Sec modified strains for improved secretion of recombinant proteins

Abstract

To provide modified strains for improving secretion of recombinantly expressed products secreted from a host organism with improved growth and productivity characteristics, and to provide methods of using the modified strains.SOLUTION: There is provided a Pichia pastoris microorganism, in which the sec72 gene has been deleted, the microorganism expressing a recombinant protein comprising a secretory signal sequence derived from yeast, the recombinant protein comprising at least one block polypeptide sequence derived from a silk protein or is a silk-like protein, and the secretory signal sequence being selected from a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and a MFα1 signal sequence.SELECTED DRAWING: Figure 12B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 673,001, filed May 17, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to methods for optimizing strains for enhanced secretion of proteins or metabolites from cells. The disclosure also relates to compositions obtained from those methods. In particular, the disclosure relates to yeast cells that have been selected or genetically engineered for enhanced secretion of recombinant proteins expressed by the yeast cells, and methods for culturing the yeast cells to produce useful compounds. [Background technology]

[0003] Background of the Invention The methylotrophic yeast Pichia pastoris is widely used for recombinant protein production. P. pastoris can grow to high cell densities, provide tightly controlled, methanol-inducible transgene expression, and has the ability to secrete recombinant proteins into synthetic media.

[0004] However, a significant portion of the expressed recombinant protein remains intracellularly, making recovery difficult and potentially adversely affecting cell growth and lifespan. Furthermore, recombinantly expressed proteins can be degraded intracellularly before being secreted, resulting in a protein mixture containing recombinantly expressed protein fragments and low yields of full-length recombinant protein. Therefore, there is a need for a means and genetically engineered strains that enhance secretion in P. pastoris while attenuating any loss of recombinant protein productivity or improving its productivity.

[0005] Thus, there is a need for modified organisms, and methods of using these organisms, that produce and secrete recombinant proteins while reducing any adverse effects on cell growth and productivity. Summary of the Invention

[0006] In some embodiments, provided herein is a Pichia pastoris microorganism in which SEC72 activity has been abolished or the sec72 gene has been deleted, wherein the microorganism expresses a recombinant protein. In some embodiments, the microorganism further comprises a recombinantly expressed SSH1 translocon complex.

[0007] In some embodiments, SEC72 comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the SEC72 comprises SEQ ID NO: 1. In some embodiments, the SEC72 is encoded by the sec72 gene. In some embodiments, the sec72 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 2. In some embodiments, the sec72 gene comprises SEQ ID NO: 2. In some embodiments, the sec72 gene is present at locus PAS_chr2-1_0448 of the microorganism.

[0008] In some embodiments, the SSH1 translocon complex comprises a first polypeptide sequence at least 95% identical to SEQ ID NO: 4, a second polypeptide sequence at least 95% identical to SEQ ID NO: 6, and a third polypeptide sequence at least 95% identical to SEQ ID NO: 8. In some embodiments, the SSH1 translocon complex comprises a first polypeptide comprising SEQ ID NO: 4, a second polypeptide comprising SEQ ID NO: 6, and a third polypeptide comprising SEQ ID NO: 8.

[0009] In some embodiments, the microorganism further comprises a recombinantly expressed translocon complex, hi some embodiments, the translocon complex is expressed from a recombinant SSH1 gene, a recombinant SSS1 gene, and a recombinant SBH2 gene.

[0010] In some embodiments, the SSH1 gene comprises SEQ ID NO: 3. In some embodiments, the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:3.

[0011] In some embodiments, the SSH1 gene comprises SEQ ID NO: 5. In some embodiments, the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:5.

[0012] In some embodiments, the SBH2 gene comprises SEQ ID NO: 7. In some embodiments, the SBH2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:7.

[0013] In some embodiments, expression of the recombinant SSH1 gene to increase the level of the SSH1 translocon in the microorganism above the level of the SSH1 translocon expressed by the native organism improves the growth rate and / or fermentation capacity of the microorganism.

[0014] In some embodiments, the translocon complex comprises an SSH1 protein, an SSS1 protein, and an SBH2 protein. In some embodiments, the SSH1 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO:4. In some embodiments, the SSH1 protein comprises SEQ ID NO:4. In some embodiments, the SSS1 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO:6. In some embodiments, the SSS1 protein comprises SEQ ID NO:6. In some embodiments, the SBH2 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO:8. In some embodiments, the SBH2 protein comprises SEQ ID NO:8.

[0015] In some embodiments, the activity of YPS1-1 protease and YPS1-2 protease in the microorganism is reduced or eliminated.

[0016] In some embodiments, the YPS1-1 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the YPS1-1 protease comprises SEQ ID NO: 10. In some embodiments, the YPS1-1 protease is encoded by the YPS1-1 gene. In some embodiments, the YPS1-1 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the YPS1-1 gene comprises SEQ ID NO: 9. In some embodiments, the YPS1-1 gene is present at locus PAS_chr4_0584 of the microorganism.

[0017] In some embodiments, the YPS1-2 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 12. In some embodiments, the YPS1-2 protease comprises SEQ ID NO: 12. In some embodiments, the YPS1-2 protease is encoded by the YPS1-2 gene. In some embodiments, the YPS1-2 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 11. In some embodiments, the YPS1-2 gene comprises SEQ ID NO: 11. In some embodiments, the YPS1-2 gene is present at locus PAS_chr3_1157 of the microorganism.

[0018] In some embodiments, the YPS1-1 gene, the YPS1-2 gene, or both of the microorganisms are mutated or knocked out, and in some embodiments, the activity of one or more additional proteases of the microorganism is reduced or eliminated.

[0019] In some embodiments, the recombinant protein comprises at least one block polypeptide sequence derived from a silk protein. In some embodiments, the recombinant protein comprises a silk-like polypeptide. In some embodiments, the silk-like polypeptide comprises one or more repeat sequences. TIFF2025061129000002.tif4128 (SEQ ID NO: 13), wherein X1 = SGGQQ (SEQ ID NO: 14) or GAGQQ (SEQ ID NO: 15) or GQGPY (SEQ ID NO: 16) or AGQQ (SEQ ID NO: 17) or SQ, n1 is 4 to 8, n2 is 6 to 20, and n3 is 2 to 20. In some embodiments, the silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO: 21.

[0020] In some embodiments, the recombinant protein comprises a secretory signal peptide. In some embodiments, the secretory signal peptide is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. In some embodiments, the secretory signal peptide is selected from the group consisting of SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, and SEQ ID NO:90.

[0021] Also provided herein, according to some embodiments, is a microorganism called Pichia pastoris, in which the activity of SEC72 of the microorganism has been eliminated or the sec72 gene comprising SEQ ID NO: 1 has been knocked out, and the microorganism comprises a recombinantly expressed SSH1 gene comprising SEQ ID NO: 3, a recombinantly expressed SSS1 gene comprising SEQ ID NO: 5, and a recombinantly expressed SBH2 gene comprising SEQ ID NO: 7, and the microorganism comprises a silk-like polypeptide comprising the polypeptide encoded by SEQ ID NO: 21.

[0022] Also provided herein, according to some embodiments, is a cell culture comprising a recombinant microorganism described herein.

[0023] Also provided herein, according to some embodiments, are cell cultures comprising a recombinant microorganism described herein, which have improved strain growth rate and fermentation capacity under standard cell culture conditions compared to cell cultures that do not contain a recombinantly expressed SSH1 translocon complex.

[0024] Also provided herein, in some embodiments, is a cell culture comprising a recombinant microorganism described herein, wherein the cell culture, under standard cell culture conditions, has an improved yield or specific productivity of the recombinant protein compared to a cell culture of the same microorganism, except that the microorganism contains a functional sec72 gene and does not contain a recombinantly expressed SSH1 translocon complex, and each microorganism has the same number of copies of the recombinant silk polypeptide gene.

[0025] Also provided herein, according to some embodiments, is a method for producing a recombinant protein, comprising culturing a recombinant microorganism described herein in a culture medium under conditions suitable for expression of the recombinantly expressed protein, and isolating the recombinant protein from the microorganism or the culture medium.

[0026] In some embodiments, the recombinant protein is secreted from the microorganism, and isolating the recombinant protein comprises recovering the medium containing the secreted recombinant protein.

[0027] In some embodiments, the yield or specific productivity of the recombinant protein is increased in the microorganism compared to an otherwise identical microorganism in which the sec72 gene has not been deleted.

[0028] In some embodiments, the yield or specific productivity of the recombinant protein is increased in the microorganism compared to an identical microorganism that does not contain the recombinantly expressed SSH1 translocon complex and does not have the sec72 gene deleted.

[0029] Also provided herein, according to some embodiments, is a method of modifying Pichia pastoris to improve secretion of a recombinantly expressed protein, the method comprising knocking out a gene encoding a SEC72 protein. In some embodiments, the method of modifying Pichia pastoris to improve secretion of a recombinantly expressed protein further comprises transforming the Pichia pastoris with a vector comprising a gene encoding a recombinantly expressed SSH1 translocon complex.

[0030] In some embodiments, the recombinantly expressed protein comprises a silk-like polypeptide. In some embodiments, the silk-like polypeptide comprises one or more repeat sequences {GGY-[GPG-X1] n1 -GPS-(A) n2} n3 (SEQ ID NO:13), where X1 = SGGQQ (SEQ ID NO:14) or GAGQQ (SEQ ID NO:15) or GQGPY (SEQ ID NO:16) or AGQQ (SEQ ID NO:17) or SQ, n1 is 4 to 8, n2 is 6 to 20, and n3 is 2 to 20. In some embodiments, the recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO:21.

[0031] [The present invention 1001] A microorganism called Pichia pastoris in which SEC72 activity has been eliminated or the sec72 gene has been deleted, said microorganism expressing a recombinant protein. [The present invention 1002] 1001. The microorganism of claim 10, wherein said SEC72 comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:1. [The present invention 1003] 1001. The microorganism of the present invention, wherein said SEC72 comprises SEQ ID NO:1. [The present invention 1004] 1001. The microorganism of the present invention, wherein said SEC72 is encoded by the sec72 gene. [The present invention 1005] 1001. The microorganism of claim 1001, wherein the sec72 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:2. [The present invention 1007] 1001. The microorganism of claim 10, wherein the sec72 gene comprises SEQ ID NO:2. [The present invention 1008] 1001. The microorganism of the present invention, wherein said sec72 gene is present at locus PAS_chr2-1_0448 of said microorganism. [The present invention 1009] The microorganism of any of the preceding inventions, further comprising a recombinantly expressed SSH1 translocon complex. [The present invention 1010] The microorganism of the present invention 1009, wherein the SSH1 translocon complex comprises a first polypeptide sequence at least 95% identical to SEQ ID NO: 4, a second polypeptide sequence at least 95% identical to SEQ ID NO: 6, and a third polypeptide sequence at least 95% identical to SEQ ID NO: 8. [The present invention 1011] 1009. The microorganism of claim 10, wherein the SSH1 translocon complex comprises a first polypeptide comprising SEQ ID NO: 4, a second polypeptide comprising SEQ ID NO: 6, and a third polypeptide comprising SEQ ID NO: 8. [The present invention 1012] The microorganism of any of the preceding inventions, further comprising a recombinantly expressed translocon complex. [The present invention 1013] The microorganism of the present invention, wherein the translocon complex is expressed from a recombinant SSH1 gene, a recombinant SSS1 gene, and a recombinant SBH2 gene. [The present invention 1014] The microorganism of the present invention, wherein the SSH1 gene comprises SEQ ID NO:3. [The present invention 1015] 1013. The microorganism of claim 10, wherein the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:3. [The present invention 1017] 1013. The microorganism of the present invention, wherein the SSH1 gene comprises SEQ ID NO:5. [The present invention 1018] 1013. The microorganism of claim 10, wherein the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:5. [The present invention 1020] 1013. The microorganism of claim 10, wherein the SBH2 gene comprises SEQ ID NO: 7. [The present invention 1021] 1013. The microorganism of claim 10, wherein the SBH2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:7. [The present invention 1023] A microorganism of the present invention 1013, wherein expression of the recombinant SSH1 gene to increase the level of the SSH1 translocon in the microorganism above the level of the SSH1 translocon expressed by the natural organism improves the growth rate and / or fermentation capacity of the microorganism. [The present invention 1024] The microorganism of the present invention, wherein the translocon complex comprises an SSH1 protein, an SSS1 protein, and an SBH2 protein. [The present invention 1025] 1024. The microorganism of claim 1024, wherein said SSH1 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:4. [The present invention 1026] 1024. The microorganism of claim 1024, wherein the SSH1 protein comprises SEQ ID NO:4. [The present invention 1027] 1024. The microorganism of claim 1024, wherein the SSS1 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6. [The present invention 1028] 1024. The microorganism of claim 1024, wherein the SSS1 protein comprises SEQ ID NO: 6. [The present invention 1029] 1024. The microorganism of claim 1024, wherein the SBH2 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:8. [The present invention 1030] 1024. The microorganism of claim 1024, wherein the SBH2 protein comprises SEQ ID NO:8. [The present invention 1031] Any of the microorganisms of the preceding inventions, in which the activity of YPS1-1 protease and YPS1-2 protease is reduced or eliminated. [The present invention 1032] 1031. The microorganism of the present invention, wherein the YPS1-1 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:10. [The present invention 1033] 1031. The microorganism of the present invention, wherein the YPS1-1 protease comprises SEQ ID NO: 10. [The present invention 1034] 1031. The microorganism of the present invention, wherein the YPS1-1 protease is encoded by the YPS1-1 gene. [This invention 1035] 1034. The microorganism of claim 1034, wherein the YPS1-1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:9. [This invention 1037] 1034. The microorganism of the present invention, wherein the YPS1-1 gene comprises SEQ ID NO:9. [The present invention 1038] 1034. The microorganism of the present invention, wherein the YPS1-1 gene is present at locus PAS_chr4_0584 of the microorganism. [This invention 1039] 12. The microorganism of any of the preceding inventions, wherein the YPS1-2 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:12. [The present invention 1040] 1031. The microorganism of the present invention, wherein the YPS1-2 protease comprises SEQ ID NO: 12. [The present invention 1041] 1031. The microorganism of the present invention, wherein the YPS1-2 protease is encoded by the YPS1-2 gene. [The present invention 1042] 1041. The microorganism of claim 10, wherein the YPS1-2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:11. [This invention 1044] 1041. The microorganism of claim 10, wherein the YPS1-2 gene comprises SEQ ID NO: 11. [This invention 1045] 1041. The microorganism of the present invention, wherein the YPS1-2 gene is present at locus PAS_chr3_1157 of the microorganism. [The present invention 1046] The microorganism of any one of claims 1031 to 1045, wherein the YPS1-1 gene or the YPS1-2 gene, or both, are mutated or knocked out. [This invention 1047] The microorganism of any one of claims 1031 to 1046, wherein the activity of one or more additional proteases is reduced or eliminated. [This invention 1048] 10. The microorganism of any of the preceding inventions, wherein the recombinant protein comprises at least one blocked polypeptide sequence derived from a silk protein. [This invention 1049] The microorganism of any of the preceding inventions, wherein said recombinant protein comprises a silk-like polypeptide. [The present invention 1050] The silk-like polypeptide comprises one or more repeat sequences Contains TIFF2025061129000003.tif4128, During the ceremony, X1=SGGQQ or GAGQQ or GQGPY or AGQQ or SQ; n1 is 4 to 8, n2 is 6 to 20, and n3 is 2 to 20, The microorganism of the present invention 1049. [This invention 1051] 1049. The microorganism of claim 1049, wherein said silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:21. [This invention 1052] Any of the preceding microorganisms of the invention, wherein said recombinant protein comprises a secretory signal peptide. [This invention 1053] The microorganism of claim 1052, wherein the secretory signal peptide is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. [This invention 1054] The microorganism of the present invention 1052, wherein the secretory signal peptide is selected from the group consisting of SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90. [This invention 1055] Pichia pastoris is a microorganism The activity of SEC72 in the microorganism is eliminated or the sec72 gene comprising SEQ ID NO: 1 is knocked out; a recombinantly expressed SSH1 gene comprising SEQ ID NO:3, a recombinantly expressed SSS1 gene comprising SEQ ID NO:5, and a recombinantly expressed SBH2 gene comprising SEQ ID NO:7; and A silk-like polypeptide comprising a polypeptide encoded by SEQ ID NO: 21, The microorganism. [This invention 1056] A cell culture comprising the microorganism of any one of 1001 to 1055 of the present invention. [This invention 1057] A cell culture comprising any of the microorganisms of the present inventions 1009 to 1055, wherein the growth rate and fermentation capacity of the strain are improved under standard cell culture conditions compared to a cell culture that does not contain a recombinantly expressed SSH1 translocon complex. [This invention 1058] A cell culture comprising any one of the microorganisms of the present invention 1009 to 1055, Under standard cell culture conditions, Identical microorganisms, each containing the same number of copies of a recombinant silk polypeptide gene, except that they contain a functional sec72 gene and do not contain a recombinantly expressed SSH1 translocon complex. the yield or specific productivity of the recombinant protein is improved compared to a cell culture of The cell culture. [This invention 1059] Cultivating the microorganism of any one of claims 1001 to 1055 in a medium under conditions suitable for expression of the recombinantly expressed protein; isolating the recombinant protein from the microorganism or the culture medium. A method for producing a recombinant protein, comprising: [The present invention 1060] 1059. The method of claim 1059, wherein said recombinant protein is secreted from said microorganism and wherein the step of isolating said recombinant protein comprises recovering the culture medium containing said secreted recombinant protein. [This invention 1061] 1059. The method of claim 1059, wherein the yield or specific productivity of said recombinant protein in said microorganism is increased compared to an otherwise identical microorganism in which the sec72 gene is not deleted. [This invention 1062] an identical microorganism except that it does not contain the recombinantly expressed SSH1 translocon complex and the sec72 gene is not deleted. 1059. The method of claim 1059, wherein the yield or specific productivity of said recombinant protein in said microorganism is increased compared to [This invention 1063] 1. A method for modifying Pichia pastoris to improve secretion of recombinantly expressed proteins, the method comprising knocking out the gene encoding the SEC72 protein. [This invention 1064] 1063. The method of claim 1063, further comprising the step of transforming said Pichia pastoris with a vector comprising a gene encoding a recombinantly expressed SSH1 translocon complex. [This invention 1065] 1064. The method of claim 1063, wherein said recombinantly expressed protein comprises a silk-like polypeptide. [The present invention 1066] The silk-like polypeptide has one or more repeat sequences {GGY-[GPG-X1] n1 -GPS-(A) n2} n3 Including, During the ceremony, X1=SGGQQ or GAGQQ or GQGPY or AGQQ or SQ; n1 is 4 to 8, n2 is 6 to 20, and n3 is 2 to 20, The method of the present invention 1063. [This invention 1067] 1063. The method of claim 1063, wherein said recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO:21. The foregoing and other objects, features, and advantages will become apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention. [Brief explanation of the drawings]

[0032] [Figure 1] 1 shows the classification of genes in the secretion complexes SEC, SEC61, SEC63, and SSH1, and also the homology scores between the S. cerevisiae and P. pastoris translocon complexes. [Figure 2] 16 is a plasmid map of a vector for SEC72 deletion containing a yeast selectable marker flanked by SEC72 homology arms. [Figure 3]

[0033] Figure 16 is a plasmid map of a vector for overexpression encoding proteins comprising the SSH1 complex, including promoters, open reading frames (ORFs), and terminators for SBH2, SSS1, and SSH1, as well as a yeast selectable marker. [Figure 4-1] FIG. 4A is a plasmid map of a vector containing the nourseothricin marker used in conjunction with homology arms for targeted protease gene deletion. [Figure 4-2]Figure 4B shows a cassette for protease knockout with homology arms targeting a desired protease gene adjacent to a nourseothricin resistance marker. Figure 4C shows a cassette for protease knockout with homology arms targeting a desired protease gene adjacent to a nourseothricin resistance marker. [Figure 5] 1 is a plasmid map of a vector containing an open reading frame (ORF) for expressing a silk-like polypeptide, including a secretion signal and a FLAG tag. The ORF is operably linked to the pGCW14 promoter and tAOX1 terminator. The vector also contains a selection marker for selecting successfully transformed cells. [Figure 6] FIG. 1 shows the secretion of recombinant 18B silk-like polypeptide expressed by wild-type (WT) and Δsec72 (Δ) strains expressing 2X, 4X, or 6X copies of 18B, as measured by enzyme-linked immunosorbent assay (i.e., ELISA). [Figure 7] Figure 1 shows secretion of recombinant proteins containing the PEP4 (sc), CPY+4 (sc), DAP2 (sc), or MFα1 (sc) signal peptides expressed by wild-type (WT) and Δsec72 (Δ) strains and measured by ELISA. (sc) indicates the signal peptide from S. cerevisiae. [Figure 8] Figure 1 shows the secretion of long silk polypeptides, Argiope bruennichi major ampullate gland spidroin 2 (i.e., Ab MaSp2) (106 kDa) and Latrodectus hesperus major ampullate gland spidroin 1 (i.e., Lh MaSp1 (55 kDa)), expressed by the wild-type (WT) and Δsec72 (Δ) strains and measured by ELISA. [Figure 9A]FIG. 1 shows secretion of recombinant 18B silk-like polypeptide expressed by wild-type (WT) strain, Δsec72 (Δ) strain, strain overexpressing sec72 under the control of pTHI11, and strain in which SEC72 was knocked down by DAmP (Decreased Abundance by mRNA Perturbation), as measured by ELISA. [Figure 9B] FIG. 1 shows the secretion of recombinant 18B silk-like polypeptide expressed by wild-type (WT) strains, strains overexpressing non-SEC72 proteins from the SEC63 complex (i.e., sec62, sec63, and sec66), and strains overexpressing sec72, as measured by ELISA. [Figure 10A] 1 shows expression normalized to the reference in the Δsec72 strain as fold change from the WT strain. Shading indicates the range, and the middle line represents the median across n=3 biological replicates (excluding n=2 for Δsec72). [Figure 10B] Projections of centered, log-scaled, reference-normalized expression data onto principal component space (left panel = PC1 vs. PC3, right panel = PC2 vs. PC3) are shown. Replicate data are color-coded by strain genotype, indicating low intra-genotype variability compared to inter-genotype variability. [Figure 11] FIG. 1 shows the growth rates observed for the WT and Δsec72 strains and a comparison of each growth rate with the growth rates of strains engineered to overexpress the SSH1 translocon complex or SEC61. [Figure 12A] Figure 1 shows the production (yield) of 18B as a function of glucose for wild-type (WT) strains expressing 4 (4x) or 6 (4+2x) copies of 18B compared to a Δsec72 strain (4x, Δsec72) and a Δsec72 strain overexpressing the SSH1 translocon complex (4x, Δsec72, 2N SSH1). [Figure 12B] FIG. 1 shows the specific productivity (production per biomass) of each strain. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description Details of various embodiments of the invention are set forth in the description that follows. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0034] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. The terms "a" and "an" include plural referents unless the context otherwise dictates. Generally, the terms used in connection with and techniques relating to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art.

[0035] Unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0036] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides at least 10 bases in length. Such terms include DNA molecules (e.g., cDNA or genomic DNA or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-natural internucleoside linkages, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially duplexed, branched, hairpinned, circular, or padlocked.

[0037] Unless otherwise indicated, for any sequence described herein in the general format of "SEQ ID NO:," as an example, a "nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid having, at least in part, (i) the sequence set forth in SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 1. The alternative is dictated by the context. For example, if the nucleic acid is being used as a probe, the alternative is dictated by the requirement that the probe be complementary to the desired target.

[0038] The term "recombinant" refers to a biological molecule, e.g., a gene or protein, that is (1) removed from its naturally occurring environment, (2) not associated with all or part of a polynucleotide with which the gene is found in nature, (3) operably linked to a polynucleotide with which it is not linked in nature, or (4) not occurring in nature. The term "recombinant" may be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized in heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0039] An endogenous nucleic acid sequence is considered "recombinant" herein when a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence in the genome of an organism such that expression of the endogenous nucleic acid sequence (or the protein product encoded by the sequence) is altered. In this case, the heterologous sequence is a sequence that does not naturally flank the endogenous nucleic acid sequence, regardless of whether the heterologous sequence is itself endogenous (from the same host cell or its progeny) or exogenous (from a different host cell or its progeny). As an example, a promoter sequence can replace (e.g., by homologous recombination) the native promoter of a gene present in the genome of a host cell, resulting in an altered expression pattern of the gene. The gene would now be considered "recombinant" because it has been separated from at least some of the sequences that naturally flank it.

[0040] A nucleic acid is also considered "recombinant" if it contains any alteration relative to the corresponding nucleic acid in a genome that does not occur in nature. For example, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion, or point mutation introduced artificially, such as by human intervention. "Recombinant nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, and a nucleic acid construct present as an episome.

[0041] As used herein, the phrase "degenerate variant" of a reference nucleic acid sequence encompasses a nucleic acid sequence that can be translated according to the standard genetic code to provide an amino acid sequence identical to the amino acid sequence translated from the reference nucleic acid sequence. The terms "degenerate oligonucleotide" or "degenerate primer" are used to refer to oligonucleotides that are capable of hybridizing to target nucleic acid sequences that are not necessarily identical in sequence but are homologous to each other within one or more specific segments.

[0042] The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison can be over a region of at least about 9 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, usually at least about 28 nucleotides, more usually at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are many different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, programs included in the Wisconsin Package version 10.0 (Genetics Computer Group (GCG), Madison, Wis.). FASTA provides alignments and percent sequence identity of the regions of the most overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated herein by reference in its entirety). For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (string size of 6 and scoring matrix factor of NOPAM) or Gap with its default parameters, as provided in GCG version 6.1, which is incorporated herein by reference.Alternatively, sequences can be compared using the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0043] The terms "substantial homology" or "substantial similarity" when referring to a nucleic acid or fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand) using appropriate insertions or deletions of nucleotides, the nucleotide sequence identity is at least about 75%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases as measured by any of the well-known sequence identity algorithms such as FASTA, BLAST or Gap, as described above.

[0044] Nucleic acids (also called polynucleotides) of the present invention may include both sense and antisense strands of RNA, cDNA, and genomic DNA, as well as synthetic forms and mixed polymers of the above. They may be chemically or biochemically modified and may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha-anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to designated sequences via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages replace phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure, such as those found in "locked" nucleic acids.

[0045] The term "mutated," as applied to a nucleic acid sequence, means that nucleotides within a nucleic acid sequence have been inserted, deleted, or changed relative to a reference nucleic acid sequence. A single change (point mutation) may be made at a locus, or multiple nucleotides may be inserted, deleted, or changed at a single locus. Furthermore, one or more changes may be made at any number of loci within a nucleic acid sequence. Nucleic acid sequences may be mutated by any method known in the art, including, but not limited to, mutagenesis techniques such as "error-prone PCR" (a method in which PCR is performed under conditions in which the fidelity of DNA polymerase replication is low, resulting in a high rate of point mutations throughout the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)) and "oligonucleotide-directed mutagenesis" (a method that allows for the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).

[0046] As used herein, the term "attenuate" generally refers to functional deletion, including mutation, partial or complete deletion, insertion, or other alteration made to a gene sequence or a sequence controlling the transcription of the gene sequence, thereby reducing or inhibiting the production of the gene product or rendering the gene product non-functional. In some instances, functional deletion is expressed as a knockout mutation. Attenuation also includes altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulating it, modifying the amino acid sequence by expressing an interfering RNA, ribozyme, or antisense sequence that targets the gene of interest, or any other technique known in the art. In one example, feedback inhibition or the sensitivity of a particular enzyme to inhibition caused by a compound that is neither a product nor a reactant (pathway non-specific feedback) is reduced, so that the enzyme's activity is not affected by the presence of a compound. In other cases, an enzyme that has been altered to have reduced activity may be said to be attenuated.

[0047] As used herein, the term "deletion" refers to the removal of one or more nucleotides from a nucleic acid molecule, or one or more amino acids from a protein, leaving the regions on either side joined to each other.

[0048] As used herein, the term "knockout" refers to a gene whose expression or activity level is reduced to zero.In some instances, a gene is knocked out by deleting part or all of its coding sequence.In other instances, a gene is knocked out by introducing one or more nucleotides into its open reading frame, thereby resulting in the translation of a nonsense protein product, or an otherwise non-functional protein product.

[0049] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which generally refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated, but also includes linear double-stranded molecules, such as those resulting from amplification by polymerase chain reaction (PCR) or from restriction enzyme treatment of circular plasmids. Other vectors include cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors may be integrated into the genome of a host cell upon introduction, thereby replicating along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0050] "Operatively linked" or "operably linked" expression control sequences refer to linkages in which the expression control sequences are adjacent to a gene of interest to control such a gene of interest, as well as expression control sequences that act in trans or at a distance to control a gene of interest.

[0051] The term "expression control sequence" refers to a polynucleotide sequence necessary to affect the expression of a coding sequence to which it is operably linked. Expression control sequences are sequences that control the transcription, post-transcriptional events, and translation of a nucleic acid sequence. Expression control sequences include appropriate sequences for transcription initiation, transcription termination, promoters, and enhancers; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and, where necessary, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include, at a minimum, all components the presence of which is essential for expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0052] The term "regulatory element" refers to any element that affects the transcription or translation of a nucleic acid molecule. Examples include, but are not limited to, regulatory proteins (e.g., transcription factors), chaperones, signal transduction proteins, RNAi molecules, antisense RNA molecules, microRNAs, and RNA aptamers. Regulatory elements may be endogenous to the host organism. Regulatory elements may be exogenous to the host organism. Regulatory elements may be synthetically generated regulatory elements.

