce protein misfolding. Each Takara plasmid carries an origin of replication (ORF) derived from pACYC and a chloramphenicol-resistance gene (Cmr) gene, which allows the use of E. coli expression systems containing ColE1-type plasmids that confer ampicillin resistance. The chaperone genes are situated downstream of the araB or Pzt-1 (tet) promoters and, as a result, expression of target proteins and chaperones can be individually induced if the target gene is placed under the control of different promoters (e.g., lac). These plasmids also contain the necessary regulator (araC or tet.sup.r) for each promoter. Takara Bio Inc. plasmids containing chaperones or sets thereof either tetracycline or arabinose inducible were coexpressed with SLP1. These include: groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, or tig in plasmids (TakaraBo Inc.). Expression of SLP1 in the presence of groES-groEL alone or with tig (groES-groEL-tig) enhanced the amount of active enzyme produced roughly to 40-60 units/cell OD. Activity was optimal at 15.degree. C. but also observed at or below 25.degree. C. At 37.degree. C., expression was more limited.

[0042] Expression of SLP1 in the Pam153 cell with concomitant GroESL chaperone expression, in LB, produces 68 micrograms of SLP1 per milliliter at a bacterial OD550 of 3, when grown in test tubes at 37.degree. C. and induced at 20.degree. C. overnight. However, SLP1 expression in an E. coli strain that is not a protease deficient strain and without chaperone expression can either not be detected at all with standard SDS PAGE analysis, or western analysis, or expresses less than 1 .mu.g per milliliter LB under similar conditions at an OD550 of 3 (see FIG. 4). In general, expression of SLP1 in E. coli strains grown and induced under optimal conditions was undetectable or less than 1 microgram per milliliter when appropriate chaperones were absent and strains were not protease deficient. However when expressing SLP1 in E. coli K12 protease deficient strains with co-expression of an appropriate chaperone, 68 micrograms of SLP1 per milliliter at a bacterial OD550 of 3 was attained.

Example 7

Purification of SPL1

[0043] Purification of SLP1 with the 6Xhis tag (SEQ ID NO 7) was highly effective using standard Ni-NTA IMAC purification. In the 424 or 444 vectors lacking the 6Xhis tag (SEQ ID NO 7), where SLP1 was encoded by the native SLP1 sequence alone, IMAC was equally efficient though under modified conditions. Nickel and zinc were each tested with similar results and calcium or other divalent metals should do as well. Buffers for IMAC were either 50 mM phosphate or Tris-HCl at pH 7-9, with 400 mM NaCl and 10% glycerol. Cells were disrupted using B-PER (Peirce) or by a homogenizer, in the presence of PMSF as a protease inhibitor. Employing Zinc-NTA, it was discovered that loading the sample in buffer with 10 mM imidazole and elution in buffer with 80 mM imidazole was effective in purification of SLP1. Other column media that effectively binds SLP1 include MonoQ and DEAE, but not negatively charged resins.

Example 8

Novel Information Provides Improved SLP1 Expression

[0044] Preliminary studies indicate that relatively poor production of SLP1 is the result of rapid proteolysis accompanied by improper folding of the enzyme. The limited soluble SLP1 and lack of insoluble protein suggests that most of the protein produced was rapidly degraded. Degradation products of SLP1 are visible in different E. coli strains with different protease deficient genetic backgrounds (see FIG. 1). An increase in both active enzyme and total protein was observed when inducing at suboptimal growth temperatures, where proteases are less functional. A relative increase in production and activity of SLP1 when protein folding is enhanced by an over-expressed chaperone.

Example 9

High Level Expression of Lipoxygenase in the E. Coli, K12,

[0045] Unless otherwise stated, all bacterial media employed in this example was Luria Broth (herein LB, consisting of 10 grams Tryptone, 5 grams Yeast Extract, and 10 grams NaCl, dissolved in 1 liter water, and sterilized for a minimum of 20 minutes in an autoclave). Soybean Lipoxygenase 1 (herein SLP1) was expressed from a plasmid transfected into E. Coli K12 cells. FIG. 5 represents an SDS-PAGE protein gel of whole cell soluble proteins extracted from the K12 cells employing the commercial B-PER Protein Extraction Reagent (Pierce, Cat#78243), following company protocols. The highest level of soluble SLP1 protein relative to total soluble protein in the cell extract was 30% or greater and approximated at 34% as estimated by the ImageJ (National Institute of Health public software) analysis software. These levels are consistent with high level production of the enzyme. M=marker, 1 Uninduced, 2 SLP1 induced with 0.5 mM IPTG 3&4 Induced with 0.5 mM IPTG and expressing a molecular chaperone.

CITED REFERENCES

[0046] 1. Permiakova, M. D. and V. A. Trufanov, Effect of soybean lipoxygenae on baking properties of wheat flour, Prikl Biokhim Mikrobiol, 2011. 47(3): p. 348-54.

[0047] 2. Permiakova, M. D., et al., [Role of lipoxygenase in the determination of wheat grain quality]. Prikl Biokhim Mikrobiol, 2010. 46(1): p. 96-102.

[0048] 3. Kanamoto, H., M. Takemura, and K. Ohyama, Cloning and expression of three lipoxygenase genes from liverwort, Marchantia polymorpha L., in Escherichia coli. Phytochemistry, 2012. 77: p. 70-8.

[0049] 4. Osipova, E. V., et al., Recombinant maize 9-lipoxygenase: expression, purification, and properties. Biochemistry Biokhimi, 2010. 75(7): p. 861-5.

[0050] 5. Hwang, I. S. and B. K. Hwang, The pepper 9-lipoxygenase gene CaLOX1 functions in defense and cell death responses to microbial pathogens. Plant Physiol, 2010. 152(2): p. 948-67.

[0051] 6. Padilla, M. N., et al., Functional characterization of two 13-lipoxygenase genes from olive fruit in relation to the biosynthesis of volatile compounds of virgin olive oil. J Agric Food Chem, 2009. 57(19): p. 9097-107.

[0052] 7. Knust, B. and D. von Wettstein, Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 1992. 37(3): p. 342-51.

[0053] 8. Steczko, J., et al., Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli. Protein Expr Purif, 1991. 2(2-3): p. 221-7.

