SURFACE DISPLAYED ENDOGLYCOSIDASES
20240026325 ยท 2024-01-25
Inventors
Cpc classification
C12N9/2402
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure provides engineered eukaryotic cells comprising a surface displayed catalytic domain of an endoglycosidase and methods of use.
Claims
1. An engineered eukaryotic cell comprising a surface displayed catalytic domain of an endoglycosidase, wherein the surface displayed catalytic domain of an endoglycosidase is a portion of a fusion protein expressed by the cell.
2. The engineered eukaryotic cell of claim 2, wherein the fusion protein further comprises an anchoring domain of a cell surface protein.
3. The engineered eukaryotic cell of claim 1, wherein the fusion protein comprises a portion of the endoglycosidase in addition to its catalytic domain.
4. The engineered eukaryotic cell of claim 1, wherein the fusion protein comprises substantially the entire amino acid sequence of the endoglycosidase.
5. The engineered eukaryotic cell of claim 1, wherein the endoglycosidase is endoglycosidase H.
6. The engineered eukaryotic cell of claim 1, wherein the fusion protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 or SEQ ID NO:2.
7. The engineered eukaryotic cell of claim 1, wherein the fusion protein comprises a portion of the cell surface protein in addition to its anchoring domain.
8. The engineered eukaryotic cell of claim 1, wherein the fusion protein comprises substantially the entire amino acid sequence of the cell surface protein.
9. The engineered eukaryotic cell of claim 1, wherein the cell surface protein is selected from Sed1p, Flo5-2, or Flo11.
10. The engineered eukaryotic cell of claim 1, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to one of SEQ ID NO: 3 to SEQ ID NO: 7 and SEQ ID NO: 20.
11. The engineered eukaryotic cell of claim 1, wherein the anchoring domain stably attaches the fusion protein to the extracellular surface of the cell.
12. The engineered eukaryotic cell of claim 1, wherein upon translation the fusion protein comprises a signal peptide and/or a secretory signal.
13. The engineered eukaryotic cell of claim 1, wherein the anchoring domain is N-terminal to the catalytic domain in the fusion protein.
14. The engineered eukaryotic cell of claim 13, wherein the fusion protein comprises a linker C-terminal to the anchoring domain.
15. The engineered eukaryotic cell of claim 1, wherein the anchoring domain is C-terminal to the catalytic domain in the fusion protein.
16. The engineered eukaryotic cell of claim 15, wherein the fusion protein comprises a linker N-terminal to the anchoring domain.
17. The engineered eukaryotic cell of claim 1, wherein the cell surface protein is Sed1p and the endoglycosidase is endoglycosidase H.
18. The engineered eukaryotic cell of claim 17, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 or SEQ ID NO:
19. The engineered eukaryotic cell of claim 1, wherein the cell surface protein is Flo5-2 or Flo11 and the endoglycosidase is endoglycosidase H.
20. The engineered eukaryotic cell of claim 19, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 11 or SEQ ID NO: 12.
21. The engineered eukaryotic cell of claim 19, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 14.
22. The engineered eukaryotic cell of claim 1, wherein the engineered eukaryotic cell comprises a mutation in its AOX1 gene and/or its AOX2 gene.
23. The engineered eukaryotic cell of claim 1, wherein the engineered eukaryotic cell is a yeast cell or a Pichia species.
24. The engineered eukaryotic cell of claim 23, wherein the yeast cell is a Pichia species.
25. The engineered eukaryotic cell of claim 1, further comprising a genomic modification that overexpresses a secretory glycoprotein.
26. The engineered eukaryotic cell of claim 25, wherein the secretory glycoprotein is an animal protein, e.g., an egg protein.
27. The engineered eukaryotic cell of claim 26, wherein the egg protein is selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
28. The engineered eukaryotic cell of claim 1, wherein the cell lacks a genomic modification that overexpresses a secretory glycoprotein.
29. The engineered eukaryotic cell of claim 1, comprising a nucleic acid sequence that encodes the fusion protein.
30. The engineered eukaryotic cell of claim 29, wherein the nucleic acid sequence that encodes the fusion protein is integrated into the cell's genome.
31. The engineered eukaryotic cell of claim 29, wherein the nucleic acid sequence that encodes the fusion protein is extrachromosomal.
32. The engineered eukaryotic cell of claim 29, wherein the nucleic acid sequence comprises an inducible promoter.
33. The engineered eukaryotic cell of claim 32, wherein the inducible promoter is an AOX1, DAK2, PEX11, FLD1, FGH1, DAS2, CAT1, MDH3, HAC1, BiP, RAD30, RVS161-2, MPP10, THP3, or GBP2 promoter.