[0053] As used herein, the terms "promoter," "promoter element," or "promoter sequence" refer to a DNA sequence that, when linked to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is generally, although not necessarily, located 5' (i.e., upstream) of the nucleotide sequence of interest whose transcription it controls into mRNA, and provides specific binding sites for RNA polymerase and other transcription factors for transcription initiation. A promoter may be endogenous to the host organism. A promoter may be exogenous to the host organism. A promoter may be a synthetically generated regulatory element.

[0054] Promoters useful for expressing the recombinant genes described herein include both constitutive promoters and inducible / repressible promoters. When multiple recombinant genes are expressed in the genetically engineered organisms of the invention, such different genes can be controlled by different promoters, by the same promoter in separate operons, or expression of two or more genes may be controlled by a single promoter as part of an operon.

[0055] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such term is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur with passage due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell, a cell line grown in culture, or a cell present in a living tissue or organism.

[0056] As used herein, the term "wild-type" (i.e., "WT") refers to a comparative strain that does not have the modification under discussion. Wild-type does not refer to a natural, unmodified strain, but rather to a strain that does not have the selected modification. For example, when comparing two versions of a recombinant strain that have been modified to express a recombinant silk polypeptide, where one of the recombinant strains is a sec72 KO, the KO strain may be referred to as a Δsec72 strain, while the other version is referred to as "WT."

[0057] As used herein, the term "peptide" refers to a short polypeptide, e.g., generally less than about 50 amino acids in length, more usually less than about 30 amino acids in length. As used herein, the term encompasses analogs as well as mimetics that mimic structure-function and thereby biological function.

[0058] The term "polypeptide" encompasses both naturally occurring and non-naturally occurring proteins, as well as fragments, variants, derivatives, and analogs thereof. Polypeptides may be monomeric or polymeric. Furthermore, a polypeptide may contain multiple different domains, each with one or more distinct activities.

[0059] The term "isolated protein" or "isolated polypeptide" refers to, in terms of its origin or source of derivation, (1) a protein or polypeptide that is not associated with naturally associated components that accompany it in its natural state; (2) a protein or polypeptide that exists to a degree of purity not found in nature, where purity can be determined with respect to the presence of other cellular material (e.g., free from other proteins from the same species); (3) a protein or polypeptide expressed by cells from a different species; or (4) a protein or polypeptide that does not occur in nature (e.g., it is a fragment of a naturally occurring polypeptide or contains amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. A polypeptide or protein may be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art. When defined in this way, "isolated" does not necessarily require that the protein, polypeptide, peptide, or oligopeptide so described be physically removed from its natural environment.

[0060] The term "polypeptide fragment" refers to a polypeptide that has a deletion compared to a full-length polypeptide, e.g., a deletion at the amino and / or carboxy terminus. In preferred embodiments, a polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to corresponding positions in the naturally occurring sequence. Fragments are generally at least 5, 6, 7, 8, 9, or 10 amino acids in length, preferably at least 12, 14, 16, or 18 amino acids in length, more preferably at least 20 amino acids in length, more preferably at least 25, 30, 35, 40, or 45 amino acids in length, even more preferably at least 50 or 60 amino acids in length, and even more preferably at least 70 amino acids in length.

[0061] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence encoding the protein has a similar sequence to the nucleic acid sequence encoding the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that two proteins have similar amino acid sequences.) As used herein, homology between two amino acid sequence regions (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.

[0062] When using "homologous" with respect to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (incorporated herein by reference).

[0063] The 20 conventional amino acids and their abbreviations follow convention. nded. 1991). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids, such as α-,α-disubstituted amino acids, N-alkylamino acids, and other unconventional amino acids may also be suitable components of the polypeptides of the invention. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminus and the right-hand end corresponds to the carboxy terminus, in accordance with standard usage and convention.

[0064] The following six groups each contain amino acids that are conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), alanine (A), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0065] Sequence homology of polypeptides, sometimes referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using homology measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG includes programs such as "Gap" and "Bestfit," which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, e.g., GCG version 6.1.

[0066] A useful algorithm for comparing a particular polypeptide sequence to a database containing many sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0067] Preferred parameters for BLASTp are expectation: 10 (default); filter: seg (default); gap opening cost: 11 (default); gap extension cost: 1 (default); maximum alignments: 100 (default); string size: 11 (default); number of results displayed: 100 (default); penalty matrix: BLOWSUM62.

[0068] Preferred parameters for BLASTp are: expectation: 10 (default); filter: seg (default); gap opening cost: 11 (default); gap extension cost: 1 (default); maximum alignment: 100 (default); string size: 11 (default); number of results displayed: 100 (default); penalty matrix: BLOWSUM62. The length of polypeptide sequences to be compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably greater than about 35 residues. When searching a database containing sequences from multiple different organisms, amino acid sequence comparison is preferred. Database searches using amino acid sequences can be performed using algorithms known in the art other than blastp. For example, polypeptide sequences can be compared using FASTA, a program in GCG version 6.1. FASTA provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990), which is incorporated herein by reference. For example, percent sequence identity between amino acid sequences can be determined using FASTA, provided in GCG version 6.1, which is incorporated herein by reference, with its default parameters (string size of 2 and scoring matrix PAM250).

[0069] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0070] Exemplary methods and materials are described below; however, methods and materials similar or equivalent to those described herein can be used in the practice of the present invention and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and are intended to be non-limiting.

[0071] overview Provided herein are recombinant strains and methods for improving the secretion and productivity of recombinantly expressed proteins in yeast strains (e.g., P. pastoris).

[0072] Proteins destined for secretion must first cross the membrane of the endoplasmic reticulum (ER translocation). Multiple targeting pathways recruit elongating ribosomes or fully translated proteins to the ER membrane, where their polypeptide chains enter through pore-forming protein complexes called translocons. Yeast, including S. cerevisiae and P. pastoris, express two translocons: the SEC61 complex and the SSH1 complex. Each complex consists of a core trimeric complex, but the SEC61 translocon also associates with the tetrameric SEC63 complex. Figure 1 shows the protein subunits contained within each of these translocon complexes.

[0073] Although SEC72 encodes a non-essential component of the SEC63 complex, deletion of SEC72 (Δsec72) results in the accumulation of some secreted precursors. Surprisingly, we demonstrate herein that the Δsec72 strain exhibits improved secretion of recombinant silk polypeptides.

[0074] In some embodiments, the strain is engineered to delete the sec72 gene (SEQ ID NO: 1). As we demonstrate and demonstrate herein, deletion of sec72, which expresses SEC72 (SEQ ID NO: 2), a cofactor for protein translocation into the ER for secretion, unexpectedly enhances silk secretion (up to +75%) in bench-scale block model assays. This holds true across expression levels, signal sequences, and different silks.

[0075] In some embodiments, deletion of sec72 is achieved using a plasmid carrying a 5' homology arm to sec72 and a 3' homology arm to sec72 flanked by yeast selectable markers, for example as shown in Figure 2. Deleting Δsec72 induced a transcriptional adaptation that can indirectly promote secretion. The sequences of the sec72 gene and SEC72 protein are shown in Table 1.

[0076] Table 1. Sequence of the sec72 open reading frame and SEC72 protein. TIFF2025061129000004.tif121149

[0077] In some cases, the deletion of sec72 slowed strain growth, and fermentation screening of Δsec72 strains struggled with glucose accumulation. Therefore, some embodiments of the present invention provide strains that recombinantly overexpress proteins comprising the SSH1 translocon complex. As demonstrated herein, overexpression of the SSH1 translocon complex in Δsec72 strains improved the strain's growth rate and fermentation capacity while maintaining improved secretion. Testing of this deletion-overexpression combination strain, which contained only four copies of the recombinant silk-like block polypeptide-expressing gene (i.e., 18B), demonstrated similar titer and specific productivity improvements (+18%) compared to strains with six copies of the silk-like block polypeptide-expressing gene (i.e., 18B).

[0078] In some embodiments, the SSH1 translocon complex is overexpressed in host cells by inserting a plasmid (FIG. 3) containing genes encoding recombinant SBH2, SSS1, and SSH1. The sequences of these genes and the expressed proteins are shown in Table 2.

[0079] Table 2. Open reading frames and protein sequences for subunits of the SSH1 translocon complex. TIFF2025061129000005.tif50149TIFF2025061129000006.tif218149TIFF2025061129000007.tif76149

[0080] Protease knockout In some embodiments, to attenuate protease activity in Pichia pastoris, the genes encoding these enzymes are inactivated or mutated to reduce or eliminate activity. This can be done through mutations or insertions in the genes themselves or through modifications to the gene's regulatory elements. This can be achieved using standard yeast genetic techniques. An example of such a technique is gene replacement by double homologous recombination, in which homologous regions flanking the gene to be inactivated are cloned into a vector adjacent to a selectable marker gene (such as an antibiotic resistance gene or a gene that complements an auxotrophy of the yeast strain). In some embodiments, the nourseothricin selection plasmid shown in Figure 4A can be used as the base plasmid, and homology arms (HA) flanking the resistance cassette (Figures 4B and 4C) can be used, with the homology arms specifically targeting the desired protease to be knocked out. Descriptions of protease knockouts and methods for modifying host cells to inhibit degradation of recombinant proteins are provided in U.S. Application No. 15 / 724,196 and PCT Application No. PCT / US2017 / 054997, each of which is incorporated herein by reference in its entirety.

[0081] Alternatively, the homologous region can be PCR amplified and linked to a selectable marker gene by overlap PCR. The DNA fragment is then transformed into Pichia pastoris by methods known in the art, for example, electroporation. Transformants grown under selective conditions are then analyzed for gene disruption events by standard techniques, for example, PCR or Southern blotting of genomic DNA. In an alternative experiment, gene inactivation can be achieved by single homologous recombination, for example, by cloning the 5' end of the gene's ORF into a promoterless vector that also contains a selectable marker gene. Once the vector is linearized by digestion with a restriction enzyme that cuts the vector only at the target gene homologous fragment, the vector is transformed into Pichia pastoris. Integration into the target gene site is confirmed by PCR or Southern blotting of genomic DNA. In this method, the gene fragment cloned onto the vector is replicated within the genome, resulting in two copies of the target locus: the first copy expresses a truncated, inactive protein (if expressed at all) due to an incomplete ORF, and the second copy lacks a promoter to direct transcription.

[0082] Alternatively, transposon mutagenesis is used to inactivate the target gene, and a library of such mutants can be screened for insertion events within the target gene by PCR.

[0083] The functional phenotype (i.e., deficiency) of the engineered / knockout strain can be assessed using techniques known in the art. For example, the deficiency of protease activity in the engineered strain can be confirmed using any of a variety of methods known in the art, including hydrolytic activity assays of chromogenic protease substrates, band shifts of substrate proteins for the protease of choice, among others.

[0084] The attenuation of protease activity described herein can be achieved by mechanisms other than knockout mutation. For example, a desired protease can be attenuated by altering its nucleic acid sequence, placing the gene under the control of a less active promoter, downregulating it, modifying its amino acid sequence by expressing an interfering RNA, ribozyme, or antisense sequence targeting the gene of interest, or by any other technique known in the art. In preferred strains, the protease activity of the proteases encoded by PAS_chr4_0584 (YPS1-1) and PAS_chr3_1157 (YPS1-2) (e.g., polypeptides comprising SEQ ID NOs: 10 and 12) is attenuated by any of the above methods. In some embodiments, the present invention is directed to methylotrophic yeast strains, particularly Pichia pastoris strains, in which the YPS1-1 and YPS1-2 genes (e.g., as set forth in SEQ ID NOs: 9 and 11) have been inactivated. In some embodiments, additional genes encoding proteases may be knocked out according to the methods provided herein to further reduce the protease activity of the desired protein product expressed by the strain.

[0085] Table 3. Open reading frame nucleotide and polypeptide sequences for proteases targeted for deletion in P. pastoris TIFF2025061129000008.tif248153TIFF2025061129000009.tif147153

[0086] Production of recombinant strains The present specification provides methods for transforming strains to reduce activity, for example, using vectors that deliver or knock out recombinant genes or other methods that attenuate the desired endogenous gene. These vectors may take the form of a vector backbone containing an origin of replication and a selectable marker (commonly antibiotic resistance, but many other methods are possible), or a linear fragment that allows integration into the target cell chromosome. The vector must be compatible with the chosen organism and method of insertion.

[0087] Once the vector elements have been selected, vector construction can be carried out in a number of different ways: In one embodiment, a DNA synthesis service may be used, or all vectors may be made individually.

[0088] Once the DNA for each vector (including any additional elements required for insertion and manipulation) is obtained, it must be assembled. There are many possible assembly methods, including, but not limited to, restriction enzyme cloning, blunt-end ligation, and overlap assembly (see, e.g., Gibson, DG, et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods, 6(5), 343-345 (2009), and the GeneArt Kit (http: / / tools.invitrogen.com / content / sfs / manuals / geneart_seamless_cloning_and_assembly_man.pdf)). Overlap assembly provides a method to ensure that all of the elements are assembled in the correct locations and that no undesired sequences are introduced.

[0089] The vectors generated above can be inserted into target cells using standard molecular biology techniques, such as molecular cloning, etc. In one embodiment, the target cells have already been genetically engineered or selected to already contain the genes required for production of the desired product, which may be done during or after the subsequent insertion of the vector.

[0090] Depending on the organism and type of library element (plasmid or genomic insertion), several known methods for inserting DNA-containing vectors into cells may be used, including, for example, transformation of microorganisms that can take up and replicate DNA from the local environment, transformation by electroporation or chemical means, transduction with viruses or phages, mating of two or more cells, or conjugation from different cells.

[0091] Several methods for introducing recombinant DNA into bacterial cells are known in the art, including, but not limited to, transformation, transduction, and electroporation (see Sambrook, et al., Molecular Cloning: A Laboratory Manual (1989), Second Edition, Cold Spring Harbor Press, Plainview, NY). Non-limiting examples of commercially available kits and bacterial host cells for transformation include NovaBlue Singles™ (EMD Chemicals Inc., NJ, USA), Max Efficiency™ DH5α™, One Shot™ BL21(DE3) E. coli cells, One Shot™ BL21(DE3)pLys E. coli cells (Invitrogen Corp., Carlsbad, Calif., USA), and XL1-Blue competent cells (Stratagene, CA, USA). Non-limiting examples of commercially available kits and bacterial host cells for electroporation include Zappers™ electrocompetent cells (EMD Chemicals Inc., NJ, USA), XL1-Blue electroporation-competent cells (Stratagene, CA, USA), and ElectroMAX™ A. tumefaciens LBA4404 cells (Invitrogen Corp., Carlsbad, Calif., USA).

[0092] Several methods for introducing recombinant nucleic acids into eukaryotic cells are known in the art. Exemplary methods include transfection, electroporation, liposome-mediated nucleic acid delivery, and microinjection into host cells (see Sambrook, et al., Molecular Cloning: A Laboratory Manual (1989), Second Edition, Cold Spring Harbor Press, Plainview, NY). Non-limiting examples of commercially available kits and reagents for transfecting eukaryotic cells with recombinant nucleic acids include Lipofectamine™ 2000, Optifect™ Reagent, calcium phosphate transfection kit (Invitrogen Corp., Carlsbad, Calif., USA), GeneJammer® transfection reagent, and LipoTAXI® transfection reagent (Stratagene, CA, USA). Alternatively, recombinant nucleic acids may be introduced into insect cells (e.g., sf9, sf21, High Five™) using baculovirus vectors.

[0093] The transformed cells are isolated so that each clone can be tested separately. In one embodiment, this is done by plating onto one or more plates of medium containing (or lacking) a selection agent that ensures that only transformed cells survive and reproduce. This specific agent may be an antibiotic (if the library contains an antibiotic resistance marker), a metabolite deprivation (for complementation of an auxotrophy), or other selection means. The cells grow into individual colonies, each containing a single clone.

[0094] Colonies are screened for the desired production of a protein, metabolite, or other product, for reduced protease activity, for growth, or for increased secretion activity. In one embodiment, recombinant cells with the highest (or sufficiently high) titer or efficiency of product production are identified by screening. This includes a reduced rate of degradation products or an increase in the total amount of desired polypeptide secreted from the cells and recovered from the cell culture.

[0095] This assay can be performed by growing individual clones in multiwell culture plates, one clone per well. Once the cells reach an appropriate biomass density, they are induced with methanol. After a period of time, typically 24-72 hours after induction, the cultures are harvested by spinning in a centrifuge to pellet the cells and removing the supernatant. The supernatant from each culture can then be tested to determine whether the desired secretion level has been achieved.

[0096] Silk arrangement In some embodiments, the engineered strains described herein recombinantly express silk-like polypeptide sequences. In some embodiments, the silk-like polypeptide sequences are 1) block copolymer polypeptide compositions produced by mixing and matching repeat domains from silk polypeptide sequences, and / or 2) recombinantly expressed block copolymer polypeptides large enough (approximately 40 kDa) to form useful fibers upon secretion from industrially scalable microorganisms. Large (approximately 40 kDa to approximately 100 kDa) block copolymer polypeptides engineered with repeat domain fragments of silk, including sequences derived from nearly all of the published amino acid sequences of spider silk polypeptides, can be expressed in the engineered microorganisms described herein. In some embodiments, the silk polypeptide sequences are matched and engineered to produce highly expressed and secreted polypeptides capable of fiber formation. In some embodiments, knocking out protease genes or reducing protease activity in the host engineered strain reduces silk-like polypeptide degradation. In some embodiments, knockout of sec72 and overexpression of the SSH1 translocon complex improves secretion while mitigating the growth defect and maintaining or improving growth of the strain.

[0097] In some embodiments, provided herein are compositions for expressing and secreting engineered block copolymers by comprehensively combinatorially mixing silk polypeptide domains spanning the entire silk polypeptide sequence space, wherein the block copolymers exhibit minimal degradation. In some embodiments, provided herein are methods for secreting block copolymers in scalable organisms (e.g., yeast, fungi, and Gram-positive bacteria) exhibiting minimal degradation. In some embodiments, the block copolymer polypeptides comprise zero or more N-terminal domains (NTDs), one or more repeat domains (REPs), and zero or more C-terminal domains (CTDs). In some aspects of the above embodiments, the block copolymer polypeptides are single-chain polypeptides of greater than 100 amino acids. In some embodiments, the block copolymer polypeptide comprises a domain that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a block copolymer polypeptide disclosed in International Publication No. WO / 2015 / 042164, "Methods and Compositions for Synthesizing Improved Silk Fibers," which is incorporated by reference in its entirety.

[0098] Several types of natural spider silk have been identified to date, and the various mechanical properties of naturally spun spider silk are thought to be closely related to the molecular composition of the silk. See, for example, Garb, JE, et al., Untangling spider silk evolution with spidroin terminal domains, BMC Evol. Biol., 10:243 (2010); Bittencourt, D., et al., Protein families, natural history and biotechnological aspects of spider silk, Genet. Mol. Res., 11:3 (2012); Rising, A., et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell. Mol. Life Sci., 68:2, pp. 169-184 (2011); and Humenik, M., et al., Spider silk: understanding the structure-function relationship of a natural fiber, Prog. Mol. Biol. Transl. Sci., 103, pp. 131-85 (2011). For example:

[0099] Grape-like (AcSp) silks tend to be tough, combining moderately high strength with moderately high elasticity. AcSp silks are characterized by large block ("repeat aggregate") sizes, often incorporating polyserine and GPX motifs. Tubular (TuSp or cylindrical) silks tend to be large in diameter, moderate in strength, and highly elastic. TuSp silks are characterized by their polyserine and polythreonine content and short polyalanine sequences. MaSp silks tend to be high in strength and moderately elastic. MaSp silks are one of two subtypes, MaSp1 and MaSp2. MaSp1 silks are generally less elastic than MaSp2 silks and are characterized by polyalanine, GX, and GGX motifs. MaSp2 silks are characterized by polyalanine, GGX, and GPX motifs. Ampullate (MiSp) silks tend to have moderate strength and moderate flexibility. MiSp silks are characterized by GGX, GA, and polyA motifs and often contain a spacer element of approximately 100 amino acids. Flagelliform (Flag) silks tend to have very high flexibility and moderate strength. Flag silks are usually characterized by GPG, GGX, and a short spacer motif.

[0100] The properties of each silk species may vary from species to species, and spiders with different lifestyles (e.g., stationary web-weaving spiders versus wandering, feeding spiders) or evolutionarily older spiders may produce silks that differ from those described above (for a description of spider diversity and classification, see Hormiga, G., and Griswold, C.E., Systematics, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, pp. 487-512 (2014); and Blackedge, T.A. et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. Natl. Acad. Sci. USA, 106:13, pp. 5229-5234 (2009)). However, using synthetic block copolymer polypeptides with sequence similarity and / or amino acid composition similarity to the repeating domains of natural silk proteins, it is possible to produce consistent silk-like fibers on a commercial scale that recapitulate properties corresponding to natural silk fibers.

[0101] In some embodiments, a list of putative silk sequences can be compiled by searching GenBank for related terms, such as "spidroin," "fibroin," or "MaSp," and these sequences can be pooled with additional sequences obtained from independent sequencing efforts. The sequences are then translated into amino acids, duplicate entries are filtered, and manually divided into each domain (NTD, REP, CTD). In some embodiments, the candidate amino acid sequences are reverse-translated into DNA sequences optimized for expression in Pichia (Komagataella) pastoris. The DNA sequences are each cloned into an expression vector and transformed into Pichia (Komagataella) pastoris. In some embodiments, the various silk domains that demonstrate successful expression and secretion are then assembled combinatorially to construct fiber-forming silk molecules.

[0102] Silk polypeptides characteristically consist of repeat domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In one embodiment, both the C-terminal and N-terminal domains are 75-350 amino acids in length. The repeat domains exhibit a hierarchical structure. The repeat domains contain a series of blocks (also called repeat units). These blocks are repeated sometimes perfectly and sometimes imperfectly (forming quasi-repetitive domains) throughout the repeat domain of the silk. The length and composition of the blocks vary between different silk species and between different species. Table 1 lists example block sequences for selected species and silk species; further examples are described in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell Mol. Life Sci., 68:2, pp. 169-184 (2011), and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pp. 2603-2605 (2001). In some cases, blocks may be arranged in a regular pattern to form larger macrorepeats that occur multiple times (usually 2-8 times) in the repeat domain of the silk sequence. Blocks repeated within the repeat domain or macrorepeat may be separated by spacing elements from macrorepeats repeated within the repeat domain. In some embodiments, the block sequence comprises a glycine-rich region followed by a poly(A) region. In some embodiments, a short (about 1-10) amino acid motif occurs multiple times within a block. For purposes of the present invention, blocks derived from different native silk polypeptides can be selected without regard to circular permutation (i.e., identified blocks may not align due to circular permutation if they are otherwise similar between silk polypeptides).Thus, for example, a "block" of SGAGG (SEQ ID NO:23) is, for purposes of the present invention, identical to GSGAG (SEQ ID NO:24), which is also identical to GGSGA (SEQ ID NO:25), which are all merely circular permutations of one another. The particular permutation chosen for a given silk sequence may be determined more by convenience than anything else (usually starting with G). Silk sequences obtained from the NCBI database can be divided into blocks and unique regions.

[0103] (Table 4) Block sequence samples TIFF2025061129000010.tif240148TIFF2025061129000011.tif181148

[0104] Fiber-forming block copolymer polypeptides derived from block and / or macrorepeat domains according to certain embodiments of the present invention are described in International Publication No. WO / 2015 / 042164, which is incorporated by reference. Natural silk sequences, obtained from protein databases such as GenBank or by de novo sequencing, are broken down into domains (N-terminal, repeat, and C-terminal). The N-terminal and C-terminal domain sequences selected for post-synthetic assembly into fibers include native amino acid sequence information and other modifications described herein. The repeat domains are broken down into repeat sequences, which contain representative blocks (usually 1-8, depending on the silk species) that capture key amino acid information, while reducing the size of the DNA encoding the amino acids to easily synthesizable fragments. In some embodiments, properly formed block copolymer polypeptides contain at least one repeat domain containing at least one repeat sequence, optionally flanked by an N-terminal and / or C-terminal domain.

[0105] In some embodiments, the repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence is 150-300 amino acid residues. In some embodiments, the repeat sequence comprises multiple blocks. In some embodiments, the repeat sequence comprises multiple macrorepeats. In some embodiments, the blocks or macrorepeats are divided across the multiple repeat sequences.

[0106] In some embodiments, repeat sequences must begin with glycine and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine ​​(C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) to meet DNA assembly requirements. In some embodiments, some repeat sequences can be altered relative to the native sequence. In some embodiments, repeat sequences can be altered, such as by adding serine to the C-terminus of the polypeptide (to avoid termination at F, Y, W, C, H, N, M, or D). In some embodiments, repeat sequences can be modified by filling in incomplete blocks with homologous sequences from another block. In some embodiments, repeat sequences can be modified by rearranging the order of the blocks or macrorepeats.

[0107] In some embodiments, unique N-terminal and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain can be obtained by removing a leading signal sequence, such as those identified by SignalP (Peterson, TN, et. al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg. 785-786 (2011)).

[0108] In some embodiments, the N-terminal domain sequence, the repeat sequence, or the C-terminal domain sequence is selected from the group consisting of Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope argentata, Argiope bruennichi, Argiope trifasciata, and the like. trifasciata, Atypoides riversi, Avicularia juruensis, Bothriocyrtum californicum, Deinopis spinosa, Diguetia canities, Dolomedes tenebrosus, Euagrus chisoseus, Euprosthenops australis, Gasteracantha mammosa, Hypochilus thorelli, Kukulcania hibernalis, Latrodectus hesperus hesperus, Megahexura fulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavata, Nephila clavipesclavipes, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

[0109] In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to a 3X FLAG nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence is operably linked to another affinity tag, such as 6-8 His residues (SEQ ID NO: 107). An example of a vector for delivering a silk polypeptide nucleotide coding sequence into a host cell is shown in Figure 5, which depicts a plasmid map of a vector containing an open reading frame (ORF) for expressing a silk-like polypeptide, including a secretion signal and a FLAG tag. The ORF is operably linked to the pGCW14 promoter and tAOX1 terminator. The vector also contains a selection marker for selecting successfully transformed cells.

[0110] In some embodiments, the silk polypeptide comprises a full-length spider silk polypeptide, hi some embodiments, the full-length spider silk polypeptide is major ampullate spidron 1 (MaSpl) or major ampullate spidroin 2 (MaSp2).

[0111] Silk-like polypeptides In some embodiments, the P. pastoris strains disclosed herein are engineered to express silk-like polypeptides. Preferred embodiment methods for producing silk-like polypeptides are provided in WO 2015 / 042164, particularly paragraphs 114-134, which are incorporated herein by reference. WO 2015 / 042164 discloses synthetic proteinaceous copolymers based on recombinant spider silk protein fragment sequences from MaSp2, such as from the species Argiope bruennichi. Silk-like polypeptides are described that contain 2 to 20 repeat units, each of which has a molecular weight greater than about 20 kDa. Within each repeat unit of the copolymer, there are more than about 60 amino acid residues organized into multiple "quasi-repeat units." In some embodiments, the repeat units of the polypeptides described herein share at least 95% sequence identity with the sequence of the MaSp2 dragline silk protein.

[0112] In some embodiments, each "repeat unit" of a silk-like polypeptide comprises 2 to 20 "quasi-repeat" units (i.e., n3 is 2 to 20). Quasi-repeats do not have to be exact repeats. Each repeat may be composed of a concatenation of quasi-repeats. Formula 1 shows the composition of a repeat unit according to this disclosure and WO2015 / 042164, which is incorporated by reference. Each silk-like polypeptide may have one or more repeat units defined by Formula 1. TIFF2025061129000012.tif4128

[0113] The variable element of the composition, X1 (called a "motif"), follows one of the amino acid sequences shown in Formula 2 below, with X1 varying randomly within each quasi-repeat unit. TIFF2025061129000013.tif11161

[0114] Referring again to Equation 1, in Equation 1 The compositional element of the quasi-repeated unit represented by TIFF2025061129000014.tif4128 is called the "first region." Some quasi-repeated units are formed by repeating the first region 4 to 8 times within the quasi-repeated unit. That is, the value of n1 indicates the number of first region units repeated within a single quasi-repeated unit, and the value of n1 is any one of 4, 5, 6, 7, or 8. "(A) n2 " (SEQ ID NO: 19), is referred to as the "second region" and is formed by repeating the amino acid sequence "A" n times within each quasi-repeat unit (SEQ ID NO: 19). That is, the value of n indicates the number of units of the second region that are repeated within a single quasi-repeat unit, and the value of n is any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the sequence identity of a repeat unit of a polypeptide of the disclosure to a sequence containing quasi-repeats according to Formulas 1 and 2 is at least 95%. In some embodiments, the sequence identity of a repeat unit of a polypeptide of the disclosure to a sequence containing quasi-repeats according to Formulas 1 and 2 is at least 80%, or at least 90%, or at least 95%, or at least 99%.