Sequence Information

TABLE-US-00001

[0054] SLP1 polypeptide sequence (SEQ ID NO 1) MFSAGHKIKGTVVLMPKNELEVNPDGSAVDNLNAFLGRSVSLQLISAT KADAHGKGKVGKDTFLEGINTSLPTLGAGESAFNIHFEWDGSMGIPGA FYIKNYMQVEFFLKSLTLEAISNQGTIRFVCNSWVYNTKLYKSVRIFF ANHTYVPSETPAPLVSYREEELKSLRGNGTGERKEYDRIYDYDVYNDL GNPDKSEKLARPVLGGSSTFPYPRRGRTGRGPTVTDPNTEKQGEVFYV PRDENLGHLKSKDALEIGTKSLSQIVQPAFESAFDLKSTPIEFHSFQD VHDLYEGGIKLPRDVISTIIPLPVIKELYRTDGQHILKFPQPHVVQVS QSAWMTDEEFAREMIAGVNPCVIRGLEEFPPKSNLDPAIYGDQSSKIT ADSLDLDGYTMDEALGSRRLFMLDYHDIFMPYVRQINQLNSAKTYATR TILFLREDGTLKPVAIELSLPHSAGDLSAAVSQVVLPAKEGVESTIWL LAKAYVIVNDSCYHQLMSHWLNTHAAMEPFVIATHRHLSVLHPIYKLL TPHYRNNMNINALARQSLINANGIIETTFLPSKYSVEMSSAVYKNWVF TDQALPADLIKRGVAIKDPSTPHGVRLLIEDYPYAADGLEIWAAIKTW VQEYVPLYYARDDDVKNDSELQHWWKEAVEKGHGDLKDKPWWPKLQTL EDLVEVCLIIIWIASALHAAVNFGQYPYGGLIMNRPTASRRLLPEKGT PEYEEMINNHEKAYLRTITSKLPTLISLSVIEILSTHASDEVYLGQRD NPHWTSDSKALQAFQKFGNKLKEIEEKLVRRNNDPSLQGNRLGPVQLP YTLLYPSSEEGLTFRGIPNSISI SLP3 polypeptide sequence (SEQ ID NO 2) MLGGLLHRGHKIKGTVVLMRKNVLDVNSVTSVGGIIGQGLDLVGSTLD TLTAFLGRSVSLQLISATKADANGKGKLGKATFLEGIITSLPTLGAGQ SAFKINFEWDDGSGIPGAFYIKNFMQTEFFLVSLTLEDIPNHGSIHFV CNSWIYNAKLFKSDRIFFANQTYLPSETPAPLVKYREEELHNLRGDGT GERKEWERIYDYDVYNDLGDPDKGENHARPVLGGNDTFPYPRRGRTGR KPTRKDPNSESRSNDVYLPRDEAFGHLKSSDFLTYGLKSVSQNVLPLL QSAFDLNFTPREFDSFDEVHGLYSGGIKLPTDIISKISPLPVLKEIFR TDGEQALKFPPPKVIQVSKSAWMTDEEFAREMLAGVNPNLIRCLKDFP PRSKLDSQVYGDHTSQITKEHLEPNLEGLTVDEAIQNKRLFLLDHHDP IMPYLRRINATSTKAYATRTILFLKNDGTLRPLAIELSLPHPQGDQSG AFSQVFLPADEGVESSIWLLAKAYVVVNDSCYHQLVSHWLNTHAVVEP FIIATNRHLSVVHPIYKLLHPHYRDTMNINGLARLSLVNDGGVIEQTF LWGRYSVEMSAVVYKDWVFTDQALPADLIKRGMAIEDPSCPHGIRLVI EDYPYTVDGLEIWDAIKTWVHEYVFLYYKSDDTLREDPELQACWKELV EVGHGDKKNEPWWPKMQTREELVEACAIIIWTASALHAAVNFGQYPYG GLILNRPTLSRRFMPEKGSAEYEELRKNPQKAYLKTITPKFQTLIDLS VIEILSRHASDEVYLGERDNPNWTSDTRALEAFKRFGNKLAQIENKLS ERNNDEKLRNRCGPVQMPYTLLLPSSKEGLTFRGIPNSISI Minilox1 polypeptide sequence (SEQ ID NO 3) MSTPIEFHSFQDVHDLYEGGIKLPRDVISTIIPLPVIKELYRTDGQHI LKFPQPHVVQVSQSAWMTDEEFAREMIAGVNPCVIRGLEEFPPKSNLD PAIYGDQSSKITADSLDLDGYTMDEALGSRRLFMLDYHDIFMPYVRQI NQLNSAKTYATRTILFLREDGTLKPVAIELSLPHSAGDLSAAVSQVVL PAKEGVESTIWLLAKAYVIVNDSCYHQLMSHWLNTHAAMEPFVIATHR HLSVLHPIYKLLTPHYRNNMNINALARQSLINANGIIETTFLPSKYSV EMSSAVYKNWVFTDQALPADLIKRGVAIKDPSTPHGVRLLIEDYPYAA DGLEIWAAIKTWVQEYVPLYYARDDDVKNDSELQHWWKEAVEKGHGDL KDKPWWPKLQTLEDLVEVCLIIIWIASALHAAVNFGQYPYGGLIMNRP TASRRLLPEKGTPEYEEMINNHEKAYLRTITSKLPTLISLSVIEILST HASDEVYLGQRDNPHWTSDSKALQAFQKFGNKLKEIEEKLVRRNNDPS LQGNRLGPVQLPYTLLYPSSEEGLTFRGIPNSISI SLP1 DNA optimized encoding sequence (with restriction sites 5' SmaI and 3' XhoI with stop codon for cloning into pET47b with 6X histidine tag (SEQ ID NO 7)) (SEQ ID NO 4) CCCGGGATGTTTAGTGCTGGTCACAAAATCAAAGGTACCGTGGTCCTG ATGCCGAAAAATGAACTGGAAGTCAACCCGGATGGTAGCGCCGTTGAT AACCTGAATGCGTTCCTGGGTCGTAGCGTGTCTCTGCAGCTGATTTCC GCCACCAAAGCAGACGCTCACGGCAAGGGTAAAGTTGGCAAAGATACG TTTCTGGAAGGTATTAATACCTCCCTGCCGACCCTGGGTGCCGGTGAA TCAGCTTTCAACATCCATTTCGAATGGGATGGTTCAATGGGCATTCCG GGCGCCTTCTACATCAAAAACTACATGCAGGTGGAATTTTTCCTGAAA AGTCTGACCCTGGAAGCAATCTCCAATCAGGGTACGATTCGTTTTGTC TGCAACTCGTGGGTGTATAATACCAAACTGTACAAAAGCGTTCGCATC