34. The engineered eukaryotic cell of claim 29, wherein the nucleic acid sequence comprises an AOX1, TDH3, RPS25A, or RPL2A terminator.
35. The engineered eukaryotic cell of claim 29, wherein the nucleic acid sequence encodes a signal peptide and/or a secretory signal.
36. The engineered eukaryotic cell of claim 29, wherein the nucleic acid sequence comprises codons that are optimized for the species of the engineered cell.
37. A method for deglycosylating a secreted glycoprotein, the method comprising contacting a secreted protein with a fusion protein anchored to an engineered eukaryotic cell of claim 1, thereby providing a deglycosylated secreted glycoprotein.
38. The method of claim 37, wherein the secreted glycoprotein is expressed by the engineered eukaryotic cell.
39. The method of claim 37, wherein the fusion protein anchored to an engineered eukaryotic cell is more effective at deglycosylating the secreted protein than an intracellular endoglycosidase.
40. The method of claim 39, wherein the intracellular endoglycosidase is located within a Golgi vesicle.
41. The method of claim 39, wherein the intracellular endoglycosidase is linked to a membrane associating domain.
42. The method of claim 41, wherein the membrane associating domain comprises an amino acid sequence of OCH1.
43. The method of claim 37, wherein the secreted protein is expressed by a cell other than the engineered eukaryotic cell.
44. The method of claim 37, further comprising a step of isolating the deglycosylated secreted protein.
45. The method of claim 44, further comprising a step of drying the deglycosylated secreted protein.
46. The method of claim 37, wherein the secreted protein is an animal protein, e.g., an egg protein.
47. The method of claim 46, wherein the egg protein is selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
48. A method for deglycosylating a plurality of secreted glycoproteins, the method comprising contacting the plurality of secreted glycoproteins with a population of engineered eukaryotic cells of claim 1, thereby providing a plurality of deglycosylated secreted glycoproteins.
49. The method of claim 48, wherein substantially every secreted glycoprotein in the plurality of secreted proteins is deglycosylated upon contact with the population of engineered eukaryotic cells.
50. The method of claim 48, wherein the amount of deglycosylation of the secreted glycoproteins is not increased by further contacting the secreted protein with an isolated endoglycosidase.
51. The method of claim 48, wherein the amount of deglycosylation of the secreted glycoproteins is more than the amount obtained from a population of cells that express an intracellular endoglycosidase.
52. The method of claim 48, further comprising a step of isolating the plurality of deglycosylated secreted proteins.
53. The method of claim 52, further comprising a step of drying the plurality of deglycosylated secreted proteins.
54. The method of claim 48, wherein the secreted protein is an animal protein, e.g., an egg protein.
55. The method of claim 54, wherein the egg protein is selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
56. A method for expressing a fusion protein comprising an anchoring domain of a cell surface protein and a catalytic domain of an endoglycosidase, the method comprising obtaining the engineered eukaryotic cell of claim 1 and culturing the engineered eukaryotic cell under conditions that promote expression of the fusion protein.
57. The method of claim 56, wherein when the engineered eukaryotic cell comprises a nucleic acid sequence that encodes the fusion protein and comprises an inducible promoter, culturing the engineered eukaryotic cell under conditions that promote expression of the fusion protein comprises contacting the cell with an agent that activates the inducible promoter.
58. The method of claim 57, wherein the inducible promoter is an AOX1, DAK2, PEX11 promoter and the agent that activates the inducible promoter is methanol.
59. A population of engineered eukaryotic cells of claim 1.
60. A bioreactor comprising the population of engineered eukaryotic cells of claim 59.
61. A composition comprising an engineered eukaryotic cell of claim 1 and a secreted glycoprotein.
62. The composition of claim 61, wherein the secreted glycoprotein is an animal protein, e.g., an egg protein.
63. The composition of claim 62, wherein the egg protein is selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
64. A composition comprising an engineered eukaryotic cell of claim 1, a secreted protein that has been deglycosylated, and one or more oligosaccharides cleaved from the secreted protein.
65. The composition of claim 64, wherein the secreted glycoprotein is an animal protein, e.g., egg protein.
66. The composition of claim 65, wherein the egg protein is selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
67. An engineered eukaryotic cell which expresses a surface displayed catalytic domain of endoglycosidase H, wherein the catalytic domain is directly or indirectly tethered to the exterior surface of the cell.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also Figure and FIG. herein), of which:
[0048]
[0049]
DETAILED DESCRIPTION
Introduction
[0050] The present disclosure provides engineered eukaryotic cells comprising a surface displayed catalytic domain of an endoglycosidase and methods of use.