[0115] In further embodiments, three "long" quasi-repeats are followed by three "short" quasi-repeat units. Short quasi-repeat units are those where n1 = 4 or 5. Long quasi-repeat units are defined as those where n1 = 6, 7, or 8. In some embodiments, all of the short quasi-repeats have the same X1 motif at the same position within each quasi-repeat unit of the repeat unit. In some embodiments, no more than three of the six quasi-repeat units share the same X1 motif.

[0116] In further embodiments, the repeat unit is made up of quasi-repeat units in which the same X1 does not occur more than two times in a row within the repeat unit. In further embodiments, the repeat unit is made up of quasi-repeat units in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the quasi-repeats do not use the same X1 more than two times in a row within a single quasi-repeat unit of the repeat unit.

[0117] Thus, in some embodiments, provided herein are yeast strains that recombinantly express a silk-like polypeptide with reduced degradation, thereby increasing the amount of full-length polypeptide present in a product isolated from a cell culture. In some embodiments, the silk-like polypeptide-expressing strain is a P. pastoris strain comprising a PAS_chr4_0584 knockout and a PAS_chr3_1157 knockout. In some embodiments, the silk-like polypeptide-expressing strain is a P. pastoris strain comprising a sec72 knockout and / or an overexpressed SSH1 translocon complex.

[0118] Table 5. 18B vectors TIFF2025061129000015.tif194139TIFF2025061129000016.tif194144

[0119] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Detailed Description, but rather is set forth in the appended claims.

[0120] In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or clear from the context. A claim or detailed description containing "or" between one or more members of a group is deemed to satisfy the requirement that one, more than one, or all of the members of the group be present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The invention includes embodiments in which one or more, or all, of the members of the group are present in, used in, or otherwise relevant to a given product or process.

[0121] It should also be noted that the term "comprising" is intended to be open-ended and permissive, but does not require the inclusion of additional elements or steps. Thus, when the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0122] When ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, it is to be understood that values ​​expressed as ranges can take any specific value or subrange within the ranges set forth in various embodiments of this invention to the nearest tenth of the lower limit of that range, unless the context clearly dictates otherwise.

[0123] All cited sources, e.g., references, publications, databases, database entries, and art cited herein, are incorporated by reference into this application, even if not explicitly stated otherwise in the citation. In the event of a conflict between the statement in the cited source and this application, the statement in this application shall control.

[0124] Subheadings and table titles are not intended to be limiting. [Example]

[0125] Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0126] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd Ed. (Plenum Press) Vols. A and B (1992).

[0127] Example 1: Production of recombinant yeast expressing 18B A strain of P. pastoris was engineered to recombinantly express the 18B silk-like polypeptide as follows.

[0128] First, a P. pastoris strain was transformed to disrupt KU70 function to facilitate subsequent editing and engineering. GS115 (NRRL Y15851), a HIS+ derivative of Pichia pastoris (Komagataella phaffii) strain, was electroporated with a DNA cassette targeting the KU70 locus, consisting of homology arms flanking a zeocin resistance marker. The sequence is listed in Table 12. Transformants were plated on YPD agar plates supplemented with zeocin, resulting in disruption of KU70 function.

[0129] This strain was then engineered to express a recombinant gene encoding a silk-like polypeptide. A HIS+ derivative of Pichia pastoris (Komagataella phaffii) strain, GS115 (NRRL Y15851), was transformed with the recombinant vector (SEQ ID NO: 20) to express and secrete the silk-like polypeptide ("18B") (SEQ ID NO: 21). Transformation was achieved by electroporation as described in PMID 15679083, which is incorporated herein by reference.

[0130] Each vector contained an 18B expression cassette with a polynucleotide sequence encoding a silk-like protein flanked by a promoter (pGCW14) and a terminator (tAOX1 pA signal) in the recombinant vector. The recombinant vectors also contained a dominant resistance marker for selection of bacterial and yeast transformants, as well as a bacterial origin of replication. The first recombinant vector contained a targeting region that directed integration of the 18B polynucleotide sequence adjacent to the 3' end of the AOX2 locus in the Pichia pastoris genome. The resistance marker in the first vector conferred resistance to G418 (also known as geneticin). The second recombinant vector contained a targeting region that directed integration of the 18B polynucleotide sequence adjacent to the 3' end of the TEF1 locus in the Pichia pastoris genome. The resistance marker in the second vector conferred resistance to hygromycin B.

[0131] Example 2: Construction of recombinant Δsec72 strain Cells engineered to express 18B were transformed with a vector containing a DNA cassette with 5' and 3' homology arms targeting sec72 (SEQ ID NO: 1). As shown in the plasmid map in Figure 2, the homology arms flank a yeast selectable marker and, when inserted into the sec72 gene, knock out the sec72 gene. Transformants were plated onto YPD agar plates supplemented with yeast selection medium and incubated at 30°C for 48 hours.

[0132] Example 3: Generation of recombinant Δsec72 strains overexpressing the SSH1 translocon complex P. pastoris (Δsec72) cells engineered to express 18B (SEQ ID NO: 21) were transformed with a vector for overexpressing the SSH1 translocon complex. The plasmid map of the vector is shown in Figure 3. The vector contains open reading frames for SSH1 (SEQ ID NO: 3), SSS1 (SEQ ID NO: 5), and SBH2 (SEQ ID NO: 7). Each open reading frame is operably linked to a promoter and terminator. Transformants were plated on YPD agar plates supplemented with yeast selective medium and incubated at 30°C for 48 hours.

[0133] Example 4: Production of a protease double knockout strain To generate the ΔΔ protease strain (i.e., double protease knockout), the selected yeast strain was transformed with a vector containing a DNA cassette with approximately 1150 bp of homology arms flanking a nourseothricin resistance marker. The plasmid map containing the nourseothricin resistance marker is shown in Figure 4A, and the sequence is listed in Table 13. The homology arms for each target were amplified with the primers listed in Table 8 and inserted into the nourseothricin resistance plasmid. The homology arms were inserted into the nourseothricin plasmid to generate a cassette containing the nourseothricin resistance marker flanked by 3' and 5' homology arms for the target protease, as shown in Figures 4B and 4C. Figure 4B shows the resistance cassette (Nour resistance cassette) flanked by homology arms (HA1 and HA2). Figure 4C shows details of the nourseothricin marker, which includes the promoter from the ILV5 gene from Saccharomyces cerevisiae (pILV5), the nourseothricin acetyltransferase gene from Streptomyces noursei (nat), and the poly(A) signal from the CYC1 gene from Saccharomyces cerevisiae.

[0134] The engineered yeast strain was transformed with a vector carrying homology arms targeting YPS1-1 (SEQ ID NO: 77). The transformants were plated on YPD agar plates supplemented with nourseothricin and incubated at 30°C for 48 hours.

[0135] Table 6. Proteases targeted for deletion in P. pastoris strains TIFF2025061129000017.tif40128

[0136] To generate a double knockout, nourseothricin resistance was eliminated from the single protease knockout strain prepared above. The single protease knockout strain was transformed with a vector carrying homology arms targeting YPS1-2 (SEQ ID NO: 80). The transformants were plated on YPD agar plates supplemented with nourseothricin and incubated at 30°C for 48 hours.

[0137] Example 5: Δsec72 improves MFα-18B secretion across integration copy numbers Pichia pastoris strains engineered to express two, four, or six copies of 18B (AbMaSp2 79 kDa) containing the MFα1(sc) prepro leader sequence were prepared using the techniques described in Example 1.

[0138] Each of the above Δsec72 strains was prepared using the techniques described in Example 2. Δsec72 and WT strains were prepared for each of the 2X, 4X, and 6X 18B-expressing strains. MFα-18B secretion from the WT and Δsec72 strains (i.e., SEC72KO strains) of the 2X, 4X, and 6X 18B-expressing strains was measured by ELISA, and the results are shown in Figure 6.

[0139] Referring to Figure 6, "Silk copies" is the number of expression cassettes for 18B (Ab MaSp2 79 kDa) using the MFα1(sc) prepro leader sequence in each strain. 18B was C-terminally tagged with a 3xFLAG epitope for ELISA detection. Error bars indicate the standard error of the mean across n > 4 biological replicates. The results demonstrate that deleting the sec72 gene improves secretion of MFα-18B across integrated copy numbers (i.e., 2X, 4X, and 6X silk copies).

[0140] Example 6: Δsec72 improves secretion from non-MFα signals P. pastoris strains were engineered according to Example 1 to express four different silk polypeptides, each containing a different secretion signal: PEP4(sc), CPY+4(sc), DAP2(sc), and MFα1(sc). Each complete leader sequence is a hybrid consisting of the indicated signal peptide and the MFα1(sc) propeptide (systematic name of the S. cerevisiae orthologue: YPL187W). (sc) indicates that the signal peptide is derived from S. cerevisiae. Strains recombinantly expressing polypeptides using PEP4(sc), CPY+4(sc), and DAP2(sc) each contain a single expression cassette (i.e., one silk copy). Strains recombinantly expressing MFα1(sc) contain two expression cassettes (i.e., two silk copies). To compare secretion from the WT and Δsec72 strains, each of the above Δsec72 strains was prepared using the techniques described in Example 2. The Δsec72 strain containing DAP2(sc) was further modified to knock out the YPS1-1 and YPS1-2 proteases as described in Example 4. Secretion of polypeptides from each secretion signal from the WT and Δsec72 strains was measured by ELISA, and the results are shown in Figure 7.

[0141] The above results demonstrate that deletion of the sec72 gene improves secretion of polypeptides containing signal peptides other than MFα1(sc). Error bars indicate the standard error of the mean across n≧4 biological replicates.

[0142] Table 7A: MFα1(sc) peptide sequences TIFF2025061129000018.tif66132

[0143] Table 7B Silk secretion signals TIFF2025061129000019.tif205132

[0144] Example 7: Δsec72 improves secretion of longer silks and distinct silk sequences A P. pastoris strain was engineered according to Example 1 to express the long silk polypeptides Argioppe bruennichi (Ab) MaSp2 (106 kDa) (SEQ ID NO: 43) and Latrodectus hesperus (Lh) MaSp1 (55 kDa) (SEQ ID NO: 44). Ab MaSp2 106 kDa (also known as 24B) is a longer concatamer of Ab MaSp2 79 kDa (18B). Lh MaSp1 55 kDa is a distinct sequence from other spidroins.

[0145] To compare secretion from the WT and Δsec72 strains, each Δsec72 strain was prepared using the techniques described in Example 2. The strain containing Lh MaSpl (55 kDa) was further modified to knock out the YPS1-1 and YPS1-2 proteases as described in Example 4. Secretion of long silk polypeptides from the WT and Δsec72 strains was measured by ELISA, and the results are shown in Figure 8. Error bars indicate the standard error of the mean among n≧4 biological replicates.

[0146] Argioppe bruennichi MaSp2 protein amino acid sequence (SEQ ID NO: 43): TIFF2025061129000020.tif210120

[0147] Latrodectus hesperus MaSp1 protein amino acid sequence (SEQ ID NO: 44): TIFF2025061129000021.tif198120

[0148] Example 8: Deletion of SEC72 improves secretion, but neither knockdown nor overexpression improves it P. pastoris strains were modified according to Example 1 to contain four copies of a DNA cassette containing a recombinant gene expressing the 18B silk-like polypeptide. The Δsec72 strain was prepared using the techniques described in Example 2. These strains were further modified to knock out the YPS1-1 and YPS1-2 proteases, as described in Example 4.

[0149] A strain overexpressing sec72 was prepared from this strain by transformation with a vector containing the recombinant sec72 gene operably linked to the THI11 promoter (pTHI11). pTHI11 is derepressed in minimal medium lacking the vitamin thiamin. Previous RNA-seq and promoter fusion studies have shown that pTHI11 is one of the strongest promoters in block and tank minimal medium lacking thiamin.

[0150] Furthermore, instead of knocking out sec72 from a P. pastoris strain engineered to express four copies of a recombinant gene expressing the 18B silk-like polypeptide, we knocked down SEC72 expression using DAmP (decreased abundance by mRNA perturbation), a transcriptional knockdown method that disrupts the 3'UTR of a gene with a marker cassette.

[0151] Secretion of 18B silk-like polypeptide from the WT strain, the Δsec72 strain, the Δsec72 strain harboring recombinantly overexpressed sec72 (operably linked to pTHI11), and the sec72 knockdown strain was measured by ELISA, and the results are shown in Figure 9A.

[0152] The Δsec72 phenotype was reverted by complementation with an inducible allele of sec72. Similarly, knockdown of SEC72 had no measurable effect on silk secretion compared to WT. Thus, secretion is improved by deleting sec72, but not by knockdown or overexpression of sec72.

[0153] Example 9: Other perturbations to the stoichiometry of the SEC63 complex do not affect silk secretion A P. pastoris strain was modified to contain four copies of a DNA cassette containing a recombinant gene expressing the 18B silk-like polypeptide according to Example 1. The Δsec72 strain was prepared using the techniques described in Example 2.

[0154] To test whether the Δsec72 phenotype was a direct effect of SEC72 removal or an indirect effect of disrupting the stoichiometry of the SEC63 complex, we simultaneously overexpressed three non-SEC72 complex members (SEC62, SEC63, and SEC66) using pTHI11 (by transforming the strain with a vector containing a triple-linked expression cassette for sec62, sec63, and sec66). Additionally, we overexpressed SEC72 using pTHI11. Secretion of 18B from each strain was measured using ELISA and compared with that of the Δsec72 strain. The results are shown in Figure 9B.

[0155] Neither stoichiometric change (overexpression of SEC62, SEC63, and SEC66, nor overexpression of SEC72) had any measurable effect on silk secretion. Error bars indicate the standard error of the mean among n≧3 biological replicates.

[0156] Example 10: RTqPCR demonstrates transcriptional adaptation to Δsec72, including at the translocon level. The Δsec72 strain was prepared using the techniques described in Example 2. After growth, mRNA was isolated and RTqPCR was performed against selected markers to analyze transcriptional adaptation to Δsec72.

[0157] Expression (ALG9 or ACT1) normalized to a reference as fold change from the "WT" strain RMs71 in minimal medium is shown in Figure 10A. Shading indicates the range, and the middle line represents the median across n=3 biological replicate experiments (except n=2 for Δsec72). SSH1 transcript levels increase 4- to 8-fold across all three perturbations. Δsec72 reduces expression of the SEC61 translocon component by 2- to 4-fold.

[0158] Figure 10B shows the projection of centered, log-scaled, reference-normalized expression data onto principal component space (left panel = PC1 vs. PC3, right panel = PC2 vs. PC3). Replicate data are color-coded by strain genotype, indicating low intragenotypic variation relative to intergenotypic variation. Examination of loading coefficients reveals that most variation along PC1 is driven by strong SSA3 repression, and most variation along PC3 is driven by repression of the SEC61 translocon complex. These three PCs reconstruct 99% of the variation from the original 20 dimensions.

[0159] Example 11: Overexpression of SSH1 improves the growth rate of the Δsec72 silk-secreting strain SSH1 also functions in ER translocation but differs from SEC61 in its non-essentiality for S. cerevisiae growth and lack of interaction with the SEC63 complex. SSH1 can also display preferences for translocation substrates, but these preferences overlap extensively with those of SEC61.

[0160] To test whether overexpression of SSH1 can promote the growth of the Δsec72 strain, a P. pastoris Δsec72 strain was prepared using the techniques described in Example 2. The regulatory and coding sequences of the SEC61 and SSH1 complexes (SSS1, SBH2, and one of SEC61 or SSH1) were assembled on an integration plasmid by PCR. The resulting integration duplicates the gene copy number of one of the SEC61 or SSH1 complexes. P. pastoris(Δsec72) cells were transformed with a vector for overexpression of the SSH1 translocon complex as described in Example 3. Similarly, P. pastoris(Δsec72) cells were transformed with a vector for overexpression of SEC61. Selected strains (denoted by "ΔΔ" in Figure 11) were further engineered to knock out the YPS1-1 and YPS1-2 proteases as described in Example 4.

[0161] Precultures in YPD were diluted 1:1600 in minimal medium (RMml7). OD600 was recorded at six time points over an 8-h exponential growth period starting 19 h after dilution. Linear model fit log(OD600) versus time to estimate growth rate and standard error of slope among n=6 biological replicates. Growth measured for each strain is shown in Figure 11. Doubling time is expressed as the reciprocal of growth rate. Error bars are asymmetric due to reciprocal transformation of estimates.

[0162] Replication of SEC61 and SSH1 promoted faster doubling of the SEC72 silk-producing strain (Figure 11, left). This effect may be due to a reduction in the intracellular silk load. However, Δsec72 exhibited a significant growth defect that was partially rescued by replication of SSH1 but not by replication of SEC61 (Figure 11, right).

[0163] Example 12: Duplication of SSH1 improves the fermentation ability of the Δsec72 strain According to Example 1, P. pastoris strains were modified to contain four or six copies of a DNA cassette containing a recombinant gene expressing the 18B silk-like polypeptide. The Δsec72 strain was prepared from recombinant cells expressing four copies of the DNA cassette using the techniques described in Example 2. A portion of the resulting P. pastoris(Δsec72) cells was also transformed with a vector for overexpression of the SSH1 translocon complex, as described in Example 3. Secretion of 18B from each strain was measured using ELISA, and the results are shown in Figures 12A and 12B.

[0164] In Figure 12A, each curve represents an independent fed-batch fermentation. The marked points are samples taken for analysis, including the concentration of silk protein secreted into the broth ("18B titer," y-axis). Because the feeding program is time-varying, the x-axis is the cumulative glucose feed. Shading indicates the full experimental range for the same strain. The Δsec72 deletion strain exhibits highly variable performance. When SSH1 is duplicated in this strain ("2N SSH1"), performance improves and variability decreases. The silk secretion yield of the Δsec72 2N SSH1 strain, which has only four silk expression cassettes, approaches the range of secretion yields of the reference strain with six silk expression cassettes.

[0165] Specific productivity (ratio of product to biomass) was measured for cultures of each strain and is shown in Figure 12B. Δsec72 background growth is slower and produces less biomass, and these metrics rise above those of the higher-expressing, highest-titer strains. Due to the high variability of the Δsec72 strains, only the top n=2 experiments are shown in the combined data. Error bars are standard error of the mean.

[0166] Other embodiments The words that have been used are words of description rather than of limitation, and it is to be understood that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

[0167] While the present invention has been described at considerable length and in considerable detail with reference to several described embodiments, it is not intended that the present invention be limited to any of such individual details or embodiments, or to any particular embodiment, but rather should be construed with reference to the appended claims in order to obtain the broadest possible interpretation of such claims in light of the prior art, so as to effectively encompass the intended scope of the invention.

[0168] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the section headings, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0169] array Table 8: Forward (F) and reverse (R) primers of 5' and 3' homology arms (HA) targeting the protease ORF TIFF2025061129000022.tif53156

[0170] Table 9: Forward and reverse primers for amplifying modified sequences TIFF2025061129000023.tif28135

[0171] Table 10. sec72KO vector TIFF2025061129000024.tif226129TIFF2025061129000025.tif226147TIFF2025061129000026.tif226147TIFF2025061129000027.tif22681

[0172] Table 11. SSH1 complex overexpression vector TIFF2025061129000028.tif22643TIFF2025061129000029.tif226149TIFF2025061129000030.tif226149TIFF20250611290 00031.tif226149TIFF2025061129000032.tif226148TIFF2025061129000033.tif226138TIFF2025061129000034.tif226142

[0173] Table 12. Zeocin cassette with HA arms for KU70 deletion in P. pastoris TIFF2025061129000035.tif219130TIFF2025061129000036.tif219146TIFF2025061129000037.tif219113

[0174] Table 13. Template nouseothricin cassette for protease deletion in P. pastoris TIFF2025061129000038.tif217132TIFF2025061129000039.tif21757

[0175] Table 14. Exemplary nourseothricin cassettes with HA arms for deletion of YPS1-1 and YPS1-2 proteases in P. pastoris TIFF2025061129000040.tif21966TIFF2025061129000041.tif219148TIFF20250611290 00042.tif219148TIFF2025061129000043.tif219149TIFF2025061129000044.tif219114

[0176] Table 15. Gene and sequence information disclosed herein TIFF2025061129000045.tif94156

[0177] Table 16. Promoter and terminator information for SSH1 complex genes TIFF2025061129000046.tif88135