TTTTTCGCGAACCACACCTATGTTCCGAGCGAAACCCCGGCACCGCTG GTTTCTTACCGTGAAGAAGAACTGAAAAGTCTGCGCGGCAATGGTACC GGCGAACGTAAAGAATATGATCGCATTTATGACTACGATGTTTACAAC GACCTGGGCAATCCGGATAAAAGCGAAAAACTGGCCCGTCCGGTCCTG GGCGGTAGCTCTACCTTCCCGTATCCGCGTCGCGGTCGTACCGGTCGT GGTCCGACCGTGACCGATCCGAACACCGAAAAACAGGGCGAAGTCTTT TATGTGCCGCGCGACGAAAATCTGGGCCATCTGAAATCTAAAGATGCC CTGGAAATCGGTACCAAAAGTCTGTCCCAGATTGTGCAACCGGCGTTT GAAAGCGCCTTCGATCTGAAATCTACGCCGATTGAATTTCACTCCTTC CAGGACGTTCATGATCTGTATGAAGGCGGTATCAAACTGCCGCGTGAC GTCATTTCAACCATTATCCCGCTGCCGGTGATCAAAGAACTGTACCGC ACGGATGGTCAGCACATTCTGAAATTTCCGCAACCGCATGTGGTTCAG GTTTCACAATCGGCGTGGATGACCGATGAAGAATTCGCGCGTGAAATG ATCGCCGGCGTTAACCCGTGCGTCATTCGCGGTCTGGAAGAATTTCCG CCGAAAAGCAATCTGGACCCGGCAATCTATGGCGATCAGAGTTCCAAA ATTACCGCTGACTCTCTGGACCTGGATGGCTACACGATGGATGAAGCC CTGGGTAGTCGTCGCCTGTTTATGCTGGACTATCACGATATCTTCATG CCGTACGTGCGTCAGATTAACCAACTGAATTCTGCAAAAACCTATGCT ACCCGTACGATCCTGTTTCTGCGCGAAGACGGCACGCTGAAACCGGTT GCAATTGAACTGAGCCTGCCGCATTCTGCTGGTGATCTGAGTGCCGCG GTGTCCCAGGTTGTGCTGCCGGCAAAAGAAGGCGTTGAAAGTACCATC TGGCTGCTGGCGAAAGCCTATGTTATTGTCAACGATTCATGTTACCAT CAACTGATGTCGCACTGGCTGAATACCCATGCAGCTATGGAACCGTTT GTTATCGCAACGCATCGCCACCTGTCTGTCCTGCACCCGATTTATAAA CTGCTGACCCCGCATTACCGTAACAATATGAACATCAATGCACTGGCT CGCCAGAGTCTGATTAACGCGAATGGTATTATCGAAACCACGTTCCTG CCGTCAAAATATTCGGTGGAAATGTCATCGGCCGTTTACAAAAACTGG GTCTTTACCGACCAGGCACTGCCGGCTGATCTGATCAAACGTGGCGTC GCGATTAAAGATCCGAGCACCCCGCATGGTGTGCGTCTGCTGATTGAA GACTATCCGTACGCGGCCGATGGCCTGGAAATCTGGGCAGCTATTAAA ACCTGGGTGCAGGAATATGTTCCGCTGTATTACGCACGCGATGACGAT GTGAAAAATGACTCCGAACTGCAACACTGGTGGAAAGAAGCTGTTGAA AAAGGTCATGGCGACCTGAAAGATAAACCGTGGTGGCCGAAACTGCAG ACCCTGGAAGATCTGGTGGAAGTTTGTCTGATTATCATTTGGATTGCC AGCGCACTGCATGCCGCGGTGAACTTTGGTCAATATCCGTACGGCGGT CTGATTATGAATCGTCCGACCGCAAGCCGTCGCCTGCTGCCGGAAAAA GGCACGCCGGAATACGAAGAAATGATCAACAACCATGAAAAAGCGTAC CTGCGCACCATCACGAGCAAACTGCCGACCCTGATTAGCCTGTCTGTT ATCGAAATTCTGTCAACGCACGCGTCGGATGAAGTCTATCTGGGTCAG CGTGACAACCCGCATTGGACCAGTGATTCCAAAGCGCTGCAGGCCTTC CAAAAATTCGGCAACAAACTGAAAGAAATCGAAGAAAAACTGGTCCGT CGCAACAATGATCCGAGCCTGCAGGGTAACCGTCTGGGTCCGGTGCAA CTGCCGTATACCCTGCTGTATCCGTCCAGTGAAGAAGGTCTGACGTTT CGTGGTATTCCGAACTCCATTTCCATCTGACTCGAG SLP3 DNA optimized encoding sequence (with restriction sites 5' NdeI and 3' EcoRI and 3' stop codon for cloning into the pJex purple 424 vector from DNA2.0 Inc. (SEQ ID NO 5) CATATGCTGGGCGGCCTGCTGCACCGTGGTCATAAAATCAAGGGCACC GTGGTCCTGATGCGTAAGAACGTCCTGGATGTGAATAGCGTGACCTCG GTCGGCGGTATTATCGGCCAGGGTCTGGACCTGGTGGGTAGCACGCTG GATACCCTGACGGCCTTTCTGGGCCGCTCAGTGTCGCTGCAACTGATC AGCGCAACCAAAGCAGATGCTAACGGCAAAGGCAAGCTGGGCAAGGCG ACGTTCCTGGAAGGCATTATCACCTCCCTGCCGACGCTGGGTGCAGGC CAGTCAGCCTTTAAAATTAATTTCGAATGGGATGACGGCTCTGGTATT CCGGGCGCCTTCTACATCAAGAACTTCATGCAGACCGAATTTTTCCTG GTCAGCCTGACGCTGGAAGATATCCCGAATCATGGCTCGATTCACTTT GTGTGCAACAGCTGGATCTACAATGCGAAACTGTTCAAGTCCGATCGC ATTTTCTTTGCCAATCAGACCTATCTGCCGTCAGAAACGCCGGCACCG CTGGTTAAATACCGTGAAGAAGAACTGCACAACCTGCGTGGTGACGGT ACCGGTGAACGTAAAGAATGGGAACGCATCTACGATTACGACGTTTAC AACGATCTGGGTGATCCGGACAAAGGCGAAAACCATGCGCGTCCGGTC CTGGGCGGTAATGACACCTTTCCGTACCCGCGTCGCGGTCGTACCGGT CGTAAACCGACGCGTAAGGATCCGAACAGCGAATCTCGCAGTAATGAT