[0051] Surface displaying a catalytic domain of an endoglycosidase provides efficient extracellular deglycosylation of glycoproteins. A glycoprotein is a protein that carries carbohydrates covalently bound to their peptide backbone. It is known that approximately half of all proteins typically expressed in a cell undergo glycosylation, which entails the covalent addition of sugar moieties (e.g., oligosaccharides) to specific amino acids. Most soluble and membrane-bound proteins expressed in the endoplasmic reticulum are glycosylated to some extent, including secreted proteins, surface receptors and ligands, and organelle-resident proteins. Additionally, some proteins that are trafficked from the Golgi to the cell wall and/or to the extracellular environment are also glycosylated. Lipids and proteoglycans can also be glycosylated, significantly increasing the number of substrates for this type of modification. In particular, many cell wall proteins are glycosylated.
[0052] Protein glycosylation has multiple functions in a cell. In the ER, glycosylation is used to monitor the status of protein folding, acting as a quality control mechanism to ensure that only properly folded proteins are trafficked to the Golgi. Oligosaccharides on soluble proteins can be bound by specific receptors in the trans Golgi network to facilitate their delivery to the correct destination. These oligosaccharides can also act as ligands for receptors on the cell surface to mediate cell attachment or stimulate signal transduction pathways. Because they can be very large and bulky, oligosaccharides can affect proteinprotein interactions by either facilitating or preventing proteins from binding to cognate interaction domains.
[0053] In general, a glycoprotein's oligosaccharides are important to the protein's function. Consequently, should a glycoprotein be deglycosylated intracellularly, once the protein has reached its final destination (if ever), and in a deglycosylated state, the protein may have a lessened and/or an absent activity.
[0054] When it is desirable to deglycosylate a recombinant glycoprotein for inclusion in composition for human or animal use (e.g., a food product, drink product, nutraceutical, pharmaceutical, or cosmetic), the recombinant glycoprotein may be contacted with an isolated endoglycosidase that is capable of cleave sugar chains from the glycoprotein. For this, the isolated endoglycosidase may be added to a culturing vessel such that the recombinant glycoprotein is deglycosylated once secreted into its culturing medium. Alternately, a recombinant glycoprotein that has been separated from its culturing medium may be subsequently incubated with the isolated endoglycosidase. Although both of these methods may have effectiveness in providing deglycosylated recombinant proteins, they both increase, at least, the time, expense, and inefficiency involved with manufacturing deglycosylated recombinant proteins. When preparing deglycosylated recombinant proteins for human or animal use, e.g., in a consumable composition, it is preferable, and in some cases, necessary due to regulatory requirements, for the final recombinant protein be free of contaminants. One such contaminant is the endoglycosidase itself. In this case, the endoglycosidase must be removed in part or completely from the final recombinant protein product. This removal would entail multiple purification steps that both increase the expense due to these additional steps and reduce the amount of recombinant protein produced, as some protein would be lost during the various purifications. Also, these purification steps would extend the time for manufacturing the recombinant protein product, thereby reducing efficiency of the process. Moreover, when a recombinant glycoprotein is combined with the endoglycosidase, either in a culturing medium or after the recombinant glycoprotein has been separated from its medium, there is no guarantee that each recombinant glycoprotein will come into contact with an endoglycosidase; to ensure sufficient deglycosylation, the glycoprotein and endoglycosidase must remain in a solution for an extended period of time. This extension of time further reduces the efficiency of the manufacturing process. Finally, purchasing the isolated endoglycosidase or manufacturing the isolated endoglycosidase in house would incur additional expenses. Together, there is an unmet need for manufacturing deglycosylated recombinant protein that is effective and efficient. The methods and systems of the present disclosure satisfy this unmet need.
[0055] In the present disclosure, an endoglycosidase is localized to the extracellular surface of a cell, i.e., is surface displayed. This way, the endoglycosidase is unlikely to contact an intracellular, membrane-associated, or cell wall glycoprotein, thereby lowering the opportunity for the endoglycosidase to remove a needed oligosaccharide from the glycoprotein. Instead, the surface displayed endoglycosidase primarily deglycosylates proteins found in the extracellular space, e.g., secreted recombinant proteins. Accordingly, the present disclosure provides recombinant cells having the means to deglycosylate secreted glycoproteins proteins and having a reduced likelihood of undesirably deglycosylating its own intracellular, membrane bound, or cell wall glycoproteins.