[0178] Sequence information SEQUENCE LISTING <110> BOLT THREADS, INC. <120> SEC MODIFIED STRAINS FOR IMPROVED SECRETION OF RECOMBINANT PROTEINS <150> US 62 / 673,001 <151> 2018-05-17 <160> 107 <170> PatentIn version 3.5 <210> 1 <211> 588 <212> DNA <213> Pichia pastoris <400> 1 atgctaccat tttcgtacga cgtgagctca aagaaactga aagtcacagg tgactacgca 60 gacttagaat atgacataca gcagctgaac accttgagcg gagagatcct ggccaataaa 120 gcagatgttc cttctccacc aagtaaggag tcgtttgaca agaaattgtc ccacatggct 180 cagaaattac acgagtcggc tgtatccaac ataaagacag gcaagtatcc tgaggctatc 240 aaattgttga cgacgggtct tgaaatggtt aacagaaggc ccaagtacga gagttttcag 300 atgacgttga gtgaaatgac gatctttat gtcactagag ctgacgctta catgatgaat 360 ggagactttg aaggggcatt caatgatgca gatttactgg taacgctcct gccatccatt 420 ccagataatt acattagaag aggggtagcc cttttcaaga tggggagata cgttgatgca 480 aaaaacaatt ttgagagagg actttcattt gacccagata atgcaaaatt gaagaaggag 540 ttagattttg tgctgaagaa gatcgacgag gagaatggag agttatag 588 <210> 2 <211> 195 <212> PRT <213> Pichia shepherds <400> 2 Met Leu Pro Phe Ser Tyr Asp Val Ser Ser Lys Lys Lys Val Thr 1 5 10 15 Gly Asp Tyr Ala Asp Leu Glu Tyr Asp With Gln Gln Leu Asn Thr Leu 20 25 30 Ser Gly Glu Ile Leu Ala Asn Lys Ala Asp Val Pro Ser Pro Pro Ser 35 40 45 Lys Glu Ser Phe Asp Lys Lys Leu Ser Met His Met Ala Gln Lys Leu His 50 55 60 Glu Ser Ala Val Ser Asn Ile Lys Thr Gly Lys Tyr Pro Glu Ala Ile 65 70 75 80 Lys Leu Leu Thr Thr Gly Leu Glu Met Val Asn Arg Arg Pro Lys Tyr 85 90 95 Glu Ser Phe Gln Met Thr Leu Ser Glu Met Thr Ile Phe Ile Val Thr 100 105 110 Arg Ala Asp Ala Tyr Met Met Asn Gly Asp Phe Glu Gly Ala Phe Asn 115 120 125 Asp Ala Asp Leu Leu Val Thr Leu Leu Pro Ser Ile Pro Asp Asn Tyr 130 135 140 Ile Arg Arg Gly Val Ala Leu Phe Lys Met Gly Arg Tyr Val Asp Ala 145 150 155 160 Lys Asn Asn Phe Glu Arg Gly Leu Ser Phe Asp Pro Asp Asn Ala Lys 165 170 175 Leu Lys Lys Glu Leu Asp Phe Val Leu Lys Lys Ile Asp Glu Glu Asn 180 185 190 Gly Glu Leu 195 <210> 3 <211> 1440 <212> DNA <213> Pichia pastoris <400> 3 atggcggggt tgcgtttttt agacattgca agaccatttg tcagctggat cccggaagtt 60 gaacttcctt atgaaaactg ggggttcgat gaaaagctga tttactcatt tttcactgct 120 gccatctatt tgattctgtc cctgcctata tacggtgtca aatcctctga agtcgtggac 180 ccagttcccc atttgcgttc tgccttaggg agtgagaagg gaacattgct ggagcttggg 240 ttactgcctg tgattacttc ggcatttatc ttgcagttgt tggctggttg gaaagttttc 300 360. aaagtaaact ttgatctggt tagtgacaga atattgttcc aaactttgca aaagatcact tcagtcgtta tcagcatcgt atatgctgtt cttctcacat tttgtgacta ctttactcca 420 ggtgtgtcca ctgataacgt cttgtggtcc caatttctga tcatcttaca gatagtggtg gtcaacttct tggttactct actcgttgaa gtcattgaca aggattacgg attttcttca ggagctctat tgttgcttgc ggtttattcc gccaccaact tcgtttttgg cacgattggt 600 cttagcaccg tcaacacctc cagatcgaac gaatctattg gtgctctgat tcaattattc 720. cgcaatttga gctctaacc aattggtgtt gccatatatg actccttctt cagagtaaac cttcctaact tgactcaatt ttatctgggg attgccatta tttgtgtttg tctgttcttg 780 father gatacgaagt accaattaag ccaaacaagg ttcgtgccat ggcctcagct tacccaatca agctactttt caatggttct ttgccacttc tgtacacgtg gactgtgctg tacaacttga accttattgg tttctttgtc ttcaagctta ccaacttttc tcttttaggg aacttcaaag tggacccatt cggcaacaac tactacgaaa ttacatctgg actgctgtat ttattgactc ctactttcaa cgctgaagct ggacttttac ccaatgttgc taagccattt 1080 gttttcattg ccttctatgt tggtgttagc actttctttg ctagatcgtg gtccaacatt 1140 aacgggtcgt caggcaagga cattgccaag ttttcaagg ctcaaggaat ctcattgtta 1200 ggaaaaagag atgcctctgt gtctaaagag tttaacaccc tagttcctgt tgcttctgcc 1260 tctggagctt tcctattgtc ttttccagtt gccgtcgctg agttattggg tggctctggt 1320 gttccaacct ctatcggaat cggtcttttg agtggtttgg ctattttgga aactgttttg 1380 caagaatggc aacgtctgg aggtgcctca cagttctccc aatacttcca gacttcttag 1440 <210> 4 <211> 479 <212> PRT <213> Pichia pastoris <400> 4 Met Ala Gly Leu Arg Phe Leu Asp Ile Ala Arg Pro Phe Val Ser Trp 1 5 10 15 Ile Pro Glu Val Glu Leu Pro Tyr Glu Asn Trp Gly Phe Asp Glu Lys 20 25 30 Leu Ile Tyr Ser Phe Phe Thr Ala Ala Ile Tyr Leu Ile Leu Ser Leu 35 40 45 Pro Ile Tyr Gly Val Lys Ser Ser Glu Val Val Asp Pro Val Pro His 50 55 60 Leu Arg Ser Ala Leu Gly Ser Glu Lys Gly Thr Leu Leu Glu Leu Gly 65 70 75 80 Leu Leu Pro Val Ile Thr Ser Ala Phe Ile Leu Gln Leu Leu Ala Gly 85 90 95 Trp Lys Val Phe Lys Val Asn Phe Asp Leu Val Ser Asp Arg Ile Leu 100 105 110 Phe Gln Thr Leu Gln Lys Ile Thr Ser Val Val Ile Ser Ile Val Tyr 115 120 125 Ala Val Leu Leu Thr Phe Cys Asp Tyr Phe Thr Pro Gly Val Ser Thr 130 135 140 Asp Asn Val Leu Trp Ser Gln Phe Leu Ile Ile Leu Gln Ile Val Val 145 150 155 160 Val Asn Phe Leu Val Thr Leu Leu Val Glu Val Ile Asp Lys Asp Tyr 165 170 175 Gly Phe Ser Ser Gly Ala Leu Leu Leu Leu Ala Val Tyr Ser Ala Thr 180 185 190 Asn Phe Val Phe Gly Thr Ile Gly Leu Ser Thr Val Asn Thr Ser Arg 195 200 205 Ser Asn Glu Ser Ile Gly Ala Leu Ile Gln Leu Phe Arg Asn Leu Ser 210 215 220 Ser Lys Pro Ile Gly Val Ala Ile Tyr Asp Ser Phe Phe Arg Val Asn 225 230 235 240 Leu Pro Asn Leu Thr Gln Phe Tyr Leu Gly Ile Ala Ile Ile Cys Val 245 250 255 Cys Leu Phe Leu Asn Asn Ala Arg Tyr Glu Val Pro Ile Lys Pro Asn 260 265 270 Lys Val Arg Ala Met Ala Ser Ala Tyr Pro Ile Lys Leu Leu Phe Asn 275 280 285 Gly Ser Leu Pro Leu Leu Tyr Thr Trp Thr Val Leu Tyr Asn Leu Asn 290 295 300 Leu Ile Gly Phe Phe Val Phe Lys Leu Thr Asn Phe Ser Leu Leu Gly 305 310 315 320 Asn Phe Lys Val Asp Pro Phe Gly Asn Asn Tyr Tyr Glu Ile Thr Ser 325 330 335 Gly Leu Leu Tyr Leu Leu Thr Pro Thr Phe Asn Ala Glu Ala Gly Leu 340 345 350 Leu Pro Asn Val Ala Lys Pro Phe Val Phe Ile Ala Phe Tyr Val Gly 355 360 365 Val Ser Thr Phe Phe Ala Arg Ser Trp Ser Asn Ile Asn Gly Ser Ser 370 375 380 Gly Lys Asp Ile Ala Lys Phe Phe Lys Ala Gln Gly Ile Ser Leu Leu 385 390 395 400 Gly Lys Arg Asp Ala Ser Val Ser Lys Glu Phe Asn Thr Leu Val Pro 405 410 415 Val Ala Ser Ala Ser Gly Ala Phe Leu Leu Ser Phe Pro Val Ala Val 420 425 430 Ala Glu Leu Leu Gly Gly Ser Gly Val Pro Thr Ser Ile Gly Ile Gly 435 440 445 Leu Leu Ser Gly Leu Ala Ile Leu Glu Thr Val Leu Gln Glu Trp Gln 450 455 460 Gln Ser Gly Gly Ala Ser Gln Phe Ser Gln Tyr Phe Gln Thr Ser 465 470 475 <210> 5 <211> 195 <212> DNA <213> Pichia pastoris <400> 5 atgtcccaaa aagtcaccga cgtccctctg gaatttgtta aggaaggttc caaattcatc 60 tctaaatgta ctaaaccctc tcagaggag tacttaaga tagtaagagc tgttggagtt 120 gggtttta tgatggggt gttggttac gttgtcaagc tcattcat tcaatcaga 180 tattgattg ttta 195 <210> 6 <211> 64 <212> PRT <213> Pichia shepherds <400> 6 Met Ser Gln Lys Val Thr Asp Val Pro Leu Glu Phe Val Lys Glu Gly 1 5 10 15 Lys Phe Ile Cys Thr Lys Pro Serves Gln Lys Glu Tyr Leu 20 25 30 Lys Ile Val Arg Ala Val Gly Val Gly Phe Leu Met Met Gly Val Val 35 40 45 Gly Tyr Val Val Lys Leu Ile His Ile Pro Ile Arg Tyr Leu Ile Val 50 55 60 <210> 7 <211> 326 <212> DNA <213> Pichia shepherds <400> 7 atggtaagtg tccagtttga tgagtgcaga atggttccaa gttttagacc agttactaat 60 atttaaagtc acagcatt ccaggaggac agaacgtt agctaaaga agagagac 120 acttggataa gaaacaggat gaaccaacct ccgccagatc tgccggtgct ggaggttctt 180 cgtctaccat gctaaagttg tacacagacg agccccaagg tttgaagtt gatcctttaa 240 tgttcttgt tcttgctgtt gtttcattt tcagtgtcat tggtttgcac gttgttgcta 300 agctgacagg aaagttgatc actaa 326 <210> 8 <211> 86 <212> PRT <213> Pichia shepherds <400> 8 Met Ser Thr Ala Ile Pro Gly Gly Gln Arg Thr Leu Ala Lys Arg Arg 1 5 10 15 Ala Ala Asn Leu Asp Lys Lys Gln Asp Glu Pro Thr Ser Ala Arg Ser 20 25 30 Ala Gly Ala Gly Gly Ser Ser Thr Met Leu Lys Leu Tyr Thr Asp 35 40 45 Glu Ala Gln Gly Lion Lys Val Asp Pro Lion Nose Val Lion Val Lion Wing 50 55 60 Val Gly Phe Ile Phe Ser Val Ile Gly Leu His Val Val Ala Lys Leu 65 70 75 80 Thr Gly Lys Ile Asn 85 <210> 9 <211> 1800 <212> DNA <213> Pichia pastoris <400> 9 atgttgaagg atcagttctt gttatgggtt gctttgatag cgagcgtacc ggtttccggc 60 gtgatggcag ctcctagcga gtccgggcat aacacggttg aaaaacgaga tgccaaaaac 120 gttgttggcg ttcaacagtt ggacttcagc gttctgaggg gtgattcctt cgaaagtgcc 180 tcttcagaga acgtgcctcg gcttgtgagg agagatgaca cgctagaagc tgagctaatc 240 aaccagcaat cattctactt gtcacgactg aaagttggat cacatcaagc ggatattgga 300 atcctagtgg acacaggatc ctctgattta tgggtaatgg actcggtaaa cccatactgc 360 agtagccgtt cccgcgtgaa gagagatata cacgatgaga agatcgccga atgggatccc 420 atcaatctca agaaaaatga aacttctcag aataaaaatt tttgggattg gctcgttgga 480 actagcacta gttctccttc caccgccacg gcaactggta gtggtagtgg tagtggtagt 540 ggtagtggta gtggtagtgc tgccacagcc gtatcggtaa gttctgcaca ggcaacattg 600 gattgctcta cgtatggaac gtttgatcac gctgattcct cgacgttcca tgacaataat 660 acagactttt tcatctcata cgctgatacc acttttgctt caggaatctg gggttatgac 720 gacgtcatta tcgacggcat agaggtgaaa gaactttcct tcgccgttgc agacatgacc 780 aattcctcta ttggtgtgtt aggtattgga ctgaaaggcc tagaatccac atatgctagt 840 gcatcttcgg tcagtgaaat gtatcagtat gacaatttgc cagccaagat ggtcaccgat 900 gggttgatca acaaaaatgc atactccttg tacttgaact ccaaggacgc ctcaagtggt 960 tccatcctct ttggaggtgt ggatcatgaa aaatattcgg gacaattgtt gacagttcca 1020 gtcatcaaca cactcgcttc cagtggttac agagaggcaa ttcgtttaca aattacttta 1080 aatggaatag atgtgaaaaa gggttctgac cagggaactc ttttacaagg gagatttgct 1140 gcattattgg actctggagc tacgctaacg tatgctcctt cttctgtttt aaattcaatt 1200 ggccggaacc tgggcggctc ctatgattcg tcaagacaag cttataccat tcgttgtgtt 1260 tctgcatcag ataccacttc tctggtattc aattttgggg gtgctacagt ggaagtttcc 1320 ctgtacgatc tacagattgc aacatattac accgggggaa gtgccacgca atgtcttatt 1380 ggaatattca gctctggaag tgatgagttt gtgctcggtg ataccttctt gaggtcagcc 1440 tacgtggttt acgatcttga tgggcttgaa gtgtcgcttg cccaagccaa cttcaacgaa 1500 accgattctg atgttgaggc tattacctcc agtgtacctt ccgctactcg tgcatccgga 1560 tacagttcta catggtctgg ttctgccagc ggtacagttt acacttcggt tcagatggaa 1620 tccggtgctg cttccagctc caactcttct ggatcgaata tgggttcctc ttcctcatcg 1680 tcctcttcat cgtcctcgac ttccagtgga gacgaagaag gagggagctc cgccaacagg 1740 gtccccttca gctacctttc tctctgtttg gtagttattc tcggcgtgtg tatagtatag 1800 <210> 10 <211> 599 <212> PRT <213> Pichia pastoris <400> 10 Met Leu Lys Asp Gln Phe Leu Leu Trp Val Ala Leu Ile Ala Ser Val 1 5 10 15 Pro Val Ser Gly Val Met Ala Ala Pro Ser Glu Ser Gly His Asn Thr 20 25 30 Val Glu Lys Arg Asp Ala Lys Asn Val Val Gly Val Gln Gln Leu Asp 35 40 45 Phe Ser Val Leu Arg Gly Asp Ser Phe Glu Ser Ala Ser Ser Glu Asn 50 55 60 Val Pro Arg Leu Val Arg Arg Asp Asp Thr Leu Glu Ala Glu Leu Ile 65 70 75 80 Asn Gln Gln Ser Phe Tyr Leu Ser Arg Leu Lys Val Gly Ser His Gln 85 90 95 Ala Asp Ile Gly Ile Leu Val Asp Thr Gly Ser Ser Asp Leu Trp Val 100 105 110 Met Asp Ser Val Asn Pro Tyr Cys Ser Ser Arg Ser Arg Val Lys Arg 115 120 125 Asp Ile His Asp Glu Lys Ile Ala Glu Trp Asp Pro Ile Asn Leu Lys 130 135 140 Lys Asn Glu Thr Ser Gln Asn Lys Asn Phe Trp Asp Trp Leu Val Gly 145 150 155 160 Thr Ser Thr Ser Ser Pro Ser Thr Ala Thr Ala Thr Gly Ser Gly Ser 165 170 175 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Ala Ala Thr Ala Val Ser 180 185 190 Val Ser Ser Ala Gln Ala Thr Leu Asp Cys Ser Thr Tyr Gly Thr Phe 195 200 205 Asp His Ala Asp Ser Ser Thr Phe His Asp Asn Asn Thr Asp Phe Phe 210 215 220 Ile Ser Tyr Ala Asp Thr Thr Phe Ala Ser Gly Ile Trp Gly Tyr Asp 225 230 235 240 Asp Val Ile Ile Asp Gly Ile Glu Val Lys Glu Leu Ser Phe Ala Val 245 250 255 Ala Asp Met Thr Asn Ser Ser Ile Gly Val Leu Gly Ile Gly Leu Lys 260 265 270 Gly Leu Glu Ser Thr Tyr Ala Ser Ala Ser Ser Val Ser Glu Met Tyr 275 280 285 Gln Tyr Asp Asn Leu Pro Ala Lys Met Val Thr Asp Gly Leu Ile Asn 290 295 300 Lys Asn Ala Tyr Ser Leu Tyr Leu Asn Ser Lys Asp Ala Ser Ser Gly 305 310 315 320 Ser Ile Leu Phe Gly Gly Val Asp His Glu Lys Tyr Ser Gly Gln Leu 325 330 335 Leu Thr Val Pro Val Ile Asn Thr Leu Ala Ser Ser Gly Tyr Arg Glu 340 345 350 Ala Ile Arg Leu Gln Ile Thr Leu Asn Gly Ile Asp Val Lys Lys Gly 355 360 365 Ser Asp Gln Gly Thr Leu Leu Gln Gly Arg Phe Ala Ala Leu Leu Asp 370 375 380 Ser Gly Ala Thr Leu Thr Tyr Ala Pro Ser Ser Val Leu Asn Ser Ile 385 390 395 400 Gly Arg Asn Leu Gly Gly Ser Tyr Asp Ser Ser Arg Gln Ala Tyr Thr 405 410 415 Ile Arg Cys Val Ser Ala Ser Asp Thr Thr Ser Leu Val Phe Asn Phe 420 425 430 Gly Gly Ala Thr Val Glu Val Ser Leu Tyr Asp Leu Gln Ile Ala Thr 435 440 445 Tyr Tyr Thr Gly Gly Ser Ala Thr Gln Cys Leu Ile Gly Ile Phe Ser 450 455 460 Ser Gly Ser Asp Glu Phe Val Leu Gly Asp Thr Phe Leu Arg Ser Ala 465 470 475 480 Tyr Val Val Tyr Asp Leu Asp Gly Leu Glu Val Ser Leu Ala Gln Ala 485 490 495 Asn Phe Asn Glu Thr Asp Ser Asp Val Glu Ala Ile Thr Ser Ser Val 500 505 510 Pro Ser Ala Thr Arg Ala Ser Gly Tyr Ser Ser Thr Trp Ser Gly Ser 515 520 525 Ala Ser Gly Thr Val Tyr Thr Ser Val Gln Met Glu Ser Gly Ala Ala 530 535 540 Ser Ser Ser Asn Ser Ser Gly Ser Asn Met Gly Ser Ser Ser Ser Ser 545 550 555 560 Ser Ser Ser Ser Ser Ser Thr Ser Ser Ser Gly Asp Glu Glu Gly Gly Ser 565 570 575 Ser Ala Asn Arg Val Pro Phe Ser Tyr Leu Ser Leu Cys Leu Val Val 580 585 590 Ile Leu Gly Val Cys Ile Val 595 <210> 11 <211> 1781 <212> DNA <213> Pichia pastoris <400> 11 atgatcatca accacttggt attgacagcc ctcagcattg cactagcaag tgcgcaactc 60 caatcgcctt tcaaggctaa caagttgcca ttcaaaaagt ttatcattcc aacgacccaa 120 aggaccgttt aattaagaga gatgactacg agtccctcga cttgagacac atcggagtct 180 tgtacactgc agagatccaa attggatctg acgaaactga aattgaggtc attgtcgaca 240 ctggttctgc cgacttgtgg gtcatcgatt ccgacgctgc cgtctgtgag ttatcctacg 300 atgagattga ggccaatagc ttttcctcgg cttctgccaa attcatggac aagatagctc 360 ctccatcaca agagctcctg gatgggctga gtgagtttgg atttgctctc gatggtgaaa 420 tttctcaata cctagccgat aaatctggac gtgtttcgaa aagagaggaa aatcaacaag 480 atttcaacat taaccgtgac gagcctgtgt gtgaacagtt tggttcctt gattctagtt 540 cttccgacac tttccaaagc aacaattcag cttttggtat tgcttacctt gatggaacca 600 ctgctaacgg aacttgggtc agggacacag tccgcatcgg cgactttgcc atcagccaac 660 agagttttgc cttagtcaac atcacagata actacatggg aatcttgggt ctcggtcctg 720 ctacccaaa aaccaccaat agtaacccaa ttgcagcaaa cagatttact tatgatggtg 780 ttgtggattc attgcggtcc caaggattta tcaattcagc atcgttttct gtttacttgt 840 ctccagatga agataacgag caggacgaat tcagcgacgg agaaatttta tttggtgcta 900 ttgatagggc caagatagac gggccattta gacttttcc atatgtcaat ccttacaaac 960 cagtttaccc cgatcaatat acttcctacg ttacagtgtc cacaattgcg gtgtcttcgt 1020 cagatgaaac tctcattatt gaaagacgtc ctcgtttggc attaatcgat acaggtgcca 1080 ccttctccta tttgccaacc tacccattga ttcgtttagc gttttccatc catggaggct 1140 ttgaatatgt ttctcaattg ggactatttg tcattcgtac aagttctctg tctgttgcta 1200 gaaataaggt gattgagttc aagtttggtg aagacgttgt gatccaatcc ccagtttctg 1260 atcatctatt ggacgtctca ggccttttta ctgatggcca acaatactcc gcattaactg 1320 tacgtgaaag tcttgacgga ctttccattc taggtgatac attcatcaaa tcggcctact 1380 tattctttga caatgaaaac agccagctgg gtattggtca gatcaacgtc actgatgacg 1440 aggatattga ggtggtcggt gatttcacta ttgaacgaga cccagcctac tcctctactt 1500 ggtctagcga tttacctcat gaaacaccca ctagggcttt gagtactgct tcagggggag 1560 gccttggtac cggaataaac acggccacaa gtcgtgcaag ttctcgttcc acatctggct 1620 ctacttcacg aacttcttct acatctggct ctgcttctgg tacttcttca ggtgcatctt 1680 ctgctactca aaatgacgaa acatccactg atcttggagc tccagctgca tctttaagtg 1740 caacgccatg tctttttgcc atcttgctgc tcatgttgta g 1781 <210> 12 <211> 562 <212> PRT <213> Pichia pastoris <400> 12 Met Ile Ile Asn His Leu Val Leu Thr Ala Leu Ser Ile Ala Leu Ala 1 5 10 15 Asn Asp Tyr Glu Ser Leu Asp Leu Arg His Ile Gly Val Leu Tyr Thr 20 25 30 Ala Glu Ile Gln Ile Gly Ser Asp Glu Thr Glu Ile Glu Val Ile Val 35 40 45 Asp Thr Gly Ser Ala Asp Leu Trp Val Ile Asp Ser Asp Ala Ala Val 50 55 60 Cys Glu Leu Ser Tyr Asp Glu Ile Glu Ala Asn Ser Phe Ser Ser Ala 65 70 75 80 Ser Ala Lys Phe Met Asp Lys Ile Ala Pro Pro Ser Gln Glu Leu Leu 85 90 95 Asp Gly Leu Ser Glu Phe Gly Phe Ala Leu Asp Gly Glu Ile Ser Gln 100 105 110 Tyr Leu Ala Asp Lys Ser Gly Arg Val Ser Lys Arg Glu Glu Asn Gln 115 120 125 Gln Asp Phe Asn Ile Asn Arg Asp Glu Pro Val Cys Glu Gln Phe Gly 130 135 140 Ser Phe Asp Ser Ser Ser Ser Asp Thr Phe Gln Ser Asn Asn Ser Ala 145 150 155 160 Phe Gly Ile Ala Tyr Leu Asp Gly Thr Thr Ala Asn Gly Thr Trp Val 165 170 175 Arg Asp Thr Val Arg Ile Gly Asp Phe Ala Ile Ser Gln Gln Ser Phe 180 185 190 Ala Leu Val Asn Ile Thr Asp Asn Tyr Met Gly Ile Leu Gly Leu Gly 195 200 205 Pro Ala Thr Gln Gln Thr Thr Asn Ser Asn Pro Ile Ala Ala Asn Arg 210 215 220 Phe Thr Tyr Asp Gly Val Val Asp Ser Leu Arg Ser Gln Gly Phe Ile 225 230 235 240 Asn Ser Ala Ser Phe Ser Val Tyr Leu Ser Pro Asp Glu Asp Asn Glu 245 250 255 His Asp Glu Phe Ser Asp Gly Glu Ile Leu Phe Gly Ala Ile Asp Arg 260 265 270 Ala Lys Ile Asp Gly Pro Phe Arg Leu Phe Pro Tyr Val Asn Pro Tyr 275 280 285 Lys Pro Val Tyr Pro Asp Gln Tyr Thr Ser Tyr Val Thr Val Ser Thr 290 295 300 Ile Ala Val Ser Ser Ser Asp Glu Thr Leu Ile Ile Glu Arg Arg Pro 305 310 315 320 Arg Leu Ala Leu Ile Asp Thr Gly Ala Thr Phe Ser Tyr Leu Pro Thr 325 330 335 Tyr Pro Leu Ile Arg Leu Ala Phe Ser Ile His Gly Gly Phe Glu Tyr 340 345 350 Val Ser Gln Leu Gly Leu Phe Val Ile Arg Thr Ser Ser Leu Ser Val 355 360 365 Ala Arg Asn Lys Val Ile Glu Phe Lys Phe Gly Glu Asp Val Val Ile 370 375 380 Gln Ser Pro Val Ser Asp His Leu Leu Asp Val Ser Gly Leu Phe Thr 385 390 395 400 Asp Gly Gln Gln Tyr Ser Ala Leu Thr Val Arg Glu Ser Leu Asp Gly 405 410 415 Leu Ser Ile Leu Gly Asp Thr Phe Ile Lys Ser Ala Tyr Leu Phe Phe 420 425 430 Asp Asn Glu Asn Ser Gln Leu Gly Ile Gly Gln Ile Asn Val Thr Asp 435 440 445 Asp Glu Asp Ile Glu Val Val Gly Asp Phe Thr Ile Glu Arg Asp Pro 450 455 460 Ala Tyr Ser Ser Thr Trp Ser Ser Asp Leu Pro His Glu Thr Pro Thr 465 470 475 480 Arg Ala Leu Ser Thr Ala Ser Gly Gly Gly Leu Gly Thr Gly Ile Asn 485 490 495 Thr Ala Thr Ser Arg Ala Ser Ser Arg Ser Thr Ser Gly Ser Thr Ser 500 505 510 Arg Thr Ser Ser Thr Ser Gly Ser Ala Ser Gly Thr Ser Ser Gly Ala 515 520 525 Ser Ser Ala Thr Gln Asn Asp Glu Thr Ser Thr Asp Leu Gly Ala Pro 530 535 540 Ala Ala Ser Leu Ser Ala Thr Pro Cys Leu Phe Ala Ile Leu Leu Leu 545 550 555 560 Met Leu <210> 13 <211> 1800 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (7)..(11) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (15)..(19) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (23)..(27) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (31)..(35) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (39)..(43) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (47)..(51) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (55)..(59) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (63)..(67) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (4)..(67) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (71)..(90) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (97)..(101) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (105)..(109) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (113)..(117) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (121)..(125) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (129)..(133) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (137)..(141) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (145)..(149) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (153)..(157) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (94)..(157) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (161)..(180) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (187)..(191) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (195)..(199) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (203)..(207) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (211)..(215) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (219)..(223) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (227)..(231) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (235)..(239) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (243)..(247) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (184)..(247) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (251)..(270) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (277)..(281) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (285)..(289) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (293)..(297) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (301)..(305) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (309)..(313) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (317)..(321) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (325)..(329) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (333)..(337) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (274)..(337) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (341)..(360) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (367)..(371) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (375)..(379) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (383)..(387) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (391)..(395) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (399)..(403) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (407)..(411) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (415)..(419) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (423)..(427) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (364)..(427) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (431)..(450) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (457)..(461) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (465)..(469) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (473)..(477) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (481)..(485) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (489)..(493) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (497)..(501) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (505)..(509) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (513)..(517) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (454)..(517) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (521)..(540) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (547)..(551) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (555)..(559) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (563)..(567) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (571)..(575) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (579)..(583) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (587)..(591) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (595)..(599) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (603)..(607) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (544)..(607) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (611)..(630) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (637)..(641) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (645)..(649) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (653)..(657) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (661)..(665) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (669)..(673) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (677)..(681) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (685)..(689) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (693)..(697) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (634)..(697) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (701)..(720) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (727)..(731) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (735)..(739) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (743)..(747) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (751)..(755) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (759)..(763) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (767)..(771) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (775)..(779) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (783)..(787) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (724)..(787) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (791)..(810) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (817)..(821) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (825)..(829) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (833)..(837) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (841)..(845) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (849)..(853) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (857)..(861) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (865)..(869) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (873)..(877) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (814)..(877) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (881)..(900) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (907)..(911) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (915)..(919) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (923)..(927) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (931)..(935) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (939)..(943) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (947)..(951) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (955)..(959) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (963)..(967) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (904)..(967) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (971)..(990) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (997)..(1001) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1005)..(1009) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1013)..(1017) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1021)..(1025) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1029)..(1033) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1037)..(1041) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1045)..(1049) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1053)..(1057) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (994)..(1057) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1061)..(1080) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1087)..(1091) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1095)..(1099) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1103)..(1107) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1111)..(1115) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1119)..(1123) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1127)..(1131) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1135)..(1139) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1143)..(1147) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1084)..(1147) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1151)..(1170) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1177)..(1181) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1185)..(1189) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1193)..(1197) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1201)..(1205) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1209)..(1213) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1217)..(1221) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1225)..(1229) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1233)..(1237) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1174)..(1237) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1241)..(1260) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1267)..(1271) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1275)..(1279) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1283)..(1287) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1291)..(1295) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1299)..(1303) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1307)..(1311) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1315)..(1319) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1323)..(1327) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1264)..(1327) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1331)..(1350) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1357)..(1361) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1365)..(1369) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1373)..(1377) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1381)..(1385) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1389)..(1393) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1397)..(1401) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1405)..(1409) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1413)..(1417) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1354)..(1417) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1421)..(1440) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1447)..(1451) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1455)..(1459) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1463)..(1467) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1471)..(1475) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1479)..(1483) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1487)..(1491) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1495)..(1499) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1503)..(1507) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1444)..(1507) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1511)..(1530) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1537)..(1541) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1545)..(1549) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1553)..(1557) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1561)..(1565) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1569)..(1573) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1577)..(1581) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1585)..(1589) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1593)..(1597) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1534)..(1597) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1601)..(1620) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1627)..(1631) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1635)..(1639) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1643)..(1647) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1651)..(1655) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1659)..(1663) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1667)..(1671) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1675)..(1679) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1683)..(1687) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1624)..(1687) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1691)..(1710) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1717)..(1721) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1725)..(1729) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1733)..(1737) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1741)..(1745) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1749)..(1753) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1757)..(1761) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1765)..(1769) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1773)..(1777) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1714)..(1777) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1781)..(1800) <223> This region may encompass 6-20 residues <220> <221> MISC_FEATURE <222> (1)..(1800) <223> This sequence may encompass 2-20 "GGY-[GPG-X1]n1-GPS-(A)n2" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," n1 is 4-8 and n2 is 6-20 and some positions may be absent <400> 13 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 1 5 10 15 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 20 25 30 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 35 40 45 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 50 55 60 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 65 70 75 80 No No No No No No No No No Gly Gly Tyr Gly Pro Gly 85 90 95 Free Free Gly Pro Gly Free Free Gly Pro Gly 100 105 110 Free Free Gly Pro Gly Free Free Gly Pro Gly 115 120 125 Free Free Gly Pro Gly Free Free Gly Pro Gly 130 135 140 Free Free Gly Pro Gly Free Free Gly Pro Ser 145 150 155 160 No No No No No No No No No No No No No No 165 170 175 No No No No Gly Gly Tyr Gly Pro Gly Yes Yes Yes Yes Yes Gly 180 185 190 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 195 200 205 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 210 215 220 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 225 230 235 240 Pro Gly Yes Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala 245 250 255 No No No No No No No No No No No No No No No No Gly Gly 260 265 270 Tyr Gly Pro Gly How Too Much How To Gly Pro Gly Too Much 275 280 285 Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes Yes 290,295,300 Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes Yes 305 310 315 320 Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes Yes 325 330 335 Xaa Gly Pro Be Not Not Not Not Not Not Not Not Not Not Not Not not not noticeable 340 345 350 No No No No No No No No Gly Gly Tyr Gly Pro Gly Stone 355 360 365 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 370 375 380 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 385 390 395 400 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 405 410 415 Free Free Gly Pro Gly Free Free Gly Pro Ser Ala Ala 420 425 430 No No No No No No No No No No No No No No 435 440 445 No No Gly Gly Tyr Gly Pro Gly Yes Yes Yes Yes Gly Pro Gly 450 455 460 Free Free Gly Pro Gly Free Free Gly Pro Gly 465 470 475 480 Free Free Gly Pro Gly Free Free Gly Pro Gly 485,490,495 Free Free Gly Pro Gly Free Free Gly Pro Gly 500 505 510 No No No No No Gly Pro Ser No No No No No No No 515,520,525 No No No No No No No No No No No No Gly Gly Tyr Gly 530 535 540 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 545 550 555 560 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 565,570,575 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 580,585,590 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 595,600,605 Pro Being Not Not Not Not Not Not Not Not Not Not Us 610 615 620 No No No No No No Gly Gly Tyr Gly Pro Gly Yes Yes Yes Yes 625 630 635 640 Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes Yes 645,650,655 Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes Yes 660,665,670 Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes Yes 675,680,685 Yes Gly Pro Gly Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala 690,695,700 No No No No No No No No No No No No No No 705 710 715 720 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 725 730 735 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 740,745,750 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 755,760,765 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 770,775,780 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 785,790,795,800 No No No No No No No No No Gly Gly Tyr Gly Pro Gly 805 810 815 Free Free Gly Pro Gly Free Free Gly Pro Gly 820 825 830 Free Free Gly Pro Gly Free Free Gly Pro Gly 835 840 845 Free Free Gly Pro Gly Free Free Gly Pro Gly 850 855 860 Free Free Gly Pro Gly Free Free Gly Pro Ser 865 870 875 880 No No No No No No No No No No No No No No 885,890,895 No No No No Gly Gly Tyr Gly Pro Gly Yes Yes Yes Yes Yes Gly 900 905 910 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 915,920,925 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 930,935,940 Pro Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 945 950 955 960 Pro Gly Yes Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala 965,970,975 No No No No No No No No No No No No No No No Gly Gly 980,985,990 Tyr Gly Pro Gly How Too Much How To Gly Pro Gly Too Much 995 1000 1005 No Gly Pro Gly No No No No Gly Pro Gly No no 1010 1015 1020 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1025 1030 1035 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1040 1045 1050 Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 1055 1060 1065 No No No No No No No No No No No Gly Gly Tyr 1070 1075 1080 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1085 1090 1095 No Gly Pro Gly No No No No Gly Pro Gly No no 1100 1105 1110 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1115 1120 1125 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1130 1135 1140 Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 1145 1150 1155 No No No No No No No No No No No Gly Gly Tyr 1160 1165 1170 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1175 1180 1185 No Gly Pro Gly No No No No Gly Pro Gly No no 1190 1195 1200 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1205 1210 1215 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1220 1225 1230 Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 1235 1240 1245 No No No No No No No No No No No Gly Gly Tyr 1250 1255 1260 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1265 1270 1275 No Gly Pro Gly No No No No Gly Pro Gly No no 1280 1285 1290 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1295 1300 1305 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1310 1315 1320 Yeah Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 1325 1330 1335 No No No No No No No No No No No Gly Gly Tyr 1340 1345 1350 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1355 1360 1365 No Gly Pro Gly No No No No Gly Pro Gly No no 1370 1375 1380 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1385 1390 1395 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1400 1405 1410 Yeah Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 1415 1420 1425 No No No No No No No No No No No Gly Gly Tyr 1430 1435 1440 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1445 1450 1455 No Gly Pro Gly No No No No Gly Pro Gly No no 1460 1465 1470 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1475 1480 1485 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1490 1495 1500 Yeah Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 1505 1510 1515 No No No No No No No No No No No Gly Gly Tyr 1520 1525 1530 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1535 1540 1545 No Gly Pro Gly No No No No Gly Pro Gly No no 1550 1555 1560 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1565 1570 1575 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1580 1585 1590 Yeah Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 1595 1600 1605 No No No No No No No No No No No Gly Gly Tyr 1610 1615 1620 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1625 1630 1635 No Gly Pro Gly No No No No Gly Pro Gly No no 1640 1645 1650 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1655 1660 1665 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1670 1675 1680 Yeah Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 1685 1690 1695 No No No No No No No No No No No Gly Gly Tyr 1700 1705 1710 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1715 1720 1725 No Gly Pro Gly No No No No Gly Pro Gly No no 1730 1735 1740 Too Much Gly Pro Gly Too Much Too Much Not Too Much Gly Pro Gly 1745 1750 1755 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1760 1765 1770 Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 1775 1780 1785 No No No No No No No No No No 1790 1795 1800 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Ser Gly Gly Gln Gln 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Gly Ala Gly Gln Gln 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Gly Gln Gly Pro Tyr 1 5 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Ala Gly Gln Gln 1 <210> 18 <211> 70 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (7)..