GTGTATCTGCCGCGTGACGAAGCCTTTGGTCACCTGAAAAGCTCTGAT TTCCTGACGTACGGCCTGAAGTCCGTTTCACAGAACGTCCTGCCGCTG CTGCAAAGCGCATTTGATCTGAATTTCACCCCGCGCGAATTTGATTCG TTCGACGAAGTTCATGGTCTGTATAGCGGCGGTATTAAGCTGCCGACC GACATTATCTCTAAAATCAGTCCGCTGCCGGTGCTGAAGGAAATTTTT CGCACGGATGGCGAACAGGCTCTGAAGTTCCCGCCGCCGAAAGTCATC CAAGTGTCGAAAAGCGCGTGGATGACCGATGAAGAATTTGCACGTGAA ATGCTGGCTGGTGTTAACCCGAATCTGATTCGCTGTCTGAAGGATTTC CCGCCGCGTTCCAAACTGGATTCACAGGTGTATGGTGACCACACCAGT CAAATCACGAAAGAACATCTGGAACCGAACCTGGAAGGCCTGACCGTT GATGAAGCTATTCAGAATAAACGTCTGTTTCTGCTGGATCATCACGAC CCGATCATGCCGTATCTGCGTCGCATTAATGCGACCTCGACGAAAGCG TACGCCACCCGCACGATCCTGTTCCTGAAGAACGATGGTACCCTGCGT CCGCTGGCCATTGAACTGAGCCTGCCGCATCCGCAGGGTGACCAATCG GGTGCGTTTAGCCAGGTTTTCCTGCCGGCCGATGAAGGCGTCGAAAGT TCCATCTGGCTGCTGGCAAAAGCTTATGTGGTTGTCAACGATTCTTGC TACCATCAGCTGGTGTCTCACTGGCTGAATACCCATGCAGTGGTTGAA CCGTTTATTATCGCTACGAACCGCCACCTGTCTGTCGTGCATCCGATC TATAAACTGCTGCATCCGCACTACCGCGACACCATGAACATTAATGGT CTGGCGCGTCTGAGTCTGGTCAACGATGGCGGTGTGATTGAACAGACG TTTCTGTGGGGCCGTTATTCTGTTGAAATGAGTGCCGTTGTCTACAAA GATTGGGTCTTCACCGACCAAGCACTGCCGGCAGACCTGATCAAGCGT GGTATGGCAATTGAAGATCCGTCCTGTCCGCACGGCATCCGTCTGGTG ATTGAAGATTATCCGTACACCGTTGACGGTCTGGAAATCTGGGATGCA ATTAAAACGTGGGTGCATGAATACGTTTTTCTGTACTACAAGTCTGAT GACACCCTGCGCGAAGACCCGGAACTGCAGGCGTGCTGGAAAGAACTG GTGGAAGTTGGTCACGGCGATAAAAAGAACGAACCGTGGTGGCCGAAA ATGCAAACCCGTGAAGAACTGGTTGAAGCGTGTGCCATTATCATTTGG ACGGCAAGCGCTCTGCATGCGGCCGTGAACTTTGGCCAGTATCCGTAC GGCGGTCTGATTCTGAATCGCCCGACCCTGTCTCGTCGCTTCATGCCG GAAAAAGGCAGTGCTGAATATGAAGAACTGCGTAAAAATCCGCAGAAG GCGTACCTGAAAACCATCACGCCGAAATTTCAAACCCTGATTGACCTG AGCGTGATCGAAATTCTGTCCCGCCATGCGTCAGATGAAGTTTATCTG GGTGAACGTGACAACCCGAATTGGACCTCCGATACGCGTGCACTGGAA GCTTTTAAGCGCTTCGGCAACAAACTGGCCCAGATCGAAAACAAGCTG TCAGAACGTAACAACGATGAAAAGCTGCGTAATCGCTGCGGCCCGGTG CAAATGCCGTATACCCTGCTGCTGCCGTCCTCAAAAGAAGGTCTGACG TTCCGTGGTATCCCGAATAGCATTAGCATCTAAGAATTC Minilox optimized encoding sequence (with 5' NdeI and 3' XhoI restriction sites and 3' stop codon for cloning into pJexpress purple 424 vector from DNA2.0 Inc.) (SEQ ID NO 6) CATATGTCTACGCCGATTGAATTTCACTCCTTCCAGGACGTTCATGAT CTGTATGAAGGCGGTATCAAACTGCCGCGTGACGTCATTTCAACCATT ATCCCGCTGCCGGTGATCAAAGAACTGTACCGCACGGATGGTCAGCAC ATTCTGAAATTTCCGCAACCGCATGTGGTTCAGGTTTCACAATCGGCG TGGATGACCGATGAAGAATTCGCGCGTGAAATGATCGCCGGCGTTAAC CCGTGCGTCATTCGCGGTCTGGAAGAATTTCCGCCGAAAAGCAATCTG GACCCGGCAATCTATGGCGATCAGAGTTCCAAAATTACCGCTGACTCT CTGGACCTGGATGGCTACACGATGGATGAAGCCCTGGGTAGTCGTCGC CTGTTTATGCTGGACTATCACGATATCTTCATGCCGTACGTGCGTCAG ATTAACCAACTGAATTCTGCAAAAACCTATGCTACCCGTACGATCCTG TTTCTGCGCGAAGACGGCACGCTGAAACCGGTTGCAATTGAACTGAGC CTGCCGCATTCTGCTGGTGATCTGAGTGCCGCGGTGTCCCAGGTTGTG CTGCCGGCAAAAGAAGGCGTTGAAAGTACCATCTGGCTGCTGGCGAAA GCCTATGTTATTGTCAACGATTCATGTTACCATCAACTGATGTCGCAC TGGCTGAATACCCATGCAGCTATGGAACCGTTTGTTATCGCAACGCAT CGCCACCTGTCTGTCCTGCACCCGATTTATAAACTGCTGACCCCGCAT TACCGTAACAATATGAACATCAATGCACTGGCTCGCCAGAGTCTGATT AACGCGAATGGTATTATCGAAACCACGTTCCTGCCGTCAAAATATTCG GTGGAAATGTCATCGGCCGTTTACAAAAACTGGGTCTTTACCGACCAG GCACTGCCGGCTGATCTGATCAAACGTGGCGTCGCGATTAAAGATCCG AGCACCCCGCATGGTGTGCGTCTGCTGATTGAAGACTATCCGTACGCG GCCGATGGCCTGGAAATCTGGGCAGCTATTAAAACCTGGGTGCAGGAA TATGTTCCGCTGTATTACGCACGCGATGACGATGTGAAAAATGACTCC GAACTGCAACACTGGTGGAAAGAAGCTGTTGAAAAAGGTCATGGCGAC CTGAAAGATAAACCGTGGTGGCCGAAACTGCAGACCCTGGAAGATCTG GTGGAAGTTTGTCTGATTATCATTTGGATTGCCAGCGCACTGCATGCC GCGGTGAACTTTGGTCAATATCCGTACGGCGGTCTGATTATGAATCGT CCGACCGCAAGCCGTCGCCTGCTGCCGGAAAAAGGCACGCCGGAATAC GAAGAAATGATCAACAACCATGAAAAAGCGTACCTGCGCACCATCACG AGCAAACTGCCGACCCTGATTAGCCTGTCTGTTATCGAAATTCTGTCA ACGCACGCGTCGGATGAAGTCTATCTGGGTCAGCGTGACAACCCGCAT TGGACCAGTGATTCCAAAGCGCTGCAGGCCTTCCAAAAATTCGGCAAC AAACTGAAAGAAATCGAAGAAAAACTGGTCCGTCGCAACAATGATCCG AGCCTGCAGGGTAACCGTCTGGGTCCGGTGCAACTGCCGTATACCCTG CTGTATCCGTCCAGTGAAGAAGGTCTGACGTTTCGTGGTATTCCGAAC TCCATTTCCATCTGACTCGAG