[0056] Additionally, since the surface displayed endoglycosidase is securely attached to the recombinant cell, it is not released into and present in a culturing medium. Thus, there is no need to separate the endoglycosidase from the secreted recombinant protein when making a generally contaminant-free recombinant protein product. In other words, the use of surface displayed endoglycosidase avoids the added expense, time, and inefficiency, as described above, that is needed to later remove the endoglycosidase when manufacturing a recombinant protein product for human or animal use, e.g., in a consumable composition.
Fusion Proteins
[0057] Aspects of the present disclosure provide an engineered eukaryotic cell comprising a surface displayed catalytic domain of an endoglycosidase. The surface displayed catalytic domain of the endoglycosidase is included in a fusion protein expressed by the cell. As used herein, the term catalytic domain comprises a portion of an endoglycosidase that provides catalytic activity.
[0058] A fusion protein is a protein consisting of at least two domains that are normally encoded by separate genes but have been joined so that they are transcribed and translated as a single unit; thereby, producing a single (fused) polypeptide.
[0059] In the present disclosure, a fusion protein comprises at least a catalytic domain of an endoglycosidase and an anchoring domain of a cell surface protein.
[0060] A fusion protein may further comprise linkers that separate the two domains. Linkers can be flexible or rigid; they can be semi-flexible or semi-rigid. Separating the two domains, may promote activity of the catalytic domain in that it reduces steric hindrance upon the catalytic site which may be present if the catalytic site is too closely positioned relative to an anchoring domain. Additionally, a linker may further project the catalytic domain into the extracellular space, thereby increasing the likelihood that the catalytic domain will encounter and cleave glycoproteins.
[0061] When a linker is present, a fusion protein may have a general structure of: N terminus -(a)-(b)-(c)-C terminus, wherein (a) is comprises a first domain, (b) is one or more linkers, and (c) is a second domain. The first domain may comprise a catalytic domain of an enzyme and the second domain may comprise an anchoring domain of a cell surface protein. Alternately, the first domain may comprise an anchoring domain of a cell surface protein and the second domain may comprise a catalytic domain of an enzyme. In some embodiments, the anchoring domain is N-terminal to the catalytic domain in the fusion protein. The fusion protein may comprise a linker C-terminal to the anchoring domain. In other embodiments, the anchoring domain is C-terminal to the catalytic domain in the fusion protein. The fusion protein may comprise a linker N-terminal to the anchoring domain.
[0062] In some embodiments, a fusion protein comprises more than one anchoring domains of a cell surface protein. In such embodiments, the fusion protein may have a general structure of: N terminus -(a)-(b)-(c)-(d)-(e)- C terminus, wherein (a) and (e) comprise anchoring domains of a cell surface protein, (b) and (d) are linkers (which may be the same linker or different) and (c) is comprises a catalytic domain of an enzyme.
[0063] Linkers useful in fusion proteins may comprise one or more sequences of SEQ ID NO: 21 to SEQ ID NO: 25. In one example, a tandem repeat (of two, three, four, five, six, or more copies) of a linker, e.g., of SEQ ID NO: 22 or SEQ ID NO: 23, is included in a fusion protein.
[0064] In embodiments, a fusion protein comprises a Glu-Ala-Glu-Ala (EAEA; SEQ ID NO: 21) spacer dipeptide repeat. The EAEA is a removable signal that promotes yields of an expressed protein in certain cell types.
[0065] Other linkers are well-known in the art and can be substituted for the linkers of SEQ ID NO: 21 to SEQ ID NO: 25. For example, In embodiments, the linker may be derived from naturally-occurring multi-domain proteins or are empirical linkers as described, for example, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contents of which are hereby incorporated by reference. In embodiments, the linker may be designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et. al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.
[0066] In embodiments, the linker comprises a polypeptide. In embodiments, the polypeptide is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long. For example, the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In some cases, the linker is about 59 amino acids long.
[0067] The length of a linker may be important to the effectiveness of a surface displayed endoglycosidase catalytic domain. For example, if a linker is too short, then the catalytic domain of the endoglycosidase may not project far enough away from the cell surface such that it is incapable of interacting with a glycoprotein. In this case, the catalytic domain may be buried in the cell wall and/or among other cell surface proteins or sugars. On the other hand, the linker may be too long and/or too rigid to allow adequate contact between a secreted glycoprotein and the catalytic domain of the endoglycosidase.