(11) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (15)..(19) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (23)..(27) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (31)..(35) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (39)..(43) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (47)..(51) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (55)..(59) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (63)..(67) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (4)..(67) <223> This region may encompass 4-8 "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent. <400> 18 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 1 5 10 15 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 20 25 30 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 35 40 45 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 50 55 60 No. No. No. Gly Pro Ser 65 70 <210> 19 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MISC_FEATURES <222> (1)..(20) <223> This sequence may encompass 6-20 residues <400> 19 No No No No No No No No No No No No No No 1 5 10 15 God God God God 20 <210> 20 <211> 2835 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 20 ggtggttacg gtccaggcgc tggtcaacaa ggtccaggaa gtggtggtca acaaggacct 60 ggcggtcaag gaccctacgg tagtggccaa caaggtccag gtggagcagg acagcagggt 120 ccgggaggcc aaggacctta cggaccaggt gctgctgctg ccgccgctgc cgctgccgga 180 ggttacggtc caggagccgg acaacagggt ccaggtggag ctggacaaca aggtccagga 240 tcacaaggtc ctggtggaca aggtccatac ggtcctggtg ctggtcaaca gggaccaggt 300 agtcaaggac ctggttcagg tggtcagcag ggtccaggag gacagggtcc ttacggccct 360 tctgccgctg cagcagcagc cgctgccgca ggaggatacg gacctggtgc tggacaacga 420 tctcaaggac caggaggaca aggtccttat ggacctggcg ctggccaaca aggacctggt 480 tctcagggtc caggttcagg aggccaacaa ggcccaggag gtcaaggacc atacggacca 540 tccgctgcgg cagctgcagc tgctgcaggt ggatatggcc caggagccgg acaacagggt 600 cctggttcac aaggtccagg atctggtggt caacagggac caggcggcca gggaccttat 660 ggtccaggag ccgctgcagc agcagcagct gttggaggtt acggccctgg tgccggtcaa 720 caaggcccag gatctcaggg tcctggatct ggaggacaac aaggtcctgg aggtcagggt 780 ccatacggac cttcagcagc agctgctgct gcagccgctg gtggttatgg acctggtgct 840 ggtcaacaag gaccgggttc tcagggtccg ggttcaggag gtcagcaggg ccctggtgga 900 caaggacctt atggacctag tgcggctgca gcagctgccg ccgcaggtgg ttacggtcca 960 ggcgctggtc aacaaggtcc aggaagtggt ggtcaacaag gacctggcgg tcaaggaccc 1020 tacggtagtg gccaacaagg tccaggtgga gcaggacagc agggtccggg aggccaagga 1080 ccttacggac caggtgctgc tgctgccgcc gctgccgctg ccggaggtta cggtccagga 1140 gccggacaac agggtccagg tggagctgga caacaaggtc caggatcaca aggtcctggt 1200 ggacaaggtc catacggtcc tggtgctggt caacagggac caggtagtca aggacctggt tcaggtggtc agcagggtcc aggaggacag ggtccttacg gcccttctgc cgctgcagca 1320 gcagccgctg ccgcaggagg atacggacct ggtgctggac aacgatctca aggaccagga ggacaaggtc cttatggacc tggcgctggc caacaaggac ctggttctca gggtccaggt tcaggaggcc aacaaggccc aggaggtcaa ggaccatacg gaccatccgc tgcggcagct gcagctgctg caggtggata tggcccagga gccggacaac agggtcctgg ttcacaaggt 1560 ccaggatctg gtggtcaaca gggaccaggc ggccagggac cttatggtcc aggagccgct gcagcagcag cagctgttgg aggttacggc cctggtgccg gtcaacaagg cccaggatct cagggtcctg gatctggagg acaacaaggt cctggaggtc agggtccata cggaccttca gcagcagctg ctgctgcagc cgctggtggt tatggacctg gtgctggtca acaaggaccg ggttctcagg gtccgggttc aggaggtcag caggggccctg gtggacaagg accttatgga cctagtgcgg ctgcagcagc tgccgccgca ggtggttacg gtccaggcgc tggtcaacaa ggtccaggaa gtggtggtca acaaggacct ggcggtcaag gaccctacgg tagtggccaa 1980 caaggtccag gtggagcagg acagcagggt ccgggaggcc aaggacctta cggaccaggt 2040 gctgctgctg ccgccgctgc cgctgccgga ggttacggtc caggagccgg acaacagggt 2100 ccaggtggag ctggacaaca aggtccagga tcacaaggtc ctggtggaca aggtccatac 2160 ggtcctggtg ctggtcaaca gggaccaggt agtcaaggac ctggttcagg tggtcagcag 2220 ggtccaggag gacagggtcc ttacggccct tctgccgctg cagcagcagc cgctgccgca 2280 ggaggatacg gacctggtgc tggacaacga tctcaaggac caggaggaca aggtccttat 2340 ggacctggcg ctggccaaca aggacctggt tctcagggtc caggttcagg aggccaacaa 2400 ggcccaggag gtcaaggacc atacggacca tccgctgcgg cagctgcagc tgctgcaggt 2460 ggatatggcc caggagccgg acaacagggt cctggttcac aaggtccagg atctggtggt 2520 caacagggac caggcggcca gggaccttat ggtccaggag ccgctgcagc agcagcagct 2580 gttggaggtt acggccctgg tgccggtcaa caaggcccag gatctcaggg tcctggatct 2640 ggaggacaac aaggtcctgg aggtcagggt ccatacggac cttcagcagc agctgctgct 2700 gcagccgctg gtggttatgg acctggtgct ggtcaacaag gaccgggttc tcagggtccg 2760 ggttcaggag gtcagcaggg ccctggtgga caaggacctt atggacctag tgcggctgca 2820 gcagctgccg ccgca 2835 <210> 21 <211> 945 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly 1 5 10 15 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 35 40 45 Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 50 55 60 Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 65 70 75 80 Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln 85 90 95 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 100 105 110 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro 130 135 140 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 145 150 155 160 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly 165 170 175 Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 180 185 190 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 195 200 205 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 210 215 220 Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln 225 230 235 240 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 245 250 255 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 260 265 270 Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 275 280 285 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 290 295 300 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 305 310 315 320 Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly 325 330 335 Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly 340 345 350 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala 355 360 365 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 370 375 380 Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly 385 390 395 400 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser 405 410 415 Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 420 425 430 Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 435 440 445 Gly Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro Gly Gly Gln Gly Pro 450 455 460 Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly 465 470 475 480 Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser 485 490 495 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly 500 505 510 Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly 515 520 525 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala 530 535 540 Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser 545 550 555 560 Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 565 570 575 Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly 580 585 590 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly 595 600 605 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala 610 615 620 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 625 630 635 640 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 645 650 655 Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 660 665 670 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala 675 680 685 Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala 690 695 700 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr 705 710 715 720 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 725 730 735 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala 740 745 750 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly 755 760 765 Gln Arg Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 770 775 780 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln 785 790 795 800 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala 805 810 815 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 820 825 830 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly 835 840 845 Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr 850 855 860 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 865 870 875 880 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala 885 890 895 Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln 900 905 910 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 915 920 925 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 930 935 940 Ala 945 <210> 22 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 22 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly 1 5 10 15 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 35 40 45 Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 50 55 60 Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 65 70 75 80 Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln 85 90 95 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 100 105 110 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro 130 135 140 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 145 150 155 160 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly 165 170 175 Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 180 185 190 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 195 200 205 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 210 215 220 Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln 225 230 235 240 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 245 250 255 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 260 265 270 Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 275 280 285 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 290 295 300 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 305 310 315 <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Ser Gly Ala Gly Gly 1 5 <210> 24 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Gly Ser Gly Ala Gly 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Gly Gly Ser Gly Ala 1 5 <210> 26 <211> 181 <212> PRT <213> Aliatypus gulosus <400> 26 Gly Ala Ala Ser Ser Ser Ser Thr Ile Ile Thr Thr Lys Ser Ala Ser 1 5 10 15 Ala Ser Ala Ala Ala Asp Ala Ser Ala Ala Ala Thr Ala Ser Ala Ala 20 25 30 Ser Arg Ser Ser Ala Asn Ala Ala Ala Ser Ala Phe Ala Gln Ser Phe 35 40 45 Ser Ser Ile Leu Leu Glu Ser Gly Tyr Phe Cys Ser Ile Phe Gly Ser 50 55 60 Ser Ile Ser Ser Ser Tyr Ala Ala Ala Ile Ala Ser Ala Ala Ser Arg 65 70 75 80 Ala Ala Ala Glu Ser Asn Gly Tyr Thr Thr His Ala Tyr Ala Cys Ala 85 90 95 Lys Ala Val Ala Ser Ala Val Glu Arg Val Thr Ser Gly Ala Asp Ala 100 105 110 Tyr Ala Tyr Ala Gln Ala Ile Ser Asp Ala Leu Ser His Ala Leu Leu 115 120 125 Tyr Thr Gly Arg Leu Asn Thr Ala Asn Ala Asn Ser Leu Ala Ser Ala 130 135 140 Phe Ala Tyr Ala Phe Ala Asn Ala Ala Ala Gln Ala Ser Ala Ser Ser 145 150 155 160 Ala Ser Ala Gly Ala Ala Ser Ala Ser Gly Ala Ala Ser Ala Ser Gly 165 170 175 Ala Gly Ser Ala Ser 180 <210> 27 <211> 126 <212> PRT <213> Plectreurys tristis <400> 27 Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Ser Thr Ser Val Ser Thr Ser Ser Ser Ser Gly Ser 20 25 30 Gly Ala Gly Ala Gly Ala Gly Ser Gly Ala Gly Ser Gly Ala Gly Ala 35 40 45 Gly Ser Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly 50 55 60 Phe Gly Ser Gly Leu Gly Leu Gly Tyr Gly Val Gly Leu Ser Ser Ala 65 70 75 80 Gln Ala Gln Ala Gln Ala Gln Ala Ala Ala Gln Ala Gln Ala Gln Ala 85 90 95 Gln Ala Gln Ala Tyr Ala Ala Ala Gln Ala Gln Ala Gln Ala Gln Ala 100 105 110 Gln Ala Gln Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 115 120 125 <210> 28 <211> 239 <212> PRT <213> Plectreurys tristis <400> 28 Gly Ala Ala Gln Lys Gln Pro Ser Gly Glu Ser Ser Val Ala Thr Ala 1 5 10 15 Ser Ala Ala Ala Thr Ser Val Thr Ser Gly Gly Ala Pro Val Gly Lys 20 25 30 Pro Gly Val Pro Ala Pro Ile Phe Tyr Pro Gln Gly Pro Leu Gln Gln 35 40 45 Gly Pro Ala Pro Gly Pro Ser Asn Val Gln Pro Gly Thr Ser Gln Gln 50 55 60 Gly Pro Ile Gly Gly Val Gly Gly Ser Asn Ala Phe Ser Ser Ser Phe 65 70 75 80 Ala Ser Ala Leu Ser Leu Asn Arg Gly Phe Thr Glu Val Ile Ser Ser 85 90 95 Ala Ser Ala Thr Ala Val Ala Ser Ala Phe Gln Lys Gly Leu Ala Pro 100 105 110 Tyr Gly Thr Ala Phe Ala Leu Ser Ala Ala Ser Ala Ala Ala Asp Ala 115 120 125 Tyr Asn Ser Ile Gly Ser Gly Ala Asn Ala Phe Ala Tyr Ala Gln Ala 130 135 140 Phe Ala Arg Val Leu Tyr Pro Leu Val Gln Gln Tyr Gly Leu Ser Ser 145 150 155 160 Ser Ala Lys Ala Ser Ala Phe Ala Ser Ala Ile Ala Ser Ser Phe Ser 165 170 175 Ser Gly Thr Ser Gly Gln Gly Pro Ser Ile Gly Gln Gln Gln Pro Pro 180 185 190 Val Thr Ile Ser Ala Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala 195 200 205 Val Gly Gly Gly Gln Val Gly Gln Gly Pro Tyr Gly Gly Gln Gln Gln 210 215 220 Ser Thr Ala Ala Ser Ala Ser Ala Ala Ala Ala Thr Ala Thr Ser 225 230 235 <210> 29 <211> 182 <212> PRT <213> Araneus gemmoides <400> 29 Gly Asn Val Gly Tyr Gln Leu Gly Leu Lys Val Ala Asn Ser Leu Gly 1 5 10 15 Leu Gly Asn Ala Gln Ala Leu Ala Ser Ser Leu Ser Gln Ala Val Ser 20 25 30 Ala Val Gly Val Gly Ala Ser Ser Asn Ala Tyr Ala Asn Ala Val Ser 35 40 45 Asn Ala Val Gly Gln Val Leu Ala Gly Gln Gly Ile Leu Asn Ala Ala 50 55 60 Asn Ala Gly Ser Leu Ala Ser Ser Phe Ala Ser Ala Leu Ser Ser Ser 65 70 75 80 Wing Wing Ser Val Wing Ser Gln Ser Wing Ser Gln Ser Gln Wing Wing Ser 85 90 95 Gln Ser Gln Ala Ala Ser Ala Phe Arg Gln Ala Ser Gln Ser 100 105 110 Ala Ser Gln Ser Asp Ser Arg Ala Gly Ser Gln Ser Ser Thr Lys Thr 115 120 125 Thr Ser Thr Ser Thr Ser Gly Ser Gln Ala Asp Ser Arg Ser Ala Ser 130 135 140 Be Be Wing Be Gln Wing Be Wing Be Wing Phe Wing Gln Gln Be Be 145 150 155 160 Ala Ser Leu Ser Ser Ser Ser Ser Phe Ser Ser Ala Phe Ser Ser Ala 165 170 175 Thr Ser Ile Ser Ala Val 180 <210> 30 <211> 180 <212> PRT <213> Argiope aurantia <400> 30 Gly Ser Leu Ala Ser Ser Phe Ala Ser Ala Leu Ser Ala Ser Ala Ala 1 5 10 15 Being Val Ala Being Being Ala Ala Ala Gln Ala Ala Being Gln Being Gln Ala 20 25 30 Wing Wing Ser Wing Phe Ser Arg Wing Wing Ser Gln Ser Wing Ser Gln Ser 35 40 45 Ala Ala Arg Ser Gly Ala Gln Ser Ile Ser Thr Thr Thr Thr Thr Ser 50 55 60 Thr Ala Gly Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ala Ala Ser 65 70 75 80 Gln Ala Ser Ala Ser Ser Phe Ala Arg Ala Ser Ser Ala Ser Leu Ala 85 90 95 Ala Ser Ser Ser Phe Ser Ser Ala Phe Ser Ser Ala Asn Ser Leu Ser 100 105 110 Ala Leu Gly Asn Val Gly Tyr Gln Leu Gly Phe Asn Val Ala Asn Asn 115 120 125 Leu Gly Ile Gly Asn Ala Ala Gly Leu Gly Asn Ala Leu Ser Gln Ala 130 135 140 Val Ser Ser Val Gly Val Gly Ala Ser Ser Ser Thr Tyr Ala Asn Ala 145 150 155 160 Val Ser Asn Ala Val Gly Gln Phe Leu Ala Gly Gln Gly Ile Leu Asn 165 170 175 Ala Ala Asn Ala 180 <210> 31 <211> 199 <212> PRT <213> Deinopis spinosa <400> 31 Gly Ala Ser Ala Ser Ala Tyr Ala Ser Ala Ile Ser Asn Ala Val Gly 1 5 10 15 Pro Tyr Leu Tyr Gly Leu Gly Leu Phe Asn Gln Ala Asn Ala Ala Ser 20 25 30 Phe Ala Ser Ser Phe Ala Ser Ala Val Ser Ser Ala Val Ala Ser Ala 35 40 45 Ser Ala Ser Ala Ala Ser Ser Ala Tyr Ala Gln Ser Ala Ala Ala Gln 50 55 60 Ala Gln Ala Ala Ser Ser Ala Phe Ser Gln Ala Ala Ala Gln Ser Ala 65 70 75 80 Ala Ala Ala Ser Ala Gly Ala Ser Ala Gly Ala Gly Ala Ser Ala Gly 85 90 95 Ala Gly Ala Val Ala Gly Ala Gly Ala Val Ala Gly Ala Gly Ala Val 100 105 110 Ala Gly Ala Ser Ala Ala Ala Ala Ala Ser Gln Ala Ala Ala Ser Ser Ser 115 120 125 Ala Ser Ala Val Ala Ser Ala Phe Ala Gln Ser Ala Ser Tyr Ala Leu 130 135 140 Ala Ser Ser Ser Ala Phe Ala Asn Ala Phe Ala Ser Ala Thr Ser Ala 145 150 155 160 Gly Tyr Leu Gly Ser Leu Ala Tyr Gln Leu Gly Leu Thr Thr Ala Tyr 165 170 175 Asn Leu Gly Leu Ser Asn Ala Gln Ala Phe Ala Ser Thr Leu Ser Gln 180 185 190 Ala Val Thr Gly Val Gly Leu 195 <210> 32 <211> 171 <212> PRT <213> Nephila clavipes <400> 32 Gly Ala Thr Ala Ala Ser Tyr Gly Asn Ala Leu Ser Thr Ala Ala Ala 1 5 10 15 Gln Phe Phe Ala Thr Ala Gly Leu Leu Asn Ala Gly Asn Ala Ser Ala 20 25 30 Leo Ala Ser Ser Phe Ala Arg Ala Phe Ser Ala Ser Ala Glu Ser Gln 35 40 45 Ser Phe Ala Gln Ser Gln Ala Phe Gln Ala Gln Ser Ala Phe Gln Gln 50 55 60 Ala Ala Ser Arg Ser Ala Ser Gln Ser Ala Ala Glu Ala Gly Ser Thr 65 70 75 80 Ser Ser Ser Thr Thr Thr Thr Thr Thr Ser Ala Ala Arg Ser Gln Ala Ala 85 90 95 Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala Phe Ala Gln Ala Ala 100 105 110 Ser Ser Ser Leu Ala Thr Ser Ser Ala Leu Ser Arg Ala Phe Ser Ser 115 120 125 Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Ser Ile Gly Leu 130 135 140 Ser Ala Ala Arg Ser Leu Gly Ile Ala Asp Ala Ala Gly Leu Ala Gly 145 150 155 160 Val Leu Ala Arg Ala Ala Gly Ala Leu Gly Gln 165 170 <210> 33 <211> 268 <212> PRT <213> Argiope trifasciata <400> 33 Gly Gly Ala Pro Gly Gly Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala 1 5 10 15 Gly Phe Gly Pro Gly Gly Gly Ala Gly Phe Gly Pro Gly Gly Gly Ala 20 25 30 Gly Phe Gly Pro Gly Gly Ala Ala Gly Gly Pro Gly Gly Pro Gly Gly 35 40 45 Pro Gly Gly Pro Gly Gly Ala Gly Gly Tyr Gly Pro Gly Gly Ala Gly 50 55 60 Gly Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Tyr Gly 65 70 75 80 Pro Gly Gly Ala Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly 85 90 95 Ala Gly Pro Gly Gly Ala Gly Gly Glu Gly Pro Val Thr Val Asp Val 100 105 110 Asp Val Thr Val Gly Pro Glu Gly Val Gly Gly Gly Pro Gly Gly Ala 115 120 125 Gly Pro Gly Gly Ala Gly Phe Gly Pro Gly Gly Gly Ala Gly Phe Gly 130 135 140 Pro Gly Gly Ala Pro Gly Ala Pro Gly Gly Pro Gly Gly Pro Gly Gly 145 150 155 160 Pro Gly Gly Pro Gly Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly 165 170 175 Gly Tyr Gly Pro Gly Gly Ala Gly Gly Val Gly Pro Ala Gly Thr Gly 180 185 190 Gly Phe Gly Pro Gly Gly Ala Gly Gly Phe Gly Pro Gly Gly Ala Gly 195 200 205 Gly Phe Gly Pro Gly Gly Ala Gly Gly Phe Gly Pro Ala Gly Ala Gly 210 215 220 Gly Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Phe Gly 225 230 235 240 Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly 245 250 255 Glu Gly Pro Val Thr Val Asp Val Asp Val Ser Val 260 265 <210> 34 <211> 420 <212> PRT <213> Nephila clavipes <400> 34 Gly Val Ser Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly 1 5 10 15 Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro Gly Gly Ala Gly Gly Pro 20 25 30 Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr 35 40 45 Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly 50 55 60 Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro 65 70 75 80 Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly 85 90 95 Gly Tyr Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly 100 105 110 Ser Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Thr 115 120 125 Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly 130 135 140 Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro 145 150 155 160 Gly Gly Phe Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly 165 170 175 Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly 180 185 190 Phe Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Ala Pro Gly Gly Ala 195 200 205 Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly 210 215 220 Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Gly 225 230 235 240 Ala Gly Gly Ala Gly Gly Ser Gly Gly Ala Gly Gly Ser Gly Gly Thr 245 250 255 Thr Ile Ile Glu Asp Leu Asp Ile Thr Ile Asp Gly Ala Asp Gly Pro 260 265 270 Ile Thr Ile Ser Glu Glu Leu Pro Ile Ser Gly Ala Gly Gly Ser Gly 275 280 285 Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro 290 295 300 Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Pro Gly 305 310 315 320 Gly Ser Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Pro Tyr 325 330 335 Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Pro 340 345 350 Gly Gly Ala Tyr Gly Pro Gly Gly Ser Tyr Gly Pro Gly Gly Ser Gly 355 360 365 Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro 370 375 380 Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr 385 390 395 400 Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Gly 405 410 415 Pro Tyr Gly Pro 420 <210> 35 <211> 376 <212> PRT <213> Latrodectus hesperus <400> 35 Gly Ile Asn Val Asp Ser Asp Ile Gly Ser Val Thr Ser Leu Ile Leu 1 5 10 15 Ser Gly Ser Thr Leu Gln Met Thr Ile Pro Ala Gly Gly Asp Asp Leu 20 25 30 Ser Gly Gly Tyr Pro Gly Gly Phe Pro Ala Gly Ala Gln Pro Ser Gly 35 40 45 Gly Ala Pro Val Asp Phe Gly Gly Pro Ser Ala Gly Gly Asp Val Ala 50 55 60 Ala Lys Leu Ala Arg Ser Leu Ala Ser Thr Leu Ala Ser Ser Gly Val 65 70 75 80 Phe Arg Ala Ala Phe Asn Ser Arg Val Ser Thr Pro Val Ala Val Gln 85 90 95 Leu Thr Asp Ala Leu Val Gln Lys Ile Ala Ser Asn Leu Gly Leu Asp 100 105 110 Tyr Ala Thr Ala Ser Lys Leu Arg Lys Ala Ser Gln Ala Val Ser Lys 115 120 125 Val Arg Met Gly Ser Asp Thr Asn Ala Tyr Ala Leu Ala Ile Ser Ser 130 135 140 Ala Leu Ala Glu Val Leu Ser Ser Ser Gly Lys Val Ala Asp Ala Asn 145 150 155 160 Ile Asn Gln Ile Ala Pro Gln Leu Ala Ser Gly Ile Val Leu Gly Val 165 170 175 Ser Thr Thr Ala Pro Gln Phe Gly Val Asp Leu Ser Ser Ile Asn Val 180 185 190 Asn Leu Asp Ile Ser Asn Val Ala Arg Asn Met Gln Ala Ser Ile Gln 195 200 205 Gly Gly Pro Ala Pro Ile Thr Ala Glu Gly Pro Asp Phe Gly Ala Gly 210 215 220 Tyr Pro Gly Gly Ala Pro Thr Asp Leu Ser Gly Leu Asp Met Gly Ala 225 230 235 240 Pro Ser Asp Gly Ser Arg Gly Gly Asp Ala Thr Ala Lys Leu Leu Gln 245 250 255 Ala Leu Val Pro Ala Leu Leu Lys Ser Asp Val Phe Arg Ala Ile Tyr 260 265 270 Lys Arg Gly Thr Arg Lys Gln Val Val Gln Tyr Val Thr Asn Ser Ala 275 280 285 Leu Gln Gln Ala Ala Ser Ser Leu Gly Leu Asp Ala Ser Thr Ile Ser 290 295 300 Gln Leu Gln Thr Lys Ala Thr Gln Ala Leu Ser Ser Val Ser Ala Asp 305 310 315 320 Ser Asp Ser Thr Ala Tyr Ala Lys Ala Phe Gly Leu Ala Ile Ala Gln 325 330 335 Val Leu Gly Thr Ser Gly Gln Val Asn Asp Ala Asn Val Asn Gln Ile 340 345 350 Gly Ala Lys Leu Ala Thr Gly Ile Leu Arg Gly Ser Ser Ala Val Ala 355 360 365 Pro Arg Leu Gly Ile Asp Leu Ser 370 375 <210> 36 <211> 200 <212> PRT <213> Argiope trifasciata <400> 36 Gly Ala Gly Tyr Thr Gly Pro Ser Gly Pro Ser Thr Gly Pro Ser Gly 1 5 10 15 Tyr Pro Gly Pro Leu Gly Gly Gly Ala Pro Phe Gly Gln Ser Gly Phe 20 25 30 Gly Gly Ser Ala Gly Pro Gln Gly Gly Phe Gly Ala Thr Gly Gly Ala 35 40 45 Ser Ala Gly Leu Ile Ser Arg Val Ala Asn Ala Leu Ala Asn Thr Ser 50 55 60 Thr Leu Arg Thr Val Leu Arg Thr Gly Val Ser Gln Gln Ile Ala Ser 65 70 75 80 Ser Val Val Gln Arg Ala Ala Gln Ser Leu Ala Ser Thr Leu Gly Val 85 90 95 Asp Gly Asn Asn Leu Ala Arg Phe Ala Val Gln Ala Val Ser Arg Leu 100 105 110 Pro Ala Gly Ser Asp Thr Ser Ala Tyr Ala Gln Ala Phe Ser Ser Ala 115 120 125 Leu Phe Asn Ala Gly Val Leu Asn Ala Ser Asn Ile Asp Thr Leu Gly 130 135 140 Ser Arg Val Leu Ser Ala Leu Leu Asn Gly Val Ser Ser Ala Ala Gln 145 150 155 160 Gly Leu Gly Ile Asn Val Asp Ser Gly Ser Val Gln Ser Asp Ile Ser 165 170 175 Ser Ser Ser Ser Phe Leu Ser Thr Ser Ser Ser Ser Ala Ser Tyr Ser 180 185 190 Gln Ala Ser Ala Ser Ser Thr Ser 195 200 <210> 37 <211> 357 <212> PRT <213> Uloborus diversus <400> 37 Gly Ala Ser Ala Ala Asp Ile Ala Thr Ala Ile Ala Ala Ser Val Ala 1 5 10 15 Thr Ser Leu Gln Ser Asn Gly Val Leu Thr Ala Ser Asn Val Ser Gln 20 25 30 Leu Ser Asn Gln Leu Ala Ser Tyr Val Ser Ser Gly Leu Ser Ser Thr 35 40 45 Ala Ser Ser Leu Gly Ile Gln Leu Gly Ala Ser Leu Gly Ala Gly Phe 50 55 60 Gly Ala Ser Ala Gly Leu Ser Ala Ser Thr Asp Ile Ser Ser Ser Val 65 70 75 80 Glu Ala Thr Ser Ala Ser Thr Leu Ser Ser Ser Ala Ser Ser Thr Ser 85 90 95 Val Val Ser Ser Ile Asn Ala Gln Leu Val Pro Ala Leu Ala Gln Thr 100 105 110 Ala Val Leu Asn Ala Ala Phe Ser Asn Ile Asn Thr Gln Asn Ala Ile 115 120 125 Arg Ile Ala Glu Leu Leu Thr Gln Gln Val Gly Arg Gln Tyr Gly Leu 130 135 140 Ser Gly Ser Asp Val Ala Thr Ala Ser Ser Gln Ile Arg Ser Ala Leu 145 150 155 160 Tyr Ser Val Gln Gln Gly Ser Ala Ser Ser Ala Tyr Val Ser Ala Ile 165 170 175 Val Gly Pro Leu Ile Thr Ala Leu Ser Ser Arg Gly Val Val Asn Ala 180 185 190 Ser Asn Ser Ser Gln Ile Ala Ser Ser Leu Ala Thr Ala Ile Leu Gln 195 200 205 Phe Thr Ala Asn Val Ala Pro Gln Phe Gly Ile Ser Ile Pro Thr Ser 210 215 220 Ala Val Gln Ser Asp Leu Ser Thr Ile Ser Gln Ser Leu Thr Ala Ile 225 230 235 240 Ser Ser Gln Thr Ser Ser Ser Val Asp Ser Ser Thr Ser Ala Phe Gly 245 250 255 Gly Ile Ser Gly Pro Ser Gly Pro Ser Pro Tyr Gly Pro Gln Pro Ser 260 265 270 Gly Pro Thr Phe Gly Pro Gly Pro Ser Leu Ser Gly Leu Thr Gly Phe 275 280 285 Thr Ala Thr Phe Ala Ser Ser Phe Lys Ser Thr Leu Ala Ser Ser Thr 290 295 300 Gln Phe Gln Leu Ile Ala Gln Ser Asn Leu Asp Val Gln Thr Arg Ser 305 310 315 320 Ser Leu Ile Ser Lys Val Leu Ile Asn Ala Leu Ser Ser Leu Gly Ile 325 330 335 Ser Ala Ser Val Ala Ser Ser Ile Ala Ala Ser Ser Ser Gln Ser Leu 340 345 350 Leu Ser Val Ser Ala 355 <210> 38 <211> 32 <212> PRT <213> Euprosthenops australis <400> 38 Gly Gly Gln Gly Gly Gln Gly Gln Gly Arg Tyr Gly Gln Gly Ala Gly 1 5 10 15 Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 20 25 30 <210> 39 <211> 42 <212> PRT <213> Tetragnatha kauaiensis <400> 39 Gly Gly Leu Gly Gly Gly Gln Gly Ala Gly Gln Gly Gly Gln Gln Gly 1 5 10 15 Ala Gly Gln Gly Gly Tyr Gly Ser Gly Leu Gly Gly Ala Gly Gln Gly 20 25 30 Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala 35 40 <210> 40 <211> 42 <212> PRT <213> Argiope aurantia <400> 40 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 1 5 10 15 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Leu Gly Pro Tyr Gly 20 25 30 Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 35 40 <210> 41 <211> 46 <212> PRT <213> Deinopis spinosa <400> 41 Gly Pro Gly Gly Tyr Gly Gly Pro Gly Gln Gln Gly Pro Gly Gln Gly 1 5 10 15 Gln Tyr Gly Pro Gly Thr Gly Gln Gln Gly Gln Gly Pro Ser Gly Gln 20 25 30 Gln Gly Pro Ala Gly Ala Ala Ala Ala Ala Ala 35 40 45 <210> 42 <211> 42 <212> PRT <213> Nephila clavata <400> 42 Gly Pro Gly Gly Tyr Gly Leu Gly Gln Gln Gly Pro Gly Gln Gln Gly 1 5 10 15 Pro Gly Gln Gln Gly Pro Ala Gly Tyr Gly Pro Ser Gly Leu Ser Gly 20 25 30 Pro Gly Gly Way Way Way Way Way Way Way Way 35 40 <210> 43 <211> 3360 <212> PRT <213> Argiope bruennichi <400> 43 Met Asn Trp Ser Ile Arg Leu Ala Leu Leu Gly Phe Val Val Leu Ser 1 5 10 15 Thr Gln Thr Val Phe Ala Val Gly Gln Ala Ala Thr Pro Trp Glu Asn 20 25 30 Ser Gln Leu Ala Glu Asp Phe Ile Asn Ser Phe Leu Arg Phe Ile Ala 35 40 45 Gln Ser Gly Ala Phe Ser Pro Asn Gln Leu Asp Asp Met Ser Ser Ile 50 55 60 Gly Asp Thr Leu Lys Thr Ala Ile Glu Lys Met Ala Gln Ser Arg Lys 65 70 75 80 Ser Ser Lys Ser Lys Leu Gln Ala Leu Asn Met Ala Phe Ala Ser Ser 85 90 95 Met Ala Glu Ile Ala Val Ala Glu Gln Gly Gly Leu Ser Leu Glu Ala 100 105 110 Lys Thr Asn Ala Ile Ala Asn Ala Leu Ala Ser Ala Phe Leu Glu Thr 115 120 125 Thr Gly Phe Val Asn Gln Gln Phe Val Ser Glu Ile Lys Ser Leu Ile 130 135 140 Tyr Met Ile Ala Gln Ala Ser Ser Asn Glu Ile Ser Gly Ser Ala Ala 145 150 155 160 Ala Ala Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gln Gly Gly 165 170 175 Tyr Gly Gln Gly Ala Ser Ala Ser Ala Ser Ala Ala Ala Ala Tyr Gly 180 185 190 Ser Ala Pro Gln Gly Ala Gly Gly Pro Ala Pro Gln Gly Pro Ser Gln 195 200 205 Gln Gly Pro Val Ser Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala 210 215 220 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro 225 230 235 240 Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln 245 250 255 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 260 265 270 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly 275 280 285 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly 290 295 300 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala 305 310 315 320 Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 325 330 335 Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly 340 345 350 Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 355 360 365 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 370 375 380 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 385 390 395 400 Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 405 410 415 Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly 420 425 430 Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln 435 440 445 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala 450 455 460 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly 465 470 475 480 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Gly 485 490 495 Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 500 505 510 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 515 520 525 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 530 535 540 Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly 545 550 555 560 Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly 565 570 575 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala 580 585 590 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser 595 600 605 Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 610 615 620 Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly 625 630 635 640 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly 645 650 655 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala 660 665 670 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 675 680 685 Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly 690 695 700 Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 705 710 715 720 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly 725 730 735 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly 740 745 750 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 755 760 765 Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 770 775 780 Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 785 790 795 800 Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln 805 810 815 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 820 825 830 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 835 840 845 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro 850 855 860 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 865 870 875 880 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly 885 890 895 Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 900 905 910 Ala Gly Arg Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln 915 920 925 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala 930 935 940 Ala Ala Ala Ala Gly Pro Gly Ala Arg Arg Gln Gly Pro Gly Ser Gln 945 950 955 960 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 965 970 975 Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 980 985 990 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala 995 1000 1005 Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Gly 1010 1015 1020 Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Gly Gln Gly 1025 1030 1035 Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 1040 1045 1050 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 1055 1060 1065 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly 1070 1075 1080 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 1085 1090 1095 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly 1100 1105 1110 Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly Ala 1115 1120 1125 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln 1130 1135 1140 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala 1145 1150 1155 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 1160 1165 1170 Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 1175 1180 1185 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala 1190 1195 1200 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 