[0055] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.

Sequence CWU 1

1

71839PRTGlycine max 1Met Phe Ser Ala Gly His Lys Ile Lys Gly Thr Val Val Leu Met Pro 1 5 10 15 Lys Asn Glu Leu Glu Val Asn Pro Asp Gly Ser Ala Val Asp Asn Leu 20 25 30 Asn Ala Phe Leu Gly Arg Ser Val Ser Leu Gln Leu Ile Ser Ala Thr 35 40 45 Lys Ala Asp Ala His Gly Lys Gly Lys Val Gly Lys Asp Thr Phe Leu 50 55 60 Glu Gly Ile Asn Thr Ser Leu Pro Thr Leu Gly Ala Gly Glu Ser Ala 65 70 75 80 Phe Asn Ile His Phe Glu Trp Asp Gly Ser Met Gly Ile Pro Gly Ala 85 90 95 Phe Tyr Ile Lys Asn Tyr Met Gln Val Glu Phe Phe Leu Lys Ser Leu 100 105 110 Thr Leu Glu Ala Ile Ser Asn Gln Gly Thr Ile Arg Phe Val Cys Asn 115 120 125 Ser Trp Val Tyr Asn Thr Lys Leu Tyr Lys Ser Val Arg Ile Phe Phe 130 135 140 Ala Asn His Thr Tyr Val Pro Ser Glu Thr Pro Ala Pro Leu Val Ser 145 150 155 160 Tyr Arg Glu Glu Glu Leu Lys Ser Leu Arg Gly Asn Gly Thr Gly Glu 165 170 175 Arg Lys Glu Tyr Asp Arg Ile Tyr Asp Tyr Asp Val Tyr Asn Asp Leu 180 185 190 Gly Asn Pro Asp Lys Ser Glu Lys Leu Ala Arg Pro Val Leu Gly Gly 195 200 205 Ser Ser Thr Phe Pro Tyr Pro Arg Arg Gly Arg Thr Gly Arg Gly Pro 210 215 220 Thr Val Thr Asp Pro Asn Thr Glu Lys Gln Gly Glu Val Phe Tyr Val 225 230 235 240 Pro Arg Asp Glu Asn Leu Gly His Leu Lys Ser Lys Asp Ala Leu Glu 245 250 255 Ile Gly Thr Lys Ser Leu Ser Gln Ile Val Gln Pro Ala Phe Glu Ser 260 265 270 Ala Phe Asp Leu Lys Ser Thr Pro Ile Glu Phe His Ser Phe Gln Asp 275 280 285 Val His Asp Leu Tyr Glu Gly Gly Ile Lys Leu Pro Arg Asp Val Ile 290 295 300 Ser Thr Ile Ile Pro Leu Pro Val Ile Lys Glu Leu Tyr Arg Thr Asp 305 310 315 320 Gly Gln His Ile Leu Lys Phe Pro Gln Pro His Val Val Gln Val Ser 325 330 335 Gln Ser Ala Trp Met Thr Asp Glu Glu Phe Ala Arg Glu Met Ile Ala 340 345 350 Gly Val Asn Pro Cys Val Ile Arg Gly Leu Glu Glu Phe Pro Pro Lys 355 360 365 Ser Asn Leu Asp Pro Ala Ile Tyr Gly Asp Gln Ser Ser Lys Ile Thr 370 375 380 Ala Asp Ser Leu Asp Leu Asp Gly Tyr Thr Met Asp Glu Ala Leu Gly 385 390 395 400 Ser Arg Arg Leu Phe Met Leu Asp Tyr His Asp Ile Phe Met Pro Tyr 405 410 415 Val Arg Gln Ile Asn Gln Leu Asn Ser Ala Lys Thr Tyr Ala Thr Arg 420 425 430 Thr Ile Leu Phe Leu Arg Glu Asp Gly Thr Leu Lys Pro Val Ala Ile 435 440 445 Glu Leu Ser Leu Pro His Ser Ala Gly Asp Leu Ser Ala Ala Val Ser 450 455 460 Gln Val Val Leu Pro Ala Lys Glu Gly Val Glu Ser Thr Ile Trp Leu 465 470 475 480 Leu Ala Lys Ala Tyr Val Ile Val Asn Asp Ser Cys Tyr His Gln Leu 485 490 495 Met Ser His Trp Leu Asn Thr His Ala Ala Met Glu Pro Phe Val Ile 500 505 510 Ala Thr His Arg His Leu Ser Val Leu His Pro Ile Tyr Lys Leu Leu 515 520 525 Thr Pro His Tyr Arg Asn Asn Met Asn Ile Asn Ala Leu Ala Arg Gln 530 535 540 Ser Leu Ile Asn Ala Asn Gly Ile Ile Glu Thr Thr Phe Leu Pro Ser 545 550 555 560 Lys Tyr Ser Val Glu Met Ser Ser Ala Val Tyr Lys Asn Trp Val Phe 565 570 575 Thr Asp Gln Ala Leu Pro Ala Asp Leu Ile Lys Arg Gly Val Ala Ile 580 585 590 Lys Asp Pro Ser Thr Pro His Gly Val Arg Leu Leu Ile Glu Asp Tyr 595 600 605 Pro Tyr Ala Ala Asp Gly Leu Glu Ile Trp Ala Ala Ile Lys Thr Trp 610 615 620 Val Gln Glu Tyr Val Pro Leu Tyr Tyr Ala Arg Asp Asp Asp Val Lys 625 630 635 640 Asn Asp Ser Glu Leu Gln His Trp Trp Lys Glu Ala Val Glu Lys Gly 645 650 655 His Gly Asp Leu Lys Asp Lys Pro Trp Trp Pro Lys Leu Gln Thr Leu 660 665 670 Glu Asp Leu Val Glu Val Cys Leu Ile Ile Ile Trp Ile Ala Ser Ala 675 680 685 Leu His Ala Ala Val Asn Phe Gly Gln Tyr Pro Tyr Gly Gly Leu Ile 690 695 700 Met Asn Arg Pro Thr Ala Ser Arg Arg Leu Leu Pro Glu Lys Gly Thr 705 710 715 720 Pro Glu Tyr Glu Glu Met Ile Asn Asn His Glu Lys Ala Tyr Leu Arg 725 730 735 Thr Ile Thr Ser Lys Leu Pro Thr Leu Ile Ser Leu Ser Val Ile Glu 740 745 750 Ile Leu Ser Thr His Ala Ser Asp Glu Val Tyr Leu Gly Gln Arg Asp 755 760 765 Asn Pro His Trp Thr Ser Asp Ser Lys Ala Leu Gln Ala Phe Gln Lys 770 775 780 Phe Gly Asn Lys Leu Lys Glu Ile Glu Glu Lys Leu Val Arg Arg Asn 785 790 795 800 Asn Asp Pro Ser Leu Gln Gly Asn Arg Leu Gly Pro Val Gln Leu Pro 805 810 815 Tyr Thr Leu Leu Tyr Pro Ser Ser Glu Glu Gly Leu Thr Phe Arg Gly 820 825 830 Ile Pro Asn Ser Ile Ser Ile 835 2857PRTGlycine max 2Met Leu Gly Gly Leu Leu His Arg Gly His Lys Ile Lys Gly Thr Val 1 5 10 15 Val Leu Met Arg Lys Asn Val Leu Asp Val Asn Ser Val Thr Ser Val 20 25 30 Gly Gly Ile Ile Gly Gln Gly Leu Asp Leu Val Gly Ser Thr Leu Asp 35 40 45 Thr Leu Thr Ala Phe Leu Gly Arg Ser Val Ser Leu Gln Leu Ile Ser 50 55 60 Ala Thr Lys Ala Asp Ala Asn Gly Lys Gly Lys Leu Gly Lys Ala Thr 65 70 75 80 Phe Leu Glu Gly Ile Ile Thr Ser Leu Pro Thr Leu Gly Ala Gly Gln 85 90 95 Ser Ala Phe Lys Ile Asn Phe Glu Trp Asp Asp Gly Ser Gly Ile Pro 100 105 110 Gly Ala Phe Tyr Ile Lys Asn Phe Met Gln Thr Glu Phe Phe Leu Val 115 120 125 Ser Leu Thr Leu Glu Asp Ile Pro Asn His Gly Ser Ile His Phe Val 130 135 140 Cys Asn Ser Trp Ile Tyr Asn Ala Lys Leu Phe Lys Ser Asp Arg Ile 145 150 155 160 Phe Phe Ala Asn Gln Thr Tyr Leu Pro Ser Glu Thr Pro Ala Pro Leu 165 170 175 Val Lys Tyr Arg Glu Glu Glu Leu His Asn Leu Arg Gly Asp Gly Thr 180 185 190 Gly Glu Arg Lys Glu Trp Glu Arg Ile Tyr Asp Tyr Asp Val Tyr Asn 195 200 205 Asp Leu Gly Asp Pro Asp Lys Gly Glu Asn His Ala Arg Pro Val Leu 210 215 220 Gly Gly Asn Asp Thr Phe Pro Tyr Pro Arg Arg Gly Arg Thr Gly Arg 225 230 235 240 Lys Pro Thr Arg Lys Asp Pro Asn Ser Glu Ser Arg Ser Asn Asp Val 245 250 255 Tyr Leu Pro Arg Asp Glu Ala Phe Gly His Leu Lys Ser Ser Asp Phe 260 265 270 Leu Thr Tyr Gly Leu Lys Ser Val Ser Gln Asn Val Leu Pro Leu Leu 275 280 285 Gln Ser Ala Phe Asp Leu Asn Phe Thr Pro Arg Glu Phe Asp Ser Phe 290 295 300 Asp Glu Val