1205 1210 1215 Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser 1220 1225 1230 Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly 1235 1240 1245 Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala 1250 1255 1260 Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Gly 1265 1270 1275 Ala Gly Gln Gln Gly Pro Glu Gly Pro Gly Ser Gln Gly Pro Gly 1280 1285 1290 Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro 1295 1300 1305 Gly Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly 1310 1315 1320 Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly 1325 1330 1335 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala 1340 1345 1350 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln 1355 1360 1365 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly 1370 1375 1380 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala 1385 1390 1395 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro 1400 1405 1410 Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 1415 1420 1425 Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 1430 1435 1440 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala 1445 1450 1455 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 1460 1465 1470 Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Gly Gln 1475 1480 1485 Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 1490 1495 1500 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 1505 1510 1515 Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 1520 1525 1530 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly 1535 1540 1545 Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly 1550 1555 1560 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala 1565 1570 1575 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala 1580 1585 1590 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln 1595 1600 1605 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala 1610 1615 1620 Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 1625 1630 1635 Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 1640 1645 1650 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala 1655 1660 1665 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 1670 1675 1680 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln 1685 1690 1695 Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly 1700 1705 1710 Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly 1715 1720 1725 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln 1730 1735 1740 Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 1745 1750 1755 Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly 1760 1765 1770 Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln 1775 1780 1785 Gln Gly Pro Gly Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 1790 1795 1800 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 1805 1810 1815 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser 1820 1825 1830 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala 1835 1840 1845 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln 1850 1855 1860 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala 1865 1870 1875 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 1880 1885 1890 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 1895 1900 1905 Tyr Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly 1910 1915 1920 Pro Gly Gly Gln Gly Pro Tyr Gly Gly Gly Tyr Gly Pro Gly Ala 1925 1930 1935 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln 1940 1945 1950 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala 1955 1960 1965 Ala Ala Ala Ala Ala Gly Pro Gly Ala Arg Arg Gln Gly Pro Gly 1970 1975 1980 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln 1985 1990 1995 Gly Pro Tyr Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln 2000 2005 2010 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala 2015 2020 2025 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln 2030 2035 2040 Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 2045 2050 2055 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly 2060 2065 2070 Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly 2075 2080 2085 Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 2090 2095 2100 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 2105 2110 2115 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln 2120 2125 2130 Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly 2135 2140 2145 Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 2150 2155 2160 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 2165 2170 2175 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly 2180 2185 2190 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 2195 2200 2205 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser 2210 2215 2220 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala 2225 2230 2235 Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly 2240 2245 2250 Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly 2255 2260 2265 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala 2270 2275 2280 Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly 2285 2290 2295 Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 2300 2305 2310 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln 2315 2320 2325 Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 2330 2335 2340 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala 2345 2350 2355 Ala Ala Gly Pro Gly Ala Gly Arg Gln Gly Pro Gly Ser Gln Gly 2360 2365 2370 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 2375 2380 2385 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ala 2390 2395 2400 Arg Arg Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln 2405 2410 2415 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly 2420 2425 2430 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 2435 2440 2445 Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 2450 2455 2460 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln 2465 2470 2475 Gly Pro Gly Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro 2480 2485 2490 Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly 2495 2500 2505 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala 2510 2515 2520 Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly 2525 2530 2535 Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln 2540 2545 2550 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala 2555 2560 2565 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly 2570 2575 2580 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 2585 2590 2595 Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro 2600 2605 2610 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala 2615 2620 2625 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro 2630 2635 2640 Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser 2645 2650 2655 Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly 2660 2665 2670 Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly 2675 2680 2685 Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 2690 2695 2700 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 2705 2710 2715 Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly 2720 2725 2730 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 2735 2740 2745 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser 2750 2755 2760 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala 2765 2770 2775 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln 2780 2785 2790 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala 2795 2800 2805 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 2810 2815 2820 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 2825 2830 2835 Tyr Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly 2840 2845 2850 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala 2855 2860 2865 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly 2870 2875 2880 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly 2885 2890 2895 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 2900 2905 2910 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln 2915 2920 2925 Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly 2930 2935 2940 Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 2945 2950 2955 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 2960 2965 2970 Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly 2975 2980 2985 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 2990 2995 3000 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser 3005 3010 3015 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala 3020 3025 3030 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln 3035 3040 3045 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala 3050 3055 3060 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 3065 3070 3075 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 3080 3085 3090 Tyr Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly 3095 3100 3105 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala 3110 3115 3120 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly 3125 3130 3135 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly 3140 3145 3150 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 3155 3160 3165 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln 3170 3175 3180 Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly 3185 3190 3195 Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 3200 3205 3210 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 3215 3220 3225 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly 3230 3235 3240 Pro Gly Ala Gly Gln Gln Gly Pro Ser Ser Gln Ala Pro Val Ala 3245 3250 3255 Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Gln Ala Ser Ala Arg 3260 3265 3270 Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Ser 3275 3280 3285 Pro Ala Ala Leu Ser Asn Ala Ile Ser Ser Val Val Ser Gln Val 3290 3295 3300 Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln 3305 3310 3315 Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile Leu Gly Ser 3320 3325 3330 Ser Ser Ile Gly Gln Ile Asn Tyr Ala Ala Ser Ser Gln Tyr Ala 3335 3340 3345 Gln Met Val Gly Asn Ser Val Ala Gln Ala Leu Gly 3350 3355 3360 <210> 44 <211> 3129 <212> PRT <213> Latrodectus hesperus <400> 44 Met Thr Trp Ser Thr Arg Leu Ala Leu Ser Phe Leu Phe Val Leu Cys 1 5 10 15 Thr Gln Ser Leu Tyr Ala Leu Ala Gln Ala Asn Thr Pro Trp Ser Ser 20 25 30 Lys Ala Asn Ala Asp Ala Phe Ile Asn Ser Phe Ile Ser Ala Ala Ser 35 40 45 Asn Thr Gly Ser Phe Ser Gln Asp Gln Met Glu Asp Met Ser Leu Ile 50 55 60 Gly Asn Thr Leu Met Ala Ala Met Asp Asn Met Gly Gly Arg Ile Thr 65 70 75 80 Pro Ser Lys Leu Gln Ala Leu Asp Met Ala Phe Ala Ser Ser Val Ala 85 90 95 Glu Ile Ala Ala Ser Glu Gly Gly Asp Leu Gly Val Thr Thr Asn Ala 100 105 110 Ile Ala Asp Ala Leu Thr Ser Ala Phe Tyr Gln Thr Thr Gly Val Val 115 120 125 Asn Ser Arg Phe Ile Ser Glu Ile Arg Ser Leu Ile Gly Met Phe Ala 130 135 140 Gln Ala Ser Ala Asn Asp Val Tyr Ala Ser Ala Gly Ser Ser Gly Gly 145 150 155 160 Gly Gly Tyr Gly Ala Ser Ser Ala Ser Ala Ala Ser Ala Ser Ala Ala 165 170 175 Ala Pro Ser Gly Val Ala Tyr Gln Ala Pro Ala Gln Ala Gln Ile Ser 180 185 190 Phe Thr Leu Arg Gly Gln Gln Pro Val Ser Tyr Gly Gln Gly Gly Ala 195 200 205 Gly Pro Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly 210 215 220 Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly 225 230 235 240 Gln Gly Gly Ala Gly Gln Gly Gly Ser Gly Ala Ala Ala Ala Ala 245 250 255 Ala Ala Ala Gly Gly Thr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala 260 265 270 Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly 275 280 285 Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly Gly Thr Gly 290 295 300 Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 305 310 315 320 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly 325 330 335 Gly Tyr Gly Gln Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala 340 345 350 Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly 355 360 365 Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala 370 375 380 Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly 385 390 395 400 Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly 405 410 415 Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly Ser 420 425 430 Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly 435 440 445 Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ser Gly Ala 450 455 460 Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly 465 470 475 480 Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly 485 490 495 Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly 500 505 510 Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly 515 520 525 Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 530 535 540 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly 545 550 555 560 Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 565 570 575 Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr 580 585 590 Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala 595 600 605 Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly Gly Ala Gly 610 615 620 Gln Gly Gly Ala Ala Ala Ala Ala Gly Ala Gly Gln Gly Gly Tyr Gly 625 630 635 640 Gly Gln Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala 645 650 655 Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly 660 665 670 Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala 675 680 685 Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr 690 695 700 Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala 705 710 715 720 Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg 725 730 735 Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala 740 745 750 Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly 755 760 765 Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly 770 775 780 Gly Gln Gly Asp Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly 785 790 795 800 Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 805 810 815 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly 820 825 830 Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ser Ala Ala Ala Ala 835 840 845 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly Gly Ala Gly Gln Gly 850 855 860 Gly Ala Ala Ala Ala Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln 865 870 875 880 Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 885 890 895 Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr 900 905 910 Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala 915 920 925 Thr Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln 930 935 940 Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala 945 950 955 960 Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly 965 970 975 Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly 980 985 990 Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly Ala 995 1000 1005 Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln 1010 1015 1020 Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln 1025 1030 1035 Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 1040 1045 1050 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln 1055 1060 1065 Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala 1070 1075 1080 Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly Gly Ala Gly 1085 1090 1095 Gln Gly Gly Ala Ala Ala Ala Ala Gly Ala Gly Gln Gly Gly Tyr 1100 1105 1110 Gly Gly Gln Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala 1115 1120 1125 Ala Ser Arg Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg 1130 1135 1140 Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala 1145 1150 1155 Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln 1160 1165 1170 Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln 1175 1180 1185 Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln 1190 1195 1200 Gly Gly Tyr Gly Arg Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala 1205 1210 1215 Ala Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly 1220 1225 1230 Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly 1235 1240 1245 Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly 1250 1255 1260 Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala 1265 1270 1275 Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly 1280 1285 1290 Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala 1295 1300 1305 Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr 1310 1315 1320 Gly Arg Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala 1325 1330 1335 Ala Ala Ala Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala 1340 1345 1350 Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly 1355 1360 1365 Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr 1370 1375 1380 Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala 1385 1390 1395 Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr 1400 1405 1410 Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala 1415 1420 1425 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly 1430 1435 1440 Gly Tyr Gly Arg Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala 1445 1450 1455 Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln 1460 1465 1470 Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 1475 1480 1485 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln 1490 1495 1500 Gly Gly Ala Gly Gln Gly Gly Ala Gly Thr Ala Ala Ala Ala Ala 1505 1510 1515 Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln 1520 1525 1530 Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala 1535 1540 1545 Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly 1550 1555 1560 Arg Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala 1565 1570 1575 Ala Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly 1580 1585 1590 Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly 1595 1600 1605 Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly 1610 1615 1620 Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala 1625 1630 1635 Ala Ala Ala Gly Gly Ala Ser Gln Gly Gly Gln Gly Gly Tyr Gly 1640 1645 1650 Gln Gly Asp Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala 1655 1660 1665 Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly 1670 1675 1680 Arg Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Gly Ala 1685 1690 1695 Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly Ala 1700 1705 1710 Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Arg Gly 1715 1720 1725 Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala 1730 1735 1740 Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly 1745 1750 1755 Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr 1760 1765 1770 Gly Gln Gly Gly Thr Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala 1775 1780 1785 Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly Gly 1790 1795 1800 Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly 1805 1810 1815 Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly 1820 1825 1830 Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln 1835 1840 1845 Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly 1850 1855 1860 Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 1865 1870 1875 Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly 1880 1885 1890 Tyr Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly 1895 1900 1905 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly 1910 1915 1920 Tyr Gly Arg Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala 1925 1930 1935 Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly 1940 1945 1950 Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly 1955 1960 1965 Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly 1970 1975 1980 Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala 1985 1990 1995 Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly 2000 2005 2010 Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala 2015 2020 2025 Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg 2030 2035 2040 Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala 2045 2050 2055 Ala Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln 2060 2065 2070 Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 2075 2080 2085 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln 2090 2095 2100 Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala 2105 2110 2115 Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln 2120 2125 2130 Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln 2135 2140 2145 Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 2150 2155 2160 Gly Gln Gly Gly Tyr Gly Arg Gly Gly Ala Gly Gln Gly Gly Ala 2165 2170 2175 Ala Ala Ala Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly 2180 2185 2190 Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 2195 2200 2205 Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly 2210 2215 2220 Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala 2225 2230 2235 Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly 2240 2245 2250 Tyr Gly Gln Gly Gly Asn Gly Gln Gly Gly Ala Gly Gln Gly Gly 2255 2260 2265 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly 2270 2275 2280 Tyr Gly Arg Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala 2285 2290 2295 Ala Ala Ala Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly 2300 2305 2310 Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 2315 2320 2325 Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly 2330 2335 2340 Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala 2345 2350 2355 Ala Ala Ala Ala Ala Gly Gly Ala Ser Gln Gly Gly Gln Gly Gly 2360 2365 2370 Tyr Gly Gln Gly Asp Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly 2375 2380 2385 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly 2390 2395 2400 Tyr Gly Arg Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala 2405 2410 2415 Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly 2420 2425 2430 Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly 2435 2440 2445 Arg Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly 2450 2455 2460 Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala 2465 2470 2475 Ala Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly 2480 2485 2490 Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala 2495 2500 2505 Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg 2510 2515 2520 Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Gly Ala Gly 2525 2530 2535 Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly Ala Gly 2540 2545 2550 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Arg Gly Gly 2555 2560 2565 Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly 2570 2575 2580 Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly 2585 2590 2595 Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly 2600 2605 2610 Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala 2615 2620 2625 Ala Val Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly Gly Ala 2630 2635 2640 Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly 2645 2650 2655 Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly 2660 2665 2670 Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln 2675 2680 2685 Gly Gly Gln Gly Gly Tyr Gly Gly Gly Gly Tyr Gly Gln Gly Gly 2690 2695 2700 Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 2705 2710 2715 Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly 2720 2725 2730 Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala 2735 2740 2745 Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly 2750 2755 2760 Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Thr Gly Ala Gly Gln 2765 2770 2775 Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly Ala Gly Ala 2780 2785 2790 Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln 2795 2800 2805 Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln 2810 2815 2820 Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 2825 2830 2835 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln 2840 2845 2850 Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala 2855 2860 2865 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Arg Gly Gly Ala Gly Gln 2870 2875 2880 Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Gly Gln 2885 2890 2895 Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Gly Ala Gly Ala 2900 2905 2910 Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Gln 2915 2920 2925 Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln 2930 2935 2940 Gly Gly Ala Gly Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly 2945 2950 2955 Gln Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly 2960 2965 2970 Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly 2975 2980 2985 Ala Gly Gln Gly Gly Tyr Gly Gly Tyr Gly Gln Gln Gly Gly Ala 2990 2995 3000 Gly Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gln Ile Tyr Tyr 3005 3010 3015 Gly Pro Gln Ser Val Ala Ala Pro Ala Ala Ala Ala Ala Ser Ala 3020 3025 3030 Leu Ala Ala Pro Ala Thr Ser Ala Arg Ile Ser Ser His Ala Ser 3035 3040 3045 Ala Leu Leu Ser Asn Gly Pro Thr Asn Pro Ala Ser Ile Ser Asn 3050 3055 3060 Val Ile Ser Asn Ala Val Ser Gln Ile Ser Ser Ser Asn Pro Gly 3065 3070 3075 Ala Ser Ala Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu Val 3080 3085 3090 Thr Ala Leu Leu Thr Ile Ile Gly Ser Ser Asn Ile Gly Ser Val 3095 3100 3105 Asn Tyr Asp Ser Ser Gly Gln Tyr Ala Gln Val Val Thr Gln Ser 3110 3115 3120 Val Gln Asn Ala Phe Ala 3125 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 45 tactacaggc tggctgttcc 20 <210> 46 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 46 ctcacttaat cttctgtact ctgaagaagt ccaactgttg aacgcc 46 <210> 47 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 47 agaagttgat tgagactttc aacgagggtc cccttcagct accttt 46 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 48 tccctgctaa gccctaatcg 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 49 ctctgattgc acgagaaggc 20 <210> 50 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 50 ctcacttaat cttctgtact ctgaagtgaa aggcgattgg agttgc 46 <210> 51 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 51 agaagttgat tgagactttc aacgagctgg ctctgcttct ggtact 46 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 52 gatgttgagg cgggcataag 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 53 acttgtcagg acgatacgga 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 54 ccggtctccc tggaaataga 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 55 agttgtccgt cattagccct 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 56 tgttcccttt cggctagaca <210> 57 <211> 4324 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 57 60. ggagttgaat cacatcttac tggatagcga gctttttgac gaagtgaaaa tttctaattt 120. taaacaagag gaaggggtca aaaacggaga tatcttatac ttggaaaaag agatgacaat cagtgatttc atcaattttg tatctagttg gccttctgtg ttttcgtgga agcagcaacg aggaaaggag ggtatcctag atgattttta caacgaactg aacgactgct ttgagggggg 300. actccgtcct agtatttgcc aaagggttgt ataggcttta gtacttatag aggaaacggg gttacgtgca agcgcgcatg cctgagcttt 360 gagggggggg actttcacat ctcttcttct cacacttagc cctaacacag agate aaagcattgc aagatgagtg ttgtcagcaa gcaatacgac atccacgaag gcattatctt tgtaattgaa ttgaccccgg agcttcacgc gccggcttca gaagggaaat ctcagctcca 540 gatcatctta gagaatgtca gtgaggttat ttctgagcta atcattacct tgcccggtac 600 aggaataggg tgttacctta ttaattacga cggtggtcaa aacgacgaaa tttaccccat 660 ttttgagtta caagacctga atttggaaat gatgaaacaa ttgtaccaag tcttggagga 720 ccatgtaagt gggcttaatc ctctcgagaa gcaattccca attgaacaca gtaaaccgtt 780 atcagccact ctgttctttc acttaaggtc tcttttttac atggcgaaga ctcataagcg 840 tactggaaga cattacaact tgaaaaagat tttcttgttc actaataacg ataaactta 900 caatggaaac tctcagctga gagttccctt gaaaaacc ctggctgatt acaatgacgt 960 agacattact ttgattccgt ttcttctgaa caagccttca ggtgtcaagt ttgacaagac 1020 ggaatactca gaaattttgt tctatgataa agatgcttgt tcgatgtcaa ttgaggagat 1080 1140 aatcgcaaat aacttgtgca tttctgtgaa aggttattct atgttttatc atgaaactcc 1200 aaagaatc aaatttgtcg tcaatgaggg ttcaactttc aaagatgtgg agacaaaatc 1260 tcagtttgtc gatccaacat ccggaaaaga gttttccagt gaacagctga tcaaagcata 1320 tcctctaggt gccgatgctt acattccttt aaactcagag caagtcaaaa caataaatcg 1380 atttaatgat atcatcaata tcccctcttt ggaaattcta ggtttcaggg atatatctaa 1440 ttggttgcca foottcagt ttggcaaagc atcgttttta tcccctaata actatggtga 1500 ttttacacat tcgcagagaa catttagttg tcttcagataa tgtcttgttt cttttgttgc 1560 agtggtgagc cattttgact tcgtgaaagt ttctttagaa tagttgtttc cagaggccaa 1620 acattccacc cgtagtaaag tgcaagcgta ggagaccaa gactggcata aatcaggtat 1680 aagtgtcgag cactggcagg tgatcttctg aaagtttcta ctagcagata agatccagta 1740 gtcatgcata tggcaacaat gtaccgtgtg gatctaagaa cgcgtcctac taaccttgc 1800 attcgttggt ccagtttgtt gttatcgatc aacgtgacaa ggttgtcgat tccgcgtaag 1860 catgcatacc caaggacgcc tgttgcaatt ccaagtgagc cagttccaac aatctttgta 1920 1980 tctcgaaacc gcgacttcaa acgccaatat gatgtgcggc acaataag cgttcatatc 2040 cgctgggtga ctttctcgct ttaaaaaatt atccgaaaaa atttttgacg gctagctcag 2100 tcctaggtac gctagcatta aagaggagaa aatggctaaa ctgacctctg ctgttccggt 2160 tctgaccgct cgtgacgttg ctggtgctgt tgagttctgg accgaccgtc tgggtttctc 2220 tcgtgacttc gttgaagacg acttcgctgg tgttgttcgt gacgacgtta ccctgttcat 2280 ctctgctgtt caggaccagg ttgttccgga caacaccctg gcttgggttt gggttcgtgg 2340 tctggacgaa ctgtacgctg aatggtctga agttgtttct accaacttcc gtgacgcttc 2400 tggtccggct atgaccgaaa tcggtgaaca gccgtggggt cgtgagttcg ctctgcgtga 2460 cccggctggt aactgcgttc acttcgttgc tgaagaacag gactaaccg tccgacggcg 2520 gcccacgggt cccaggcctc ggagatccgt cccccttttc ctttgtcgat atcatgtaat 2580 tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag 2640 gagttagaca acctgaagtc taggtcccta tttattttt tatagttatg ttagtattaa 2700 gaacgttatt tatatttcaa attttcttt ttttctgta cagacgcgtg tacgcatgta 2760 acattact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgca 2820 agctgtatta gtttcacttt tcagcaacct ggtcggaaag atccacatca agaatggata 2880 ccaaccccaa gagtatgaaa atccttccct acaatggcac ttcaaaatgt tacgtgacga 2940 ttaccttcaa ttggaacacg atatcgacat cagtgacccc cttgagaaac aaaagtacat 3000 aaacagcctc gatgagacaa aaaccaagat catgaaacta cgggactatg tcaaggaaac 3060 tgccgatgat gacgaccctt cacggcttgc caacactctc aaagagctca accaagagct 3120 gaacaaatt tccaactttg atatcatcgc caataagaag ccaaagaccc ccacgacagt 3180 agaccctgtt cctactgatg atgacatcat caacgcctgg aaggcaggaa ctctgaacgg 3240 tttcaaggtg gatcaattac gaaaatacgt aaggtcacga aacaactttc tggagacggc 3300 ctccaaaaag gcagatctca tcgccaacat tgacaagtac tttcagcaga agttcaaaga 3360 gactaaggcc tgattcgtgt tccttacttt ttcctcgcaa cgtgtttttt tcccaccaca 3420 ttgcctatgt tgtaatgcaa tgcagatgct ggcccagttt ttgacgattc tcgaaaattg 3480 gcattttcgt cgatgccatt ggccaaactg aaaattcaag acaaaataga ttggatttta 3540 tctgcaacgt cttccaccta cacaaccact ctacaaactt cagacaaaca tgtttataaa 3600 agcagctact agatccaaaa tgacaagttc gttattctct actacgtttg ttgtggcatt 3660 tggattggtg gctagcaaca acctcttgcc atgtcctgtt gaccactcta tgaataacga 3720 gactccgcaa gaattgaaac cattgcaggc tgaatcttct actagaaagt tgaactcttc 3780 cgcttaagtc aaataaaact actgacacag atgatgcaca gaaacaacgg atcacgctct 3840 tgactgatta gtcccgtcat tttggttctc attttcttca cagtcaccta tcaatgtatg 3900 atcacctgga aggatttccc tacgatactt caaatctttt acttgataat attactcatt 3960 atggctcagg aatgcagact gcctgattca agacgctgct cttcttattt aacacttgta 4020 cactaacccc atggaagcca gggaagggaa taaccatctc tctggtaata aatcggtctt 4080 tatttatgca tagaaaagga atctattata tttcgttcat ttggcactct gctaactgta 4140 gattaacggg tctcgtaaat tcaaaatctt cttccgatca aaccggggtg aaatattact 4200 tctcgtgcat agctaatttt caaataaccg tcctaaaatg aacggtcatt tacctggact 4260 ctcttgccaa atgggcaaca aaacataaag ctgatcagaa cgtaactagt ctctcggaat 4320 ccat 4324 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 58 ggagttgaat cacatcttac tg 22 <210> 59 <211> 1099 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 59 gacaactaaa tgttctctgc gaatgtgtaa aatcaccata gttattaggg gataaaaacg 60 atgctttgcc aaactgatac tgtggcaacc aattagatat atccctgaaa ccctagaattt 120 ccaaagagggg gatattgatg atatcattaa atcgatttat tgttttgact tgctctgagt 180 ttaaaggaat gtaagcatcg gcacctagag gatatgcttt gatcagctgt tcactggaaa 240 actcttttcc ggatgttgga tcgacaaact gagattttgt ctccacatct ttgaaagttg 300 aaccctcatt gacgacaaat ttgatcttcc ttggagtttc atgataaaac atagaataac 360 ctttcacaga aatgcacaag ttatttgcga ttttcaaagg acaggtgaag taaacccgct 420 tgatctcctt atgtctagaa attcgttggc ggatctcctc aattgacatc gaacaagcat 480 ctttatcata gaacaaaatt tctgagtatt ccgtcttgtc aaacttgaca cctgaaggct 540 tgttcagaag aaacggaatc aaagtaatgt ctacgtcatt gtaatcagcc agggttttct 600 tcaagggaac tctcagctga gagtttccat tgtaaggttt atcgttatta gtgaacaaga 660 aaatcttttt caagttgtaa tgtcttccag tacgcttatg agtcttcgcc atgtaaaaaa 720 gagaccttaa gtgaaagaac agagtggctg ataacggttt actgtgttca attgggaatt 780 gcttctcgag aggattaagc ccacttacat ggtcctccaa gacttggtac aattgtttca 840 tcatttccaa attcaggtct tgtaactcaa aaatggggta aatttcgtcg ttttgaccac 900 cgtcgtaatt aataaggtaa caccctattc ctgtaccggg caaggtaatg attagctcag 960 aaataacctc actgacattc tctaagatga tctggagctg agatttccct tctgaagccg 1020 gcgcgtgaag ctccggggtc aattcaatta caaagataat gccttcgtgg atgtcgtatt 1080 gcttgctgac aacactcat 1099 <210> 60 <211> 549 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 60 tcaggcctta gtctctttga acttctgctg aaagtacttg tcaatgttgg cgatgagatc 60 tgcctttttg gaggccgtct ccagaaagtt gtttcgtgac cttacgtatt ttcgtaattg 120 atccaccttg aaaccgttca gagttcctgc cttccaggcg ttgatgatgt catcatcagt 180 aggaacaggg tctactgtcg tgggggtctt tggcttctta ttggcgatga tatcaaagtt 240 ggaaattttg ttcagctctt ggttgagctc tttgagagtg ttggcaagcc gtgaagggtc 300 gtcatcatcg gcagtttcct tgacatagtc ccgtagtttc atgatcttgg tttttgtctc 360 atcgaggctg tttatgtact tttgtttctc aagggggtca ctgatgtcga tatcgtgttc 420 caattgaagg taatcgtcac gtaacatttt gaagtgccat tgtagggaag gattttcata 480 ctcttggggt tggtatccat tcttgatgtg gatctttccg accaggttgc tgaaaagtga 540 aactaatac 549 <210> 61 <211> 552 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 61 ttcagtaatg tcttgtttct tttgttgcag tggtgagcca ttttgacttc gtgaaagttt 60 ctttagaata gttgtttcca gaggccaaac attccacccg tagtaaagtg caagcgtagg 120 aagaccaaga ctggcataaa tcaggtataa gtgtcgagca ctggcaggtg atcttctgaa 180 agtttctact agcagataag atccagtagt catgcatatg gcaacaatgt accgtgtgga 240 tctaagaacg cgtcctacta accttcgcat tcgttggtcc agtttgttgt tatcgatcaa 300 cgtgacaagg ttgtcgattc cgcgtaagca tgcataccca aggacgcctg ttgcaattcc 360 aagtgagcca gttccaacaa tctttgtaat attagagcac ttcattgtgt tgcgcttgaa 420 agtaaaatgc gaacaaatta agagataatc tcgaaaccgc gacttcaaac gccaatatga 480 tgtgcggcac acaataagcg ttcatatccg ctgggtgact ttctcgcttt aaaaaattat 540 ccgaaaaaat tt 552 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 62 cagaggccaa acattccacc 20 <210> 63 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 63 ttaaagagga gaaa 14 <210> 64 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 64 atggctaaac tgacctctgc tgttccggtt ctgaccgctc gtgacgttgc tggtgctgtt 60 gagttctgga ccgaccgtct gggtttctct cgtgacttcg ttgaagacga cttcgctggt 120 gttgttcgtg acgacgttac cctgttcatc tctgctgttc aggaccaggt tgttccggac 180 aacaccctgg cttgggtttg ggttcgtggt ctggacgaac tgtacgctga atggtctgaa 240 gttgtttcta ccaacttccg tgacgcttct ggtccggcta tgaccgaaat