His Gly Leu Tyr Ser Gly Gly Ile Lys Leu Pro Thr Asp 305 310 315 320 Ile Ile Ser Lys Ile Ser Pro Leu Pro Val Leu Lys Glu Ile Phe Arg 325 330 335 Thr Asp Gly Glu Gln Ala Leu Lys Phe Pro Pro Pro Lys Val Ile Gln 340 345 350 Val Ser Lys Ser Ala Trp Met Thr Asp Glu Glu Phe Ala Arg Glu Met 355 360 365 Leu Ala Gly Val Asn Pro Asn Leu Ile Arg Cys Leu Lys Asp Phe Pro 370 375 380 Pro Arg Ser Lys Leu Asp Ser Gln Val Tyr Gly Asp His Thr Ser Gln 385 390 395 400 Ile Thr Lys Glu His Leu Glu Pro Asn Leu Glu Gly Leu Thr Val Asp 405 410 415 Glu Ala Ile Gln Asn Lys Arg Leu Phe Leu Leu Asp His His Asp Pro 420 425 430 Ile Met Pro Tyr Leu Arg Arg Ile Asn Ala Thr Ser Thr Lys Ala Tyr 435 440 445 Ala Thr Arg Thr Ile Leu Phe Leu Lys Asn Asp Gly Thr Leu Arg Pro 450 455 460 Leu Ala Ile Glu Leu Ser Leu Pro His Pro Gln Gly Asp Gln Ser Gly 465 470 475 480 Ala Phe Ser Gln Val Phe Leu Pro Ala Asp Glu Gly Val Glu Ser Ser 485 490 495 Ile Trp Leu Leu Ala Lys Ala Tyr Val Val Val Asn Asp Ser Cys Tyr 500 505 510 His Gln Leu Val Ser His Trp Leu Asn Thr His Ala Val Val Glu Pro 515 520 525 Phe Ile Ile Ala Thr Asn Arg His Leu Ser Val Val His Pro Ile Tyr 530 535 540 Lys Leu Leu His Pro His Tyr Arg Asp Thr Met Asn Ile Asn Gly Leu 545 550 555 560 Ala Arg Leu Ser Leu Val Asn Asp Gly Gly Val Ile Glu Gln Thr Phe 565 570 575 Leu Trp Gly Arg Tyr Ser Val Glu Met Ser Ala Val Val Tyr Lys Asp 580 585 590 Trp Val Phe Thr Asp Gln Ala Leu Pro Ala Asp Leu Ile Lys Arg Gly 595 600 605 Met Ala Ile Glu Asp Pro Ser Cys Pro His Gly Ile Arg Leu Val Ile 610 615 620 Glu Asp Tyr Pro Tyr Thr Val Asp Gly Leu Glu Ile Trp Asp Ala Ile 625 630 635 640 Lys Thr Trp Val His Glu Tyr Val Phe Leu Tyr Tyr Lys Ser Asp Asp 645 650 655 Thr Leu Arg Glu Asp Pro Glu Leu Gln Ala Cys Trp Lys Glu Leu Val 660 665 670 Glu Val Gly His Gly Asp Lys Lys Asn Glu Pro Trp Trp Pro Lys Met 675 680 685 Gln Thr Arg Glu Glu Leu Val Glu Ala Cys Ala Ile Ile Ile Trp Thr 690 695 700 Ala Ser Ala Leu His Ala Ala Val Asn Phe Gly Gln Tyr Pro Tyr Gly 705 710 715 720 Gly Leu Ile Leu Asn Arg Pro Thr Leu Ser Arg Arg Phe Met Pro Glu 725 730 735 Lys Gly Ser Ala Glu Tyr Glu Glu Leu Arg Lys Asn Pro Gln Lys Ala 740 745 750 Tyr Leu Lys Thr Ile Thr Pro Lys Phe Gln Thr Leu Ile Asp Leu Ser 755 760 765 Val Ile Glu Ile Leu Ser Arg His Ala Ser Asp Glu Val Tyr Leu Gly 770 775 780 Glu Arg Asp Asn Pro Asn Trp Thr Ser Asp Thr Arg Ala Leu Glu Ala 785 790 795 800 Phe Lys Arg Phe Gly Asn Lys Leu Ala Gln Ile Glu Asn Lys Leu Ser 805 810 815 Glu Arg Asn Asn Asp Glu Lys Leu Arg Asn Arg Cys Gly Pro Val Gln 820 825 830 Met Pro Tyr Thr Leu Leu Leu Pro Ser Ser Lys Glu Gly Leu Thr Phe 835 840 845 Arg Gly Ile Pro Asn Ser Ile Ser Ile 850 855 3563PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 3Met Ser Thr Pro Ile Glu Phe His Ser Phe Gln Asp Val His Asp Leu 1 5 10 15 Tyr Glu Gly Gly Ile Lys Leu Pro Arg Asp Val Ile Ser Thr Ile Ile 20 25 30 Pro Leu Pro Val Ile Lys Glu Leu Tyr Arg Thr Asp Gly Gln His Ile 35 40 45 Leu Lys Phe Pro Gln Pro His Val Val Gln Val Ser Gln Ser Ala Trp 50 55 60 Met Thr Asp Glu Glu Phe Ala Arg Glu Met Ile Ala Gly Val Asn Pro 65 70 75 80 Cys Val Ile Arg Gly Leu Glu Glu Phe Pro Pro Lys Ser Asn Leu Asp 85 90 95 Pro Ala Ile Tyr Gly Asp Gln Ser Ser Lys Ile Thr Ala Asp Ser Leu 100 105 110 Asp Leu Asp Gly Tyr Thr Met Asp Glu Ala Leu Gly Ser Arg Arg Leu 115 120 125 Phe Met Leu Asp Tyr His Asp Ile Phe Met Pro Tyr Val Arg Gln Ile 130 135 140 Asn Gln Leu Asn Ser Ala Lys Thr Tyr Ala Thr Arg Thr Ile Leu Phe 145 150 155 160 Leu Arg Glu Asp Gly Thr Leu Lys Pro Val Ala Ile Glu Leu Ser Leu 165 170 175 Pro His Ser Ala Gly Asp Leu Ser Ala Ala Val Ser Gln Val Val Leu 180 185 190 Pro Ala Lys Glu Gly Val Glu Ser Thr Ile Trp Leu Leu Ala Lys Ala 195 200 205 Tyr Val Ile Val Asn Asp Ser Cys Tyr His Gln Leu Met Ser His Trp 210 215 220 Leu Asn Thr His Ala Ala Met Glu Pro Phe Val Ile Ala Thr His Arg 225 230 235 240 His Leu Ser Val Leu His Pro Ile Tyr Lys Leu Leu Thr Pro His Tyr 245 250 255 Arg Asn Asn Met Asn Ile Asn Ala Leu Ala Arg Gln Ser Leu Ile Asn 260 265 270 Ala Asn Gly Ile Ile Glu Thr Thr Phe Leu Pro Ser Lys Tyr Ser Val 275 280 285 Glu Met Ser Ser Ala Val Tyr Lys Asn Trp Val Phe Thr Asp Gln Ala 290 295 300 Leu Pro Ala Asp Leu Ile Lys Arg Gly Val Ala Ile Lys Asp Pro Ser 305 310 315 320 Thr Pro His Gly Val Arg Leu Leu Ile Glu Asp Tyr Pro Tyr Ala Ala 325 330 335 Asp Gly Leu Glu Ile Trp Ala Ala Ile Lys Thr Trp Val Gln Glu Tyr 340 345 350 Val Pro Leu Tyr Tyr Ala Arg Asp Asp Asp Val Lys Asn Asp Ser Glu 355 360 365 Leu Gln His Trp Trp Lys Glu Ala Val Glu Lys Gly His Gly Asp Leu 370 375 380 Lys Asp Lys Pro Trp Trp Pro Lys Leu Gln Thr Leu Glu Asp Leu Val 385 390 395 400 Glu Val Cys Leu Ile Ile Ile Trp Ile Ala Ser Ala Leu His Ala Ala 405 410 415 Val Asn Phe Gly Gln Tyr Pro Tyr Gly Gly Leu Ile Met Asn Arg Pro 420 425 430 Thr Ala Ser Arg Arg Leu Leu Pro Glu Lys Gly Thr Pro Glu Tyr Glu 435 440 445 Glu Met Ile Asn Asn His Glu Lys Ala Tyr Leu Arg Thr Ile Thr Ser 450 455 460 Lys Leu Pro Thr Leu Ile Ser Leu Ser Val Ile Glu Ile Leu Ser Thr 465 470 475 480 His Ala Ser Asp Glu Val Tyr Leu Gly Gln Arg Asp Asn Pro His Trp 485 490 495 Thr Ser Asp Ser Lys Ala Leu Gln Ala Phe Gln Lys Phe Gly Asn Lys 500 505 510 Leu Lys Glu Ile Glu Glu Lys Leu Val Arg Arg Asn Asn Asp Pro Ser 515 520 525 Leu Gln Gly Asn Arg Leu Gly Pro Val Gln Leu Pro Tyr Thr Leu Leu 530 535 540 Tyr Pro Ser Ser Glu Glu Gly Leu Thr Phe Arg Gly Ile Pro Asn Ser 545 550 555 560 Ile Ser Ile 42532DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 4cccgggatgt ttagtgctgg tcacaaaatc aaaggtaccg tggtcctgat gccgaaaaat 60gaactggaag tcaacccgga tggtagcgcc gttgataacc tgaatgcgtt cctgggtcgt