cggtgaacag 300 ccgtggggtc gtgagttcgc tctgcgtgac ccggctggta actgcgttca cttcgttgct 360 gaagaacagg actaa 375 <210> 65 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 65 cacgtccgac ggcggcccac gggtcccagg cctcggagat ccgtccccct tttcctttgt 60 cgatatcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct 120 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt 180 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg 240 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa 300 ggctttaatt tgcaagct 318 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 66 aggagttaga caacctgaag 20 <210> 67 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 67 gtaactagtc tctcggaatc cat 23 <210> 68 <211> 1623 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 68 cttcagagta cagaagatta agtgagagaa ttctaccgtt cgtatagcat acattatacg 60 aagttatttc agtaatgtct tgtttctttt gttgcagtgg tgagccattt tgacttcgtg 120 aaagtttctt tagaatagtt gtttccagag gccaaacatt ccacccgtag taaagtgcaa 180 gcgtaggaag accaagactg gcataaatca ggtataagtg tcgagcactg gcaggtgatc 240 ttctgaaagt ttctactagc agataagatc cagtagtcat gcatatggca acaatgtacc 300 gtgtggatct aagaacgcgt cctactaacc ttcgcattcg ttggtccagt ttgttgttat 360 cgatcaacgt gacaaggttg tcgattccgc gtaagcatgc atacccaagg acgcctgttg 420 caatccaag tgagccagtt ccaacaatct ttgtaatatt agagcacttc attgtgttgc 480 gcttgaaagt aaatgcgaa caattaaga gatactcg aaaccgcgac ttcaacgcc 540 ataatgatgt gcggcacaca ataagcgttc atatccgctg ggtgactttc tcgctttaaa 600 aaattatccg aaaaaatttt tgacggctag ctcagtccta ggtacgctag cattaagag 660 gagaaaatga ctactcttga tgacacagcc tacagatata ggacatcagt tccggtgac 720 gcagaggcta tcgaagcctt ggacggttca ttcactactg atacggtgtt tagagtcacc 780 gctacaggtg atggcttcac cttgagagag gttcctgtag acccacctt aacgaaagtt 840 ttccctgatg acgaatcgga tgacgagtct gatgctggtg aggacggtga ccctgattcc 900 agaacatttg tcgcatacgg agatgatggt gacctggctg gctttgttgt ggtgtcctac 960 agcggatgga atcgtagact cacagttgag gatatcgaag ttgcacctga acatcgtggt 1020 cacggtgttg gtcgtgcact gatgggactg gcacagagt tgctagaga aagaggagcc 1080 ggacattgt ggttagaagt gaccaatgtc aacgctccctg ctattcaccc atataggcga 1140 atgggtttca ctttgtgcgg tcttgatact gctttgtatg acggaactgc ttctgatggt 1200 gaacaagctc tttacatgag tatgccatgt ccatagcacg tccgacggcg gcccacgggt 1260 cccaggcctc ggagatccgt cccccttttc ctttgtcgat atcatgtaat tagttatgtc 1320 acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca 1380 acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt 1440 tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact 1500 gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgca agctataact 1560 tcgtatagca tacattatac cttgttatgc ggccgcaaga agttgattga gactttcaac 1620 gag 1623 <210> 69 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 69 cttcagagta cagaagatta agtgaga 27 <210> 70 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 70 taccgttcgt atagcataca ttatacgaag ttat 34 <210> 71 <211> 552 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 71 ttcagtaatg tcttgtttct tttgttgcag tggtgagcca ttttgacttc gtgaaagttt 60 ctttagaata gttgtttcca gaggccaaac attccacccg tagtaaagtg caagcgtagg 120 aagaccaaga ctggcataaa tcaggtataa gtgtcgagca ctggcaggtg atcttctgaa 180 agtttctact agcagataag atccagtagt catgcatatg gcaacaatgt accgtgtgga 240 tctaagaacg cgtcctacta accttcgcat tcgttggtcc agtttgttgt tatcgatcaa 300 cgtgacaagg ttgtcgattc cgcgtaagca tgcataccca aggacgcctg ttgcaattcc 360 aagtgagcca gttccaacaa tctttgtaat attagagcac ttcattgtgt tgcgcttgaa 420 agtaaaatgc gaacaaatta agagataatc tcgaaaccgc gacttcaaac gccaatatga 480 tgtgcggcac acaataagcg ttcatatccg ctgggtgact ttctcgcttt aaaaaattat 540 ccgaaaaaat tt 552 <210> 72 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 72 ttaaagagga gaaa 14 <210> 73 <211> 570 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 73 atgactactc ttgatgacac agcctacaga tataggacat cagttccggg tgacgcagag 60 gctatcgaag ccttggacgg ttcattcact actgatacgg tgtttagagt caccgctaca 120 ggtgatggct tcaccttgag agaggttcct gtagacccac ccttaacgaa agttttccct 180 gatgacgaat cggatgacga gtctgatgct ggtgaggacg gtgaccctga ttccagaaca 240 tttgtcgcat acggagatga tggtgacctg gctggctttg ttgtggtgtc ctacagcgga 300 tggaatcgta gactcacagt tgaggacatc gaagttgcac ctgaacatcg tggtcacggt 360 gttggtcgtg cactgatggg actggcaaca gagtttgcta gagaaagagg agccggacat 420 ttgtggttag aagtgaccaa tgtcaacgct cctgctattc acgcatatag gcgaatgggt 480 ttcactttgt gcggtcttga tactgctttg tatgacggaa ctgcttctga tggtgaacaa 540 gctctttaca tgagtatgcc atgtccatag 570 <210> 74 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 74 cacgtccgac ggcggcccac gggtcccagg cctcggagat ccgtccccct tttcctttgt 60 cgatatcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct 120 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt 180 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg 240 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa 300 ggctttaatt tgcaagct 318 <210> 75 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 75 ataacttcgt atagcataca ttataccttg ttat 34 <210> 76 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 76 gcggccgcaa gaagttgatt gagactttca acgag 35 <210> 77 <211> 3869 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 77 tactacaggc tggctgttcc tcgcatggtg tttaatgtcc tgactgggtt ttcgtttatc ggtattaccg gagccacctt gactgtaagg gaacgatact ggactaagag agtaatgcga 180. aaggcaacag cgtttctggc gacctaatc aatgacggtt acgagtttac tactcctaaa gccagtctta ttttgctaga gcgagtcaac gcttacttaa agggccaggg acctaattat gacatcgatt ttgacgagca ggaggcgttc attaagaaa tggaggagtt gaggacctct ggtggatatg agaacagata ctcatattca ggaaccgatg aaacacccag agatccgggt tgcctgtttc ttcccattgc tttaataaa tggcactttg atgtgctaga ctgcctgagg 420 atatacggta ctcaggaaga tctggaatct aaattattaa gtgttcagca attggtgtta 540. caatgttgca tgaagcacag tggcatgact ccagacatgg tctttgcaac ggaagtagct cagaagccga ccttcgaaga cgacatagtt tgtgatgata ttgacgctta tgcccagggg 600 ggtgattgtc tagattattg ttacacgcca agcaattact ccagaacttt agaaattcat 660 720 gaccgttttt ccgattaagg tttttagctc cattgcgcca accccccgctc tccagactcc 780 ttcgttatcc agcattcagc atggacaggt tcaaaaaata aaatttcttg atatgggtcc 840 acttcaaaca tgcgcctacc tgtaggaaaa aaaaagagaa cataaatatg ccgcgaacag 900 aaaacgtaat gtactgttct atatataaac tgttcagatc aatcataaat tctcagtttc 960 aaactttccg ctcagccaga ttttattcgt aaagaacgca tcattggctc tatgttgaag 1020 gatcagttct tgttatgggt tgctttgata gcgagcgtac cggtttccgg cgtgatggca 1080 gctcctagcg agtccgggca taacacggtt gaaaaacgag atgccaaaaa cgttgttggc 1140 gttcaacagt tggacttctt cagagtacag aagattaagt gagagaattc taccgttcgt 1200 atagcataca ttatacgaag ttattcagt aatgtcttgt ttcttttgtt gcagtggtga 1260 gccattttga cttcgtgaaa gttctttag atagttgtt tccagaggcc aacattcca 1320 cccgtagtaa agtgcaagcg taggagacc aagactggca taatcaggt ataagtgtcg 1380 agcactggca gtgatcttc tgaagttttc tactagcaga tagatccag tagtcatgca 1440 tatggcaaca atgtaccgtg tggatctaag aacgcgtcct actaccttc gcattcgttg 1500 gtccagtttg ttgttatcga tcaacgtgac aaggttgtcg attccgcgta agcatgcata 1560 cccaaggacg cctgttgcaa ttccaagtga gccagttcca acatctttg taatattaga 1620 gcacttcatt gtgttgcgct tgaaagtaaa atgcgaacaa attaagagat aatctcgaaa 1680 ccgcgacttc aaacgccaat atgatgtgcg gcacacaata agcgttcata tccgctgggt 1740 gactttctcg ctttaaaaaa ttatccgaaa aaattttga cggctagctc agtcctaggt 1800 acgctagcat aagaggag aaatgacta ctcttgatga cacagcctac agatatagga 1860 catcagttcc gggtgacgca gaggctatcg aagccttgga cggttcattc actactgata 1920 cggtgtttag agtcaccgct acaggtgatg gctcacctt gagaggtt cctgtagacc 1980 cacccttaac gaaagttttc cctgatgacg aatcggatga cgagtctgat gctggtgagg 2040 acggtgaccc tgattccaga acatttgtcg catacggaga tgatggtgac ctggctggct 2100 ttgttgtggt gtcctacagc ggatggaatc gtagactcac agttgaggac atcgaagttg 2160 cacctgaaca tcgtggtcac ggtgttggtc gtgcactgat gggactggca acagagtttg 2220 ctagagaaag aggagccgga catttgtggt tagaagtgac caatgtcaac gctcctgcta 2280 ttcacgcata taggcgaatg ggtttcactt tgtgcggtct tgatactgct ttgtatgacg 2340 gaactgcttc tgatggtgaa caagctcttt acatgagtat gccatgtcca tagcacgtcc 2400 gacggcggcc cacgggtccc aggcctcgga gatccgtccc ccttttcctt tgtcgatatc 2460 atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa 2520 aaggaaggag ttagacaacc tgaagtctag gtccctattt attttttat agttatgtta 2580 gtattaagaa cgttatttat atttcaaatt tttcttttt ttctgtacag acgcgtgtac 2640 gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta 2700 atttgcaagc fatheracttcg fathercatac attatacctt gttatgcggc cgcaagaagt 2820. tgattgagac tttcaacgag ggtccccttc agctaccttt ctctctgttt ggtagttatt ctcggcgtgt gtatagtata gtataaaagg gcctacattg gataggcttc aacattcctc aataaacaaa catccaacat cgcgcattcc gcatttcgca tttcacattt cgcgcctgcc ttcctttagg ttctttgaat catcatcaat cgtcgccgtc catcagag caggacttat ctttgccttc cccaaaaatt gccactccgt caaatagatt cttttgaatc cttgactatt tttgcctaaa tagtttttg ttagtttttc ttcaaagccc aaaagaact ctatttagat tcatccagaa acaatctttt tctcacccca tttcgaagtg ccgtggagca cagacataaa aagatgacta ccgttcaacc tacagggcca gacaggctca ccctgccgca tattctactg gaattcaacg atggctcctc gcagcatgca gtgatcgagc tagcatgaa cgaggggatt aatatatcca cccatgagtg gaatccatcc actaatgagc aatcgccacg ggaagagaga gcaccacccc aacaatccaa tccatcgcat catcagat catcgacat agctactcaa agtcccgctc aggaaccga gactcagccc ggcattccag gactagatag gcctgccttt 3480 gatcctcgg caacggggtc gtcagacag gttgaccag tacagggag gatcctgt 3540 gatattag gccaatcatt aaggactcc gagagacg ataccgaatc ccgccagaga 3600 ccacgagacc agagacaat tatgatcacc gtgaattact tgtacgcaga cgacacaat 3660 tccagaagtg ctaatacaaa caaccagacg cccaataca cttctagaac ttccgacagt 3720 gaacgtgtgg gctccttatc gttgcacgtt ccgatctac cagataatgc cgacgattac 3780 tatatcgatg tactcattaa actaccaca agcattgccc tcagcgtcat cacgtccatg 3840 atcaagaaac gattaggggct tag3869 <210> 78 <211> 1157 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 78 tactacaggc tggctgttcc tcgcatggtg tttaatgtcc tgactggtt tcgtttatc 60 ggtattaccg gagccacctt gactgtaagg gaacgatact ggactaagag agtaatgcga 120 aaggcaacag cgtttctggc gaacctaatc aatgacggtt acgagtttac tactcctaaa 180 gccagtctta ttttgctaga gcgagtcaac gcttacttaa agggccaggg acctaattat 240 gacatcgatt ttgacgagca ggaggcgttc attaaagaaa tggaggagtt gaggacctct 300 ggtggatatg agaacagata ctcatattca ggaaccgatg aaacacccag agatccgggt 360 tgcctgtttc ttcccattgc tttaaataaa tggcactttg atgtgctaga ctgcctgagg 420 atatacggta ctcaggaaga tctggaatct aaattattaa gtgttcagca attggtgtta 480 caatgttgca tgaagcacag tggcatgact ccagacatgg tctttgcaac ggaagtagct 540 cagaagccga ccttcgaaga cgacatagtt tgtgatgata ttgacgctta tgcccagggg 600 ggtgattgtc tagattattg ttacacgcca agcaattact ccagaacttt agaaattcat 660 720 gaccgttttt ccgattaagg tttttagctc cattgcgcca accccccgctc tccagactcc 780 ttcgttatcc agcattcagc atggacaggt tcaaaaaata aaatttcttg atatgggtcc 840 acttcaaaca tgcgcctacc tgtaggaaaa aaaaagagaa cataaatatg ccgcgaacag 900 aaaacgtaat gtactgttct atatataaac tgttcagatc aatcataaat tctcagtttc 960 aaactttccg ctcagccaga ttttattcgt aaagaacgca tcattggctc tatgttgaag 1020 gatcagttct tgttatgggt tgctttgata gcgagcgtac cggtttccgg cgtgatggca 1080 gctcctagcg agtccgggca taacacggtt gaaaaacgag atgccaaaaa cgttgttggc 1140 gttcaacagt tggactt 1157 <210> 79 <211> 1089 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 79 ggtccccttc agctaccttt ctctctgttt ggtagttatt ctcggcgtgt gtatagtata 60 gtataaaagg gcctacattg gataggcttc aacattcctc aataaacaaa catccaacat 120 cgcgcattcc gcatttcgca tttcacattt cgcgcctgcc ttcctttagg ttctttgaat 180 catcatcaat cgtcgccgtc tacatcagag caggacttat ctttgccttc cccaaaaatt 240 gccactccgt caaatagatt cttttgaatc cttgactatt tttgcctaaa taggtttttg 300 ttagtttttc ttcaaagccc aaaagaaact ctatttagat tcatccagaa acaatctttt 360 tctcacccca tttcgaagtg ccgtggagca cagacataaa aagatgacta ccgttcaacc 420 tacagggcca gacaggctca ccctgccgca tattctactg gaattcaacg atggctcctc 480 gcagcatgca gtgatcgagc taagcatgaa cgaggggatt aatatatcca cccatgagtg 540 gaatccatcc actaatgagc aatcgccacg ggagaagaga gcaccacccc aacaatccaa 600 tccatcgcat catccagaat catcgaacat agctactcaa agtcccgctc aggaaaccga 660 gactcagccc ggcattccag gactagatag gcctgccttt gatacctcgg caacggggtc 720 gtcagaacag gttgacccag tacagggaag gatcctggat gatattatag gccaatcatt 780 aagacttcc gaagaagcg ataccgaatc ccgccaagaga ccacgagacc agaagaacat 840 tatgatcacc gtgaattact tgtacgcaga cgacacaaat tccagaagtg ctaatacaaa 900 caaccagacg cccaataaca cttctagaac ttccgacagt gaacgtgtgg gctccttatc 960 gttgcacgtt ccggatctac cagataatgc cgacgattac tatatcgatg tactcattaa 1020 actaaccaca agcattgccc tcagcgtcat cacgtccatg atcaagaaac gattagggct 1080 tagcaggga 1089 <210> 80 <211> 3964 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 80 gccttctcgt gcaatcagag ctgttgaaag agagaagagg gcacacggaa gctgctgttc 60 aattgtgtga attgaccgga ttacaacctg ctggagtgat aggagagctg gttcgtgacg 120 aggacggctc tatgatgcga ttagacgact gtgttcagtt tggtctccgc cacaacgtaa 180 aaattatcaa ccttgaccag atcattgaat acatggattc caagaacagc tagatacgat 240 ggataggaat acagagatat catgattgag gaacgtaaga gctttttcga aagtgtgagt 300 ttgtggtgag ggccaggcgg tggggaggtg gtggggagcc tccttggtcg aatgtagata 360 tagtaagcaa gacacaagag cgcgcgaagt cttcaacgag gcggcgttgg gtcttgtacg 420 caacgtaatg actacacagt tgagcttgtc gcgaaccggt cgacattttg atcatgcata 480 ctatgttgag acaccatctc gtactattgc ggcaaccagc tgtaaatttg actaattaaa 540 gctgatgaag gatgcagggc gtcgtcaatt ttttgattga ttgcatttaa ttgtttgagc 600 cattcaaggc tgaatgcccg gcaccctaga cccttcttgt gagtactata aacccgcagg 660 cagggtaccc ttggccttct gcgagactac cagtcataac gtatatccac aatgtactag 720 taatagcccc ggaaaactct aatcccacag aacgtctaac gcctcctatg tcatcgatac 780 ccattcgcac tactgccatg gcccccctta cgtgatcatt tcacttactc ccgcctaagc 840 ttcgcccaca tgcctgcgtt ttgccaagat ttactgacga gtttggttta ctcatcctct 900 atttataact actagacttt caccattctt caccaccctc gtgccaatga tcatcaacca 960 cttggtattg acagccctca gcattgcact agcaagtgcg caactccaat cgcctttcac 1020 ttcagagtac agaagattaa gtgagagaat tctaccgttc gtatagcata cattatacga 1080 agttattca gtaatgtctt gtttctttg ttgcagtggt gagccatttt gacttcgtga 1140 aagtttcttt agaatagttg tttccagagg ccaaacattc cacccgtagt aaagtgcaag 1200 cgtaggaaga ccaagactgg cataaatcag gtataagtgt cgagcactgg caggtgatct 1260 tctgaaagtt tctactagca gataagatcc agtagtcatg catatggcaa caatgtaccg 1320 tgtggatcta agaacgcgtc ctactaacct tcgcattcgt tggtccagtt tgttgttac 1380 gatcaacgtg acaaggttgt cgattccgcg taagcatgca tacccaagga cgcctgttgc 1440 aattccaagt gagccagttc caacaatctt tgtaatatta gagcacttca ttgtgttgcg 1500 cttgaaagta aaatgcgaac aaattaagag ataatctcga aaccgcgact tcaaacgcca 1560 atatgatgtg cggcacacaa taagcgttca tatccgctgg gtgactttct cgctttaaaa 1620 aattatccga aaaaattttt gacggctagc tcagtcctag gtacgctagc attaaagagg 1680 agaaaatgac tactcttgat gacacagcct acagatatag gacatcagtt ccgggtgacg 1740 cagaggctat cgaagccttg gacggttcat tcactactga tacggtgttt agagtcaccg 1800 ctacaggtga tggcttcacc ttgagagagg ttcctgtaga cccaccctta acgaaagttt 1860 tccctgatga cgaatcggat gacgagtctg atgctggtga ggacggtgac cctgattcca 1920 gaacatttgt cgcatacgga gatgatggtg acctggctgg ctttgttgtg gtgtcctaca 1980 gcggatggaa tcgtagactc acagttgagg acatcgaagt tgcacctgaa catcgtggtc 2040 acggtgttgg tcgtgcactg atgggactgg caacagagtt tgctagagaa agaggagccg 2100 gacatttgtg gttagaagtg accaatgtca acgctcctgc tattcacgca tataggcgaa 2160 tgggtttcac tttgtgcggt cttgatactg ctttgtatga cggaactgct tctgatggtg 2220 aacaagctct ttacatgagt atgccatgtc catagcacgt ccgacggcgg cccacgggtc 2280 ccaggcctcg gagatccgtc ccccttttcc tttgtcgata tcatgtaatt agttatgtca 2340 cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa 2400 cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt 2460 atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg 2520 aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctataactt 2580 cgtatagcat acattatacc ttgttatgcg gccgcaagaa gttgattgag actttcaacg 2640 agctggctct gcttctggta cttcttcagg tgcatcttct gctactcaaa atgacgaaac 2700 atccactgat cttggagctc cagctgcatc tttaagtgca acgccatgtc tttttgccat 2760 cttgctgctc atgttgtagt agactttttt tttcactgag tttttatgta ctactgatta 2820 cattgtgtag gtgtaatgat gtgcactata atactaatat agtcaaaatg ctacagagga 2880 aagtgcaggt tgcctgtggt ggtttttctt attagcaccc tctgaacact ctttacctct 2940 aacatcctca gccatgctaa tcgcgcataa aataaatctt cgaacttttt tccattttat 3000 gctcataaag cttccttact gtcaccttat caaaagagct tttgccacta aagtagtcac 3060 acccagaatt gctcccgaat atcgtccaac aatgctagga tctgtggaaa gtttgacaaa 3120 taatttgaac accttgagct tgaagcttcc tgaagttaat atccaaggct cctttccaga 3180 aagtaaccca gtggaccttt tgagaaacta catcactcaa gaacttagta aaatttctgg 3240 agttgacaaa gaattgattt tcccagcctt ggaatggggt accacactgg aaaaaggtga 3300 tcttttgatc ccagttcctc gtctgagaat aaagggtgct aatcctaaag atttagccga 3360 acaatgggct gctgcattcc caaagggtgg atatcttaaa gacgttattg cgcaaggacc 3420 tttcttgcag ttctttttta acacatcggt tctgtacaag ttggtgatat ctgatgctct 3480 ggagagaggc gatgactttg gtgcacttcc tctaggaaag ggacaaaaag ttatagtgga 3540 gttttcttct ccaaatattg ccaaaccttt ccacgctggc catcttagaa gtacaatcat 3600 cggtggttt atttccaatc tgtatgaaaa gctgggtcat gaagttatga ggatgaatta 3660 tttgggagac tggggaaac aatttggtgt tcttgcagta ggatttgagc gttacggtga 3720 tgaggcaaaa ttaaagactg atccaatcaa ccatttgttt gaggtctatg ttaaaatcaa 3780 ccaagatatt aaggctcaat cagagtctac tgaggagatt gcagaagggc aatcattaga 3840 tgaccaggca agagcttttt tcaagaaaat ggaaaatggc gacgaatcgg ctgtaagctt 3900 gtggaaaaga ttccgtgagt tatccattga gaagtacatt gatacttatg cccgcctcaa 3960 catc 3964 <210> 81 <211> 1019 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 81 gccttctcgt gcaatcagag ctgttgaaag agagaagagg gcacacggaa gctgctgttc 60 aattgtgtga attgaccgga ttacaacctg ctggagtgat aggagagctg gttcgtgacg 120 aggacggctc tatgatgcga ttagacgact gtgttcagtt tggtctccgc cacaacgtaa 180 aaattatcaa ccttgaccag atcattgaat acatggattc caagaacagc tagatacgat 240 ggataggaat acagagatat catgattgag gaacgtaaga gctttttcga aagtgtgagt 300 ttgtggtgag ggccaggcgg tggggaggtg gtggggagcc tccttggtcg aatgtagata 360 tagtaagcaa gacacaagag cgcgcgaagt cttcaacgag gcggcgttgg gtcttgtacg 420 caacgtaatg actacacagt tgagcttgtc gcgaaccggt cgacattttg atcatgcata 480 ctatgttgag acaccatctc gtactattgc ggcaaccagc tgtaaatttg actaattaaa 540 gctgatgaag gatgcagggc gtcgtcaatt ttttgattga ttgcatttaa ttgtttgagc 600 cattcaaggc tgaatgcccg gcaccctaga cccttcttgt gagtactata aacccgcagg 660 cagggtaccc ttggccttct gcgagactac cagtcataac gtatatccac aatgtactag 720 taatagcccc ggaaaactct aatcccacag aacgtctaac gcctcctatg tcatcgatac 780 ccattcgcac tactgccatg gcccccctta cgtgatcatt tcacttactc ccgcctaagc 840 ttcgcccaca tgcctgcgtt ttgccaagat ttactgacga gtttggttta ctcatcctct 900 atttataact actagacttt caccattctt caccaccctc gtgccaatga tcatcaacca 960 cttggtattg acagccctca gcattgcact agcaagtgcg caactccaat cgcctttca 1019 <210> 82 <211> 1322 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 82 ctggctctgc ttctggtact tcttcaggtg catcttctgc tactcaaaat gacgaaacat 60 ccactgatct tggagctcca gctgcatctt taagtgcaac gccatgtctt tttgccatct 120 tgctgctcat gttgtagtag actttttttt tcactgagtt tttatgtact actgattaca 180 ttgtgtaggt gtaatgatgt gcactataat actaatatag tcaaaatgct acagaggaaa 240 gtgcaggttg cctgtggtgg tttttcttat tagcaccctc tgaacactct ttacctctaa 300 catcctcagc catgctaatc gcgcataaaa taaatcttcg aacttttttc cattttatgc 360 tcataaagct tccttactgt caccttatca aaagagcttt tgccactaaa gtagtcacac 420 ccagaattgc tcccgaatat cgtccaacaa tgctaggatc tgtggaaagt ttgacaaata 480 atttgaacac cttgagcttg aagcttcctg aagttaatat ccaaggctcc tttccagaaa 540 gtaacccagt ggaccttttg agaaactaca tcactcaaga acttagtaaa atttctggag 600 ttgacaaaga attgattttc ccagccttgg aatggggtac cacactggaa aaaggtgatc 660 ttttgatccc agttcctcgt ctgagaataa agggtgctaa tcctaaagat ttagccgaac 720 aatgggctgc tgcattccca aagggtggat atcttaaaga cgttattgcg caaggacctt 780 tcttgcagtt cttttttaac acatcggttc tgtacaagtt ggtgatatct gatgctctgg 840 agagaggcga tgactttggt gcacttcctc taggaaaggg acaaaaagtt atagtggagt 900 tttcttcc aaatattgcc aaacctttcc acgctggcca tcttagaagt acaatcatcg 960 gtggtttat ttccaatctg tatgaaaagc tgggtcatga agttatgagg atgaattatt 1020 tgggagactg gggaaaacaa tttggtgttc ttgcagtagg atttgagcgt tacggtgatg 1080 aggcaaaatt aaagactgat ccaatcaacc atttgtttga ggtctatgtt aaaatcaacc 1140 aagatattaa ggctcaatca gagtctactg aggagattgc agaagggcaa tcattagatg 1200 accaggcaag agctttttc aagaaaatgg aaaatggcga cgaatcggct gtaagcttgt 1260 ggaaaagatt ccgtgagtta tccattgaga agtacattga tacttatgcc cgcctcaaca 1320 tc 1322 <210> 83 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 83 atgttcagct tgaaagcatt attgccattg gccttgttgt tggtcagcgc caaccaagtt 60 gctgca 66 <210> 84 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Met Phe Ser Leu Lys Ala Leu Leu Pro Leu Ala Leu Leu Leu Val Ser 1 5 10 15 Ala Asn Gln Val Ala Ala 20 <210> 85 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 85 atgaaagcat tcctgttgtt actactttta ctaggcctgt ccactacact cgctaaggca 60 <210> 86 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 Met Lys Ala Phe Leu Leu Leu Leu Leu Leu Leu Gly Leu Ser Thr Thr 1 5 10 15 Leu Ala Lys Ala 20 <210> 87 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 87 atggaaggtg gcgaagaaga agttgagcgc attcctgatg aacttttcga tacaaaaaag 60 aagcatttgt tagataagct cataagggtc ggaataatcc ttgtactcct gatatggggc 120 actgttttgt tgctaaaaag tatt 144 <210> 88 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 88 Met Glu Gly Gly Glu Glu Glu Val Glu Arg Ile Pro Asp Glu Leu Phe 1 5 10 15 Asp Thr Lys Lys Lys His Leu Leu Asp Lys Leu Ile Arg Val Gly Ile 20 25 30 Ile Leu Val Leu Leu Ile Trp Gly Thr Val Leu Leu Leu Lys Ser Ile 35 40 45 <210> 89 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 89 atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagct 57 <210> 90 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala <210> 91 <211> 5679 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 91 ggcgcgccgt ttaaaccctc caccagccat ataccactac aacaccacag aagagaaaga 60 gctcatatca tccgtcatga gagagtacca gcacagaaat acagtcaagt aaaactagta 120 tgcaagcatt acgtaataat agcaacttta tgacaaatca ttccattttt ttccactgga 180 gcgtgcactg cgtaaatcat tctctttgga aggcaaggga agaacaacaa aatttttcct 240 tccgttatac aaacattgaa tcatgtctac tgaacccact tttaaattgg tccttgtcgg 300 tgatggtggt accggtaaag taagtgcaaa ttatttgatg agtcggataa tgttttccgc 360 cccttagttc ccctcatgat tactaacaat tcatagacca ccttcgttaa gagacacctt 420 actggagagt tccgtaagaa gtacattgct actttgggag tcgaagttca tcccttgtca 480 ttccacacta actgtggtcc tatcacattc aacgtttggg acactgctgg acaagagaag 540 tttggtggac tgagagatgg ttattacatt aacggtgact gtggtatcat catgttcgac 600 gttacatcga gaattactta caagaacgtt caaactggc accgtgactt ggtcagagtg 660 tgtgagaaca ttccaattgt gctttgtggt aacaaggttg atgtcaagga aagaaaggtc 720 aaggctaaga ccatcacttt ccacagaaag aagaacttgc aatactttga cattctgcc 780 aagtccaact acaactttga gaagccattc ttgtggttag ctagaaagtt gtctggtgag 840 ccccaattag agttcgttgc tgctcccgac ttgcaagccc cagaggttca aattgatgcc 900 gatttaataa agaagtacga gcaagagaac gccgaggctg ccgctatgcc attgcctgat 960 gaagatgatg ccgacttgta agcttttact tacagtacat tgagaaccat acatagggca 1020 cgtatcgtaa gtttagttgt ttgctgatgt aagctagtttt gtttctgtag tgtttcgagg 1080 tcgcagaggg atctctctag ccttagacaa aaaaaaaaag gttgacacgt tgatacactc 1140 tctgtttcat ccgatctttc acctacgagt cccactcctc ttcagagtac agaagattaa 1200 gtgagagaat tctaccgttc gtatagcata cattatacga agttatttca gtaatgtctt 1260 gtttcttttg ttgcagtggt gagccatttt gacttcgtga aagtttcttt agaatagttg 1320 tttccagagg ccaaacattc cacccgtagt aaagtgcaag cgtaggaagt ccaagactgg 1380 cataaatcag gtataagtgt cgagcactgg caggtgatct tctgaaagtt tctactagca 1440 gataagatcc agtagtcatg catatggcaa caatgtaccg tgtggatcta agaacgcgtc 1500 ctactaacct tcgcattcgt tggtccagtt tgttgttatc gatcaacgtg acaaggttgt 1560 cgattccgcg taagcatgca tacccaagga cgcctgttgc aattccaagt gagccagttc 1620 caacaatctt tgtaatatta gagcacttca ttgtgttgcg cttgaaagta aaatgcgaac 1680 aaattaagag ataatctcga aaccgcgact tcaaacgcca atatgatgtg cggcacacaa 1740 taagcgttca tatccgctgg gtgactttct cgctttaaaa aattatccga aaaaattttt 1800 gacggctagc tcagtcctag gtacgctagc attaaagagg agaaaatggc taaactgacc 1860 tctgctgttc cggttctgac cgctcgtgac gttgctggtg ctgttgagtt ctggaccgac 1920 cgtctgggtt tctctcgtga cttcgttgag gacgacttcg ctggtgttgt tcgtgacgac 1980 gttaccctgt tcatctctgc tgttcaggac caggttgttc cggacaacac cctggcttgg 2040 gtttgggtc gtggtctgga cgaactgtac gctgaatggt ctgaagttgt ttctaccaac 2100 ttccgtgacg cttctggtcc ggctatgacc gaaatcggtg aacagccgtg gggtcgtgag 2160 ttcgctctgc gtgacccggc tggtaactgc gttcacttcg ttgctgaaga acaggactaa 2220 cacgtccgac ggcggcccac gggtcccagg cctcggagat ccgtccccct tttccttgtgt 2280 cgatatcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct 2340 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt 2400 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg 2460 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa 2520 ggctttaatt tgcaagctat aacttcgtat agcatacatt ataccttgtt atgcggccgc 2580 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 2640 gagagttata gagctgctat gcgtgtaaa atagtttaaa tcttcgtaaa gtatgttagt 2700 ccatgtaatt tgctatgaat cgatacgcta atctggatgc tgaacggatg cttactggca 2760 tgcattattc attackccatc taagctgcgc cacaacccag taaattgcag tgaggaagc 2820 ttccctgtaa ccgtcctgtc cctttagga ccatcgatcc ccaacgatca atcgcgata 2880 catctatcaa ctgtccctt ccatctatct atgcaagta atgacagact ctgttaactc 2940 tgatgattct gatctggaaa tcatagaggt gactgagcct actcaaag tggacctttt 3000 ggccccaat ccagcattta attactgc ccccataagc aacagtaacg gcacaactcc 3060 aaaggaga aaacttgatg accaatccaa ctccaatttct tttgccagac tggaatcgtt 3120 acgggaatca tcagtgaaac cacaagctag tacgttcaat agtagtaggt tcatccccca 3180 agccgaccaa ttttccaata atcagaataa tgaacttgat aacaacaatg gattcgccga 3240 ctggattct aagtcccac ctgaatttcc ctttccactt aatgatggac caaaaagtc 3300 cagcaatca cctacaact caatttga agagatcatc catcaatg agaatcga 3360 gataaataca tctgtccccg catctacatc atctctacc ccagttccct ccagcacaca 3420 gatcagagc catcatatag cacacaca cacacaca gatgcgcata tcttccaagg 3480 gaaacgacct ctccaatcat attcagatga tgagacgaa gatttgcaa ttgtaggatc 3540 caatattgtt cagcagccctc taggaatt gccaggaact ttcaaccccc ctgcaacat 3600 actccatttt gacggttcaa accagaatga acaagccaga tggctggact tgcggataaa 3660 agatttgtta gataatctc acatctcg agttcatgct cagtcgaata ttatggagat 3720 caataggttc atttccactt tggggcattt aaacagagaa gtttaaaccc tgcaggggcgc 3780 tcggtcgttc ggctgcggcg agcggtatca gctcactca aggcggtaat acggttatcc 3840 acagaatcag gggataacgc aggaagac atgtgagca aaggccagca aaaggccagg 3900 aaccgtaaaa aggccgcgtt gctggcgtttt ttccataggc tccgcccccc tgacgagcat 3960 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4020 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4080 tacctgtccg ccttctccc ttcgggaagc gtggcgctt ctcatagctc acgctgtagg 4140 tatctcagtt cggtgtaggt cgtcgctcc aagctgggct gtgtgcacga accccccgtt 4200 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4260 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 4320 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 4380 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 4440 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 4500 agaaaaaaag gatctcaaga agatccttg atcttttcta cggggtctga cgctcagtgg 4560 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 4620 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 4680 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 4740 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacgggga gggcttacca 4800 tctggcccca gtgctgcaat gataccgcgc gacccacgct caccggctcc agatttatca 4860 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5040 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 5460 ttcaccagcg tttctgggtg agcaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 5640 ataggggttc cgcgcacatt tccccgaaaa gtgccacct 5679 <210> 92 <211> 1157 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 92 aaaccctcca ccagccatat accactacaa caccacagaa gagaaagagc tcatatcatc 60 cgtcatgaga gagtaccagc acagaaatac agtcaagtaa aactagtatg caagcattac 120 gtaataatag caactttatg acaaatcatt ccattttttt ccactggagc gtgcactgcg 180 taaatcattc tctttggaag gcaagggaag aacaacaaaa tttttccttc cgttatacaa 240 acattgaatc atgtctactg aacccacttt taaattggtc cttgtcggtg atggtggtac 300 cggtaaagta agtgcaaatt atttgatgag tcggataatg ttttccgccc cttagttccc 360 ctcatgatta ctaacaattc atagaccacc ttcgttaaga gacaccttac tggagagttc 420 cgtaagaagt acattgctac tttgggagtc gaagttcatc ccttgtcatt ccacactaac 480 tgtggtccta tcacattcaa cgtttgggac actgctggac aagagaagtt tggtggactg 540 agagatggtt attacattaa cggtgactgt ggtatcatca tgttcgacgt tacatcgaga 600 attacttaca agaacgttcc aaactggcac cgtgacttgg tcagagtgtg tgagaacatt 660 720 780 aactttgaga agccattctt gtggttagct agaaagttgt ctggtgagcc ccaattagag 840 ttcgttgctg ctcccgactt gcaagcccca gaggttcaaa ttgatgccga tttaataaag 900 aagtacgagc aagagaacgc cgaggctgcc gctatgccat tgcctgatga agatgatgcc 960 1020 ttagttgttt gctgatgtaa gctagtttgt ttctgtagtg tttcgaggtc gcagagggat 1080 ctctctagcc ttagacaaaa aaaaaaaggt tgacacgttg atacactctc tgtttcatcc 1140 gatctttcac ctacgag 1157 <210> 93 <211> 1125 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 93 ggagagttat agagctgcta tgcgtgtaaa aatagtttaa atcttcgtaa agtatgttag 60 tccatgtaat ttgctatgaa tcgatacgct aatctggatg ctgaacggat gcttactggc 120 atgcattatt cattacccat ctaagctgcg ccacaaccca gtaaattgca gtgagggaag 180 cttccctgta accgtcctgt ccctttaggg accatcgatc cccaacgatc aaatcgcgat 240 acatctatca actgtccctt tccatctatc tatgcaaggt aatgacagac tctgttaact 300 ctgatgattc tgatctggaa atcatagagg tgactgagcc tactccaaaa gtggaccttt 360 tggcccccaa tccagcattt aattttactg cccccataag caacagtaac ggcacaactc 420 caataaggag aaaacttgat gaccaatcca actccaattc ttttgccaga ctggaatcgt 480 tacgggaatc atcagtgaaa ccacaagcta gtacgttcaa tagtaggg ttcatccccc 540 aagccgacca atttccaat aatcagaata atgaacttga taacacaat ggattcgccg 600 actggatttc taagtcccca cctgaatttc cctttccact taatgatgga ccaaaaagt 660 ccagcaatca acctacaaac tcaatttg aagagatcat cgatttaact gagatatcg 720 agataaatac atctgtcccc gcatctacat catctcc cccagttccc tccccc 780 agaatcagag ccatcatata gccaaaca acacagcaca agatgcgcat atcttccaag 840 ggaaacgacc tctccaatca tattcagatg atgagacga ragatttgcaa attgtaggat 900 ccaatattgt tcagcagcct ctaggaatta tgccaggaac ttcaacgcc cctgcaaaca 960 tactccattt tgacggttca aaccagaatg aacaagccag atggctggac ttgcggataa 1020 aagatttgtt agataatctt cacaatctc gagttcatgc tcagtcgaat attatggaga 1080 tcaataggtt catttccact tggggcatt taaacagaga agttt 1125 <210> 94 <211> 1292 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 94 ttcagtaatg tcttgtttct tttgttgcag tggtgagcca tttgacttc gtgaaagttt 60 ctttagaata gttgtttcca gaggccaaac attccacccg tagtaaagtg caagcgtagg 120 aagtccaaga ctggcataaa tcaggtataa gtgtcgagca ctggcaggtg atcttctgaa 180 agtttctact agcagataag atccagtagt catgcatatg gcaacaatgt accgtgtgga 240 tctaagaacg cgtcctacta accttcgcat tcgttggtc agtttgttgt tatcgatcaa 300 cgtgacaagg ttgtcgattc cgcgtaagca tgcataccca aggacgcctg ttgcaattcc 360 aagtgagcca gttccaacaa tctttgtaat attagagcac ttcattgtgt tgcgcttgaa 420 agtaaaatgc gaacaaatta agagataatc tcgaaaccgc gacttcaaac gccaatatga 480 tgtgcggcac acaataagcg ttcatatccg ctgggtgact ttctcgcttt aaaaaattat 540 ccgaaaaaat ttttgacggc tagctcagtc ctaggtacgc tagcattaaa gaggagaaaa 600 tggctaaact gacctctgct gttccggttc tgaccgctcg tgacgttgct ggtgctgttg 660 agttctggac cgaccgtctg ggtttctctc gtgacttcgt tgaggacgac ttcgctggtg 720 ttgttcgtga cgacgttacc ctgttcatct ctgctgttca ggaccaggtt gttccggaca 780 acaccctggc ttgggtttgg gttcgtggtc tggacgaact gtacgctgaa tggtctgaag 840 ttgtttctac caacttccgt gacgcttctg gtccggctat gaccgaaatc ggtgaacagc 900 cgtggggtcg tgagttcgct ctgcgtgacc cggctggtaa ctgcgttcac ttcgttgctg 960 aagaacagga ctaacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc 1020 cccttttcct ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc 1080 cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt 1140 tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt 1200 tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt 1260 ttgggacgct cgaaggcttt aatttgcaag ct 1292 <210> 95 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 95 Met Ala Lys Leu Thr Ser Ala Val Pro Val Leu Thr Ala Arg Asp Val 1 5 10 15 Ala Gly Ala Val Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg Asp 20 25 30 Phe Val Glu Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr Leu 35 40 45 Phe Ile Ser Ala Val Gln Asp Gln Val Val Pro Asp Asn Thr Leu Ala 50 55 60 Trp Val Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser Glu 65 70 75 80 Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala Met Thr Glu 85 90 95 Ile Gly Glu Gln Pro Trp Gly Arg Glu Phe Ala Leu Arg Asp Pro Ala 100 105 110 Gly Asn Cys Val His Phe Val Ala Glu Glu Gln Asp 115 120 <210> 96 <211> 11322 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 96 ccagccagga cagaaatgcc tcgacttcgc tgctgcccaa ggttgccggg tgacgcacac 60 cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt 120 aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg 180 gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta 240 tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga 300 gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac atcatgaggg 360 aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc atcgagcgcc 420 atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga 480 agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc 540 ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc 600 tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag 660 ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca ggtatcttcg 720 agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg 780 ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat 840 ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggtgatg 900 agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg 960 cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag tatcagcccg 1020 tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg 1080 cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca 1140 aataacatta ctcgcatcca ttctcaggct gtctcgtctc gtctccaact ttctgccgcc 1200 aatctccttt ccattcaaat tctctacaac aaggcaggct tcgtccgttg atgtgaagtt 1260 tgcaaaagcc aaaccacgaa aaacaccatt gtcaaaatgg tagttgaagg cataaggcaa 1320 aggcaagcta aactttgtca taacgtctaa aagttgctct ttttttatgg caaaagggat 1380 gttctttatc acaatagcag tgggaatgac atcttcatca tcattcgtat catcaatacc 1440 atcgagttct gaatccaaag gtttctgttg ttgggataag gcatcagatt ctttgagcag 1500 gtcgtccttg gcgtcctcct ctagatgaaa tttgccatcg ttaggaggtg agtattggga 1560 aacgttgact tgttgctcaa tccaaggatc aacgttgacc atcggattgc cgttctgagg 1620 agacagccag gcattatacc gtggttgacc ggcgtttacg cgcagaggat tgttgctggt 1680 gttgttgaca gaagacgcat acccagacgc tgcggatgac atcgacgaaa ttgaagggcg 1740 acgttgcata cccatttgtt gctgatccaa gaaacttggt gtttcagtca attgtctcag 1800 gtccatattg atcaatgtgt tcaactgttt caatctggtt ccggagggtg gctattacga 1860 caagctgtgg ctactatcta agtgggagaa gtaacggaac acattgatga gacacaggaa 1920 ttacagggcg tgcatccacc aataacaatt agtcgagatt tcaaccaatt acgtaagcgc 1980 tcaacccttt ttcgaacac gtatcgagca aagtccaggt gaaaccttca tccattatat 2040 ccaaagtcga ccgaagcttt aacaacatcc ataatggtaa gtgtccagtt tgatgagtgc 2100 agaatggttc caagttttag accagttact aatatttaaa gtctacagca attccaggag 2160 gacagagaac gttagctaaa agaagagcag caaacttgga taagaaacag gatgaaccaa 2220 cctccgccag atctgccggt gctggaggtt cttcgtctac catgctaaag ttgtacacag 2280 acgaggccca aggttgaaa gttgatcctt taattgttct tgttcttgct gttggtttca 2340 ttttcagtgt cattggttg cacgttgttg ctaagctgac aggaaagttg atcaactaag 2400 acctatattt aaacaggttt catcatatct gtactatatt tacaagtcca ctgcgtttag 2460 gtatatacta aagacattca agaagcacat ccacaacttg tgcaagtcct gtcaaatgta 2520 ctagatgctt ttcagaacat cctgcggttt gaggagattc ctgaatttcc cagtcccaag 2580 tctttctctt gtagaggtct ttgagttctt gtgaatgctg aattggggtt cttacctcaa 2640 tttctattag tgggaaatgc tttcccacaa ttatttgcaa tgggatcccg gcaactttac 2700 tttgcttcaa cttatgtccc atactg...

Claims

1. A microorganism called Pichia pastoris, in which the sec72 gene is deleted, The microorganism expresses a recombinant protein comprising a secretory signal sequence derived from yeast, The recombinant protein comprises at least one block polypeptide sequence derived from a silk protein or is a silk-like protein; and The secretory signal sequence is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. The microorganism.

2. The microorganism of claim 1 , wherein the sec72 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

1.

3. The microorganism of claim 1 , wherein the sec72 gene comprises SEQ ID NO:

1.

4. 2. The microorganism of claim 1, wherein the sec72 gene is present at locus PAS_chr2-1_0448 of the microorganism.

5. The microorganism according to any one of claims 1 to 4, further comprising a recombinantly expressed SSH1 translocon complex.

6. The microorganism of claim 5, wherein the SSH1 translocon complex comprises a first polypeptide sequence at least 95% identical to SEQ ID NO:4, a second polypeptide sequence at least 95% identical to SEQ ID NO:6, and a third polypeptide sequence at least 95% identical to SEQ ID NO:

8.

7. The microorganism of claim 5, wherein the SSH1 translocon complex comprises a first polypeptide comprising SEQ ID NO:4, a second polypeptide comprising SEQ ID NO:6, and a third polypeptide comprising SEQ ID NO:

8.

8. The microorganism according to any one of claims 1 to 7, further comprising a recombinantly expressed translocon complex.

9. The microorganism of claim 8 , wherein the translocon complex is expressed from a recombinant SSH1 gene, a recombinant SSS1 gene, and a recombinant SBH2 gene.

10. The microorganism of claim 9, wherein the SSH1 gene comprises SEQ ID NO:

3.

11. The microorganism of claim 9, wherein the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

3.

12. The microorganism of claim 9, wherein the SSH1 gene comprises SEQ ID NO:

5.

13. The microorganism of claim 9, wherein the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

5.

14. The microorganism of claim 9, wherein the SBH2 gene comprises SEQ ID NO:

7.

15. The microorganism of claim 9, wherein the SBH2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

7.

16. The microorganism described in claim 9, wherein expression of the recombinant SSH1 gene to increase the level of the SSH1 translocon in the microorganism beyond the level of the SSH1 translocon expressed by the natural organism improves the growth rate and / or fermentation capacity of the microorganism.

17. The microorganism of claim 8 , wherein the translocon complex comprises an SSH1 protein, an SSS1 protein, and an SBH2 protein.

18. The microorganism of claim 17, wherein the SSH1 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

4.

19. The microorganism of claim 17, wherein the SSH1 protein comprises SEQ ID NO:

4.

20. The microorganism described in claim 17, wherein the SSS1 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

6.

21. The microorganism described in claim 17, wherein the SSS1 protein comprises SEQ ID NO:

6.

22. 18. The microorganism of claim 17, wherein the SBH2 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

8.

23. The microorganism of claim 17 , wherein the SBH2 protein comprises SEQ ID NO:

8.

24. The microorganism according to any one of claims 1 to 23, wherein the activities of YPS1-1 protease and YPS1-2 protease are weakened or eliminated.

25. 25. The microorganism of claim 24, wherein the YPS1-1 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

10.

26. 25. The microorganism of claim 24, wherein the YPS1-1 protease comprises SEQ ID NO:

10.

27. The microorganism of claim 24, wherein the YPS1-1 protease is encoded by the YPS1-1 gene.

28. The microorganism of claim 27, wherein the YPS1-1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

9.

29. The microorganism of claim 27, wherein the YPS1-1 gene comprises SEQ ID NO:

9.

30. 28. The microorganism of claim 27, wherein the YPS1-1 gene is present at locus PAS_chr4_0584 of the microorganism.

31. The microorganism of any one of claims 24 to 30, wherein the YPS1-2 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

12.

32. 25. The microorganism of claim 24, wherein the YPS1-2 protease comprises SEQ ID NO:

12.

33. The microorganism of claim 24, wherein the YPS1-2 protease is encoded by the YPS1-2 gene.

34. The microorganism of claim 33, wherein the YPS1-2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

11.

35. The microorganism of claim 33, wherein the YPS1-2 gene comprises SEQ ID NO:

11.

36. The microorganism of claim 33, wherein the YPS1-2 gene is present at locus PAS_chr3_1157 of the microorganism.

37. 37. The microorganism according to any one of claims 24 to 36, wherein the YPS1-1 gene or the YPS1-2 gene, or both, are mutated or knocked out.

38. The microorganism according to any one of claims 24 to 37, wherein the activity of one or more further proteases is reduced or eliminated.

39. The silk-like polypeptide comprises one or more repeat sequences (SEQ ID NO: 13), During the ceremony, X1=SGGQQ (SEQ ID NO:14) or GAGQQ (SEQ ID NO:15) or GQGPY (SEQ ID NO:16) or AGQQ (SEQ ID NO:17) or SQ; n1 is 4 to 8; n2 is 6 to 20, and n3 is 2 to 20; The microorganism described in claim 1.

40. The microorganism of claim 1 , wherein the silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:

21.

41. The microorganism according to any one of claims 1 to 40, wherein the secretory signal sequence is selected from the group consisting of SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, and SEQ ID NO:

90.

42. A microorganism called Pichia pastoris, The sec72 gene comprising SEQ ID NO:1 of the microorganism is knocked out, a recombinantly expressed SSH1 gene comprising SEQ ID NO:3, a recombinantly expressed SSS1 gene comprising SEQ ID NO:5, and a recombinantly expressed SBH2 gene comprising SEQ ID NO:7; a recombinantly expressed protein comprising a silk-like polypeptide comprising a yeast-derived secretory signal sequence and a polypeptide encoded by SEQ ID NO:21; and The secretory signal sequence is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. The microorganism.

43. A cell culture comprising a microorganism according to any one of claims 1 to 42.

44. 43. A cell culture comprising a microorganism according to any one of claims 5 to 42, wherein the growth rate and fermentation capacity of the strain is improved under standard cell culture conditions compared to a cell culture not comprising a recombinantly expressed SSH1 translocon complex.

45. A cell culture comprising the microorganism according to any one of claims 5 to 42, Under standard cell culture conditions, microorganisms which are otherwise identical except that they contain a functional sec72 gene and do not contain a recombinantly expressed SSH1 translocon complex, each of which has the same number of copies of a recombinant silk polypeptide gene. the yield or specific productivity of the recombinant protein is improved compared to a cell culture of The cell culture.

46. Cultivating the microorganism according to any one of claims 1 to 42 in a medium under conditions suitable for the expression of the recombinantly expressed protein; and isolating said recombinant protein from said microorganism or from said culture medium. A method for producing a recombinant protein comprising the steps of:

47. 47. The method of claim 46, wherein the recombinant protein is secreted from the microorganism and the step of isolating the recombinant protein comprises recovering the medium containing the secreted recombinant protein.

48. 47. The method of claim 46, wherein the yield or specific productivity of the recombinant protein is increased in the microorganism compared to an otherwise identical microorganism in which the sec72 gene has not been deleted.

49. an identical microorganism except that it does not contain the recombinantly expressed SSH1 translocon complex and the sec72 gene is not deleted.

47. The method of claim 46, wherein the yield or specific productivity of the recombinant protein in the microorganism is increased compared to

50. 1. A method for modifying Pichia pastoris to improve secretion of a recombinantly expressed protein that contains a yeast-derived secretory signal sequence, comprising: the method comprising knocking out a gene encoding a SEC72 protein, the recombinantly expressed protein comprises at least one blocked polypeptide sequence derived from a silk protein or comprises a silk-like protein; and The secretory signal sequence is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. The method.

51. 51. The method of claim 50, further comprising transforming the Pichia pastoris with a vector comprising a gene encoding a recombinantly expressed SSH1 translocon complex.

52. The silk-like polypeptide has one or more repeat sequences {GGY-[GPG-X １ ] ｎ１ -GPS-(A) ｎ２ } ｎ３ (SEQ ID NO: 13), During the ceremony, X １ = SGGQQ (SEQ ID NO: 14) or GAGQQ (SEQ ID NO: 15) or GQGPY (SEQ ID NO: 16) or AGQQ (SEQ ID NO: 17) or SQ, n1 is 4 to 8; n2 is 6 to 20, and n3 is 2 to 20; 51. The method of claim 50.

53. 51. The method of claim 50, wherein the recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO:21.

Images