120agcgtgtctc tgcagctgat ttccgccacc aaagcagacg ctcacggcaa gggtaaagtt 180ggcaaagata cgtttctgga aggtattaat acctccctgc cgaccctggg tgccggtgaa 240tcagctttca acatccattt cgaatgggat ggttcaatgg gcattccggg cgccttctac 300atcaaaaact acatgcaggt ggaatttttc ctgaaaagtc tgaccctgga agcaatctcc 360aatcagggta cgattcgttt tgtctgcaac tcgtgggtgt ataataccaa actgtacaaa 420agcgttcgca tctttttcgc gaaccacacc tatgttccga gcgaaacccc ggcaccgctg 480gtttcttacc gtgaagaaga actgaaaagt ctgcgcggca atggtaccgg cgaacgtaaa 540gaatatgatc gcatttatga ctacgatgtt tacaacgacc tgggcaatcc ggataaaagc 600gaaaaactgg cccgtccggt cctgggcggt agctctacct tcccgtatcc gcgtcgcggt 660cgtaccggtc gtggtccgac cgtgaccgat ccgaacaccg aaaaacaggg cgaagtcttt 720tatgtgccgc gcgacgaaaa tctgggccat ctgaaatcta aagatgccct ggaaatcggt 780accaaaagtc tgtcccagat tgtgcaaccg gcgtttgaaa gcgccttcga tctgaaatct 840acgccgattg aatttcactc cttccaggac gttcatgatc tgtatgaagg cggtatcaaa 900ctgccgcgtg acgtcatttc aaccattatc ccgctgccgg tgatcaaaga actgtaccgc 960acggatggtc agcacattct gaaatttccg caaccgcatg tggttcaggt ttcacaatcg 1020gcgtggatga ccgatgaaga attcgcgcgt gaaatgatcg ccggcgttaa cccgtgcgtc 1080attcgcggtc tggaagaatt tccgccgaaa agcaatctgg acccggcaat ctatggcgat 1140cagagttcca aaattaccgc tgactctctg gacctggatg gctacacgat ggatgaagcc 1200ctgggtagtc gtcgcctgtt tatgctggac tatcacgata tcttcatgcc gtacgtgcgt 1260cagattaacc aactgaattc tgcaaaaacc tatgctaccc gtacgatcct gtttctgcgc 1320gaagacggca cgctgaaacc ggttgcaatt gaactgagcc tgccgcattc tgctggtgat 1380ctgagtgccg cggtgtccca ggttgtgctg ccggcaaaag aaggcgttga aagtaccatc 1440tggctgctgg cgaaagccta tgttattgtc aacgattcat gttaccatca actgatgtcg 1500cactggctga atacccatgc agctatggaa ccgtttgtta tcgcaacgca tcgccacctg 1560tctgtcctgc acccgattta taaactgctg accccgcatt accgtaacaa tatgaacatc 1620aatgcactgg ctcgccagag tctgattaac gcgaatggta ttatcgaaac cacgttcctg 1680ccgtcaaaat attcggtgga aatgtcatcg gccgtttaca aaaactgggt ctttaccgac 1740caggcactgc cggctgatct gatcaaacgt ggcgtcgcga ttaaagatcc gagcaccccg 1800catggtgtgc gtctgctgat tgaagactat ccgtacgcgg ccgatggcct ggaaatctgg 1860gcagctatta aaacctgggt gcaggaatat gttccgctgt attacgcacg cgatgacgat 1920gtgaaaaatg actccgaact gcaacactgg tggaaagaag ctgttgaaaa aggtcatggc 1980gacctgaaag ataaaccgtg gtggccgaaa ctgcagaccc tggaagatct ggtggaagtt 2040tgtctgatta tcatttggat tgccagcgca ctgcatgccg cggtgaactt tggtcaatat 2100ccgtacggcg gtctgattat gaatcgtccg accgcaagcc gtcgcctgct gccggaaaaa 2160ggcacgccgg aatacgaaga aatgatcaac aaccatgaaa aagcgtacct gcgcaccatc 2220acgagcaaac tgccgaccct gattagcctg tctgttatcg aaattctgtc aacgcacgcg 2280tcggatgaag tctatctggg tcagcgtgac aacccgcatt ggaccagtga ttccaaagcg 2340ctgcaggcct tccaaaaatt cggcaacaaa ctgaaagaaa tcgaagaaaa actggtccgt 2400cgcaacaatg atccgagcct gcagggtaac cgtctgggtc cggtgcaact gccgtatacc 2460ctgctgtatc cgtccagtga agaaggtctg acgtttcgtg gtattccgaa ctccatttcc 2520atctgactcg ag 253252583DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 5catatgctgg gcggcctgct gcaccgtggt cataaaatca agggcaccgt ggtcctgatg 60cgtaagaacg tcctggatgt gaatagcgtg acctcggtcg gcggtattat cggccagggt 120ctggacctgg tgggtagcac gctggatacc ctgacggcct ttctgggccg ctcagtgtcg 180ctgcaactga tcagcgcaac caaagcagat gctaacggca aaggcaagct gggcaaggcg 240acgttcctgg aaggcattat cacctccctg ccgacgctgg gtgcaggcca gtcagccttt 300aaaattaatt tcgaatggga tgacggctct ggtattccgg gcgccttcta catcaagaac 360ttcatgcaga ccgaattttt cctggtcagc ctgacgctgg aagatatccc gaatcatggc 420tcgattcact ttgtgtgcaa cagctggatc tacaatgcga aactgttcaa gtccgatcgc 480attttctttg ccaatcagac ctatctgccg tcagaaacgc cggcaccgct ggttaaatac 540cgtgaagaag aactgcacaa cctgcgtggt gacggtaccg gtgaacgtaa agaatgggaa 600cgcatctacg attacgacgt ttacaacgat ctgggtgatc cggacaaagg cgaaaaccat 660gcgcgtccgg tcctgggcgg taatgacacc tttccgtacc cgcgtcgcgg tcgtaccggt 720cgtaaaccga cgcgtaagga tccgaacagc gaatctcgca gtaatgatgt gtatctgccg 780cgtgacgaag cctttggtca cctgaaaagc tctgatttcc tgacgtacgg cctgaagtcc 840gtttcacaga acgtcctgcc gctgctgcaa agcgcatttg atctgaattt caccccgcgc 900gaatttgatt cgttcgacga agttcatggt ctgtatagcg gcggtattaa gctgccgacc 960gacattatct ctaaaatcag tccgctgccg gtgctgaagg aaatttttcg cacggatggc 1020gaacaggctc tgaagttccc gccgccgaaa gtcatccaag tgtcgaaaag cgcgtggatg 1080accgatgaag aatttgcacg tgaaatgctg gctggtgtta acccgaatct gattcgctgt 1140ctgaaggatt tcccgccgcg ttccaaactg gattcacagg tgtatggtga ccacaccagt 1200caaatcacga aagaacatct ggaaccgaac ctggaaggcc tgaccgttga tgaagctatt 1260cagaataaac gtctgtttct gctggatcat cacgacccga tcatgccgta tctgcgtcgc 1320attaatgcga cctcgacgaa agcgtacgcc acccgcacga tcctgttcct gaagaacgat 1380ggtaccctgc gtccgctggc cattgaactg agcctgccgc atccgcaggg tgaccaatcg 1440ggtgcgttta gccaggtttt cctgccggcc gatgaaggcg tcgaaagttc catctggctg 1500ctggcaaaag cttatgtggt tgtcaacgat tcttgctacc atcagctggt gtctcactgg 1560ctgaataccc atgcagtggt tgaaccgttt attatcgcta cgaaccgcca cctgtctgtc 1620gtgcatccga tctataaact gctgcatccg cactaccgcg acaccatgaa cattaatggt 1680ctggcgcgtc tgagtctggt caacgatggc ggtgtgattg aacagacgtt tctgtggggc 1740cgttattctg ttgaaatgag tgccgttgtc tacaaagatt gggtcttcac cgaccaagca 1800ctgccggcag acctgatcaa gcgtggtatg gcaattgaag atccgtcctg tccgcacggc 1860atccgtctgg tgattgaaga ttatccgtac accgttgacg gtctggaaat ctgggatgca 1920attaaaacgt gggtgcatga atacgttttt ctgtactaca agtctgatga caccctgcgc 1980gaagacccgg aactgcaggc gtgctggaaa gaactggtgg aagttggtca cggcgataaa 2040aagaacgaac cgtggtggcc gaaaatgcaa acccgtgaag aactggttga agcgtgtgcc 2100attatcattt ggacggcaag cgctctgcat gcggccgtga actttggcca gtatccgtac 2160ggcggtctga ttctgaatcg cccgaccctg tctcgtcgct tcatgccgga aaaaggcagt 2220gctgaatatg aagaactgcg taaaaatccg cagaaggcgt acctgaaaac catcacgccg 2280aaatttcaaa ccctgattga cctgagcgtg atcgaaattc tgtcccgcca tgcgtcagat 2340gaagtttatc tgggtgaacg tgacaacccg aattggacct ccgatacgcg tgcactggaa 2400gcttttaagc gcttcggcaa caaactggcc cagatcgaaa acaagctgtc agaacgtaac 2460aacgatgaaa agctgcgtaa tcgctgcggc ccggtgcaaa tgccgtatac cctgctgctg 2520ccgtcctcaa aagaaggtct gacgttccgt ggtatcccga atagcattag catctaagaa 2580ttc 258361701DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 6catatgtcta cgccgattga atttcactcc ttccaggacg ttcatgatct gtatgaaggc 60ggtatcaaac tgccgcgtga cgtcatttca accattatcc cgctgccggt gatcaaagaa 120ctgtaccgca cggatggtca gcacattctg aaatttccgc aaccgcatgt ggttcaggtt 180tcacaatcgg cgtggatgac cgatgaagaa ttcgcgcgtg aaatgatcgc cggcgttaac 240ccgtgcgtca ttcgcggtct ggaagaattt ccgccgaaaa gcaatctgga cccggcaatc 300tatggcgatc agagttccaa aattaccgct gactctctgg acctggatgg ctacacgatg 360gatgaagccc tgggtagtcg tcgcctgttt atgctggact atcacgatat cttcatgccg 420tacgtgcgtc agattaacca actgaattct gcaaaaacct atgctacccg tacgatcctg 480tttctgcgcg aagacggcac gctgaaaccg gttgcaattg aactgagcct gccgcattct 540gctggtgatc tgagtgccgc ggtgtcccag gttgtgctgc cggcaaaaga aggcgttgaa 600agtaccatct ggctgctggc gaaagcctat gttattgtca acgattcatg ttaccatcaa 660ctgatgtcgc actggctgaa tacccatgca gctatggaac cgtttgttat cgcaacgcat 720cgccacctgt ctgtcctgca cccgatttat aaactgctga ccccgcatta ccgtaacaat 780atgaacatca atgcactggc tcgccagagt ctgattaacg cgaatggtat tatcgaaacc 840acgttcctgc cgtcaaaata ttcggtggaa atgtcatcgg ccgtttacaa aaactgggtc 900tttaccgacc aggcactgcc ggctgatctg atcaaacgtg gcgtcgcgat taaagatccg 960agcaccccgc atggtgtgcg tctgctgatt gaagactatc cgtacgcggc cgatggcctg 1020gaaatctggg cagctattaa aacctgggtg caggaatatg ttccgctgta ttacgcacgc 1080gatgacgatg tgaaaaatga ctccgaactg caacactggt ggaaagaagc tgttgaaaaa 1140ggtcatggcg acctgaaaga taaaccgtgg tggccgaaac tgcagaccct ggaagatctg 1200gtggaagttt gtctgattat catttggatt gccagcgcac tgcatgccgc ggtgaacttt 1260ggtcaatatc cgtacggcgg tctgattatg aatcgtccga ccgcaagccg tcgcctgctg 1320ccggaaaaag gcacgccgga atacgaagaa atgatcaaca accatgaaaa agcgtacctg 1380cgcaccatca cgagcaaact gccgaccctg attagcctgt ctgttatcga aattctgtca 1440acgcacgcgt cggatgaagt ctatctgggt cagcgtgaca acccgcattg gaccagtgat 1500tccaaagcgc tgcaggcctt ccaaaaattc ggcaacaaac tgaaagaaat cgaagaaaaa 1560ctggtccgtc gcaacaatga tccgagcctg cagggtaacc gtctgggtcc ggtgcaactg 1620ccgtataccc tgctgtatcc gtccagtgaa gaaggtctga cgtttcgtgg tattccgaac 1680tccatttcca tctgactcga g 170176PRTArtificial SequenceDescription of Artificial Sequence Synthetic 6xHis tag 7His His His His His His 1 5

Claims

1. A method of producing lipoxygenase enzyme comprising: providing a nucleic acid expression construct within a host microorganism, wherein the construct encodes a plant-derived lipoxygenase enzyme; providing one or more chaperone plasmids within the host microorganism; and inducing expression of a lipoxygenase polypeptide encoded by the construct.

2. The method of claim 1, further comprising purifying the expressed lipoxygenase polypeptide and collecting isolated lipoxygenase polypeptide.

3. The method of claim 1, wherein the nucleic acid expression construct encodes all or a functional portion of the sequence of SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 3.

4. The method of claim 1, wherein the nucleic acid expression construct contains all or a functional portion of the sequence of SEQ ID NO 4, SEQ ID NO 5, or SEQ ID NO 6.

5. The method of claim 1, wherein the host microorganism is a bacterial cell containing one or more protease deficiencies.

6. The method of claim 5, wherein the bacterial cell is a strain of K12 cells, E. coli cells, Bacillus cells, Lactoccocus or yeast cells.

7. The method of claim 5, wherein the bacterial cell is an organism generally recognized as safe for the production of food enzymes.

8. The method of claim 1, wherein the one or more chaperone plasmids are simultaneously co-expressed with the lipoxygenase polypeptide.

9. The method of claim 1, wherein inducing comprises maintaining the heterologous microorganism at from 10-37.degree. C. for a period of time.

10. The method of claim 9, wherein inducing comprises maintaining the heterologous microorganism at from 25-35.degree. C. for a period of time.

11. The method of claim 9, wherein inducing comprises maintaining the heterologous microorganism at from 10-25.degree. C. for a period of time.

12. The method of claim 9, wherein inducing comprises maintaining the heterologous microorganism at from 20-25.degree. C. for a period of time.

13. The method of claim 9, wherein the period of time is from 1 hour to 2 days.

14. The method of claim 1, wherein purification comprises contacting the expressed lipoxygenase to immobilized-metal affinity chromatography media.

15. The method of claim 1, wherein the expressed lipoxygenase polypeptide does not contain a histidine tag.

16. The method of claim 1, wherein the collected lipoxygenase polypeptide comprises a plant lipoxygenase.

17. The method of claim 1, wherein the expressed lipoxygenase polypeptide does not contain a histidine tag.

18. The method of claim 1, wherein soluble lipoxygenase polypeptide relative to total soluble protein in the cell extract is 30% or greater.

19. A composition comprising lipoxygenase polypeptide made by the method of claim 1.

20. A method for the manufacture of a bread product, comprising adding the composition of claim 19 to a dough.

21. The method of claim 20, wherein the dough contains unsaturated fatty acids, carotinoids or both unsaturated fatty acids and carotinoids.

22. A bread product made by the method of claim 21.

23. A method of producing lipoxygenase enzyme comprising: providing a nucleic acid expression construct within a host microorganism, wherein the construct encodes a plant-derived lipoxygenase enzyme and the host microorganism is generally recognized as safe for the production of food enzymes; providing one or more chaperone plasmids within the host microorganism wherein the one or more chaperone plasmids are simultaneously co-expressed with the lipoxygenase polypeptide; inducing expression of a lipoxygenase polypeptide encoded by the construct; purifying the expressed lipoxygenase polypeptide by contacting the expressed lipoxygenase to immobilized-metal affinity chromatography media, wherein soluble lipoxygenase polypeptide relative to total soluble protein in the cell extract is 30% or greater; and collecting isolated lipoxygenase polypeptide.

24. An artificial and functional lipoxygenase enzyme produced by the method of claim 23.

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