[0068] The secondary structure of a linker may also be important to the effectiveness of a surface displayed endoglycosidase catalytic domain. More specifically, a linker designed to have a plurality of distinct regions may provide additional flexibility to the fusion protein. As examples, a linker having one or more alpha helices may be superior to a linker having no alpha helices.
[0069] The longer linker of (SEQ ID NO: 25) comprises three subsections: an N-terminal flexible GS linker with higher S content (SEQ ID NO: 295), a rigid linker that forms four turns of an alpha helix (SEQ ID NO: 24), and a flexible GS linker with much higher G content (SEQ ID NO: 296) on its C-terminus. Linkers containing only G's and S's in repetitive sequences are commonly used in fusion proteins as flexible spacers that do not introduce secondary structure. In some cases, the ratio of G to S determines the flexibility of the linker. Linkers with higher G content may be more flexible than linkers with higher S content. The structure of the linker of SEQ ID NO: 25 is designed to mimic multi-domain proteins in nature, which often uses alpha helices (sometimes multiple) to separate as well as orient their domains spatially. In fusion proteins of the present disclosure, a complex linker, such as that of SEQ ID NO: 25 can be viewed as a multi-domain protein with the catalytic domain of an endoglycosidase and an anchoring domain of a cell surface protein being separate functional domains.
[0070] In various embodiments, the fusion protein comprises a linker having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25.
[0071] In embodiments, the linker is substantially comprised of glycine and serine residues (e.g. about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines).
Endoglycosidases
[0072] An Endoglycosidase is an enzyme that releases oligosaccharides from glycoproteins or glycolipids. Unlike exoglycosidases, endoglycoidases cleave polysaccharide chains between residues that are not the terminal residue and break the glycosidic bonds between two sugar monomer in the polymer. When an endoglycosidase cleaves, it releases an oligosaccharide product.
[0073] Numerous endoglycosidases have been characterized, cloned, and/or purified. These include Endoglycosidase D, Endoglycosidase F1, Endoglycosidase F2, Endoglycosidase F3, Endoglycosidase H, Endoglycosidase Hf, Endoglycosidase S, Endoglycosidase T, Endoglycoceramidase I, O-Glycosidase, Peptide-N-Glycosidase A (PNGaseA), and PNGaseF.
[0074] Normally, an endoglycosidase comprises at least a catalytic domain which is responsible for cleaving an oligonucleotide from a glycoprotein. The endoglycosidase may also comprise domains that help recognize an oligosaccharide and/or the glycoprotein itself. The endoglycosidase may further comprise domains that help facilitate, e.g., positioning of the oligosaccharide and/or glycoprotein itself, cleavage of the oligosaccharide.
[0075] In various embodiments, a fusion protein comprises at least the catalytic domain of the endoglycosidase. In some cases, a fusion protein comprises a portion of the endoglycosidase in addition to its catalytic domain. In some embodiments, a fusion protein comprises substantially the entire amino acid sequence of the endoglycosidase.
Endoglycosidase H
[0076] In some cases, the endoglycosidase is endoglycosidase H.
[0077] Endoglycosidase H (Endo H); Endo-beta-N-acetylglucosaminidase H (EC:3.2.1.96); DI-N-acetylchitobiosyl beta-N-acetylglucosaminidase H; Mannosyl-glycoprotein endo-beta-N-acetyl-glucosaminidase H is a highly specific endoglycosidase which cleaves asparagine-linked mannose rich oligosaccharides, but not highly processed complex oligosaccharides from glycoproteins. EndoH hydrolyzes (cleaves) the bond in the diacetylchitobiose core of the oligosaccharide between two N-acetylglucosamine (GlcNAc) subunits directly proximal to the asparagine residue, generating a truncated sugar molecule that is released intact and one N-acetylglucosamine residue remaining on the asparagine.
[0078] Variants of the known amino acid sequence of endoH may be determined by consulting the literature. e.g. Robbins et al., Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H. J. Biol. Chem. 259:7577-7583 (1984); Rao et al., Crystal structure of endo-beta-N-acetylglucosaminidase H at 1.9-A resolution: active-site geometry and substrate recognition. Structure 3:449-457 (1995); Rao et al., Mutations of endo-beta-N-acetylglucosaminidase H active site residue Asp130 and Glu132: activities and conformations. Protein Sci. 8:2338-2346 (1999); the contents of which are incorporated by reference in their entirety. For example, Rao et al., (1999) teaches specific mutations that reduce (e.g., from 1.25% to 0.05% of wild-type activity) or completely obliterate enzymatic activity. Thus, a variant of endoH which comprises a substitution at Asp172 and/or Glu174 (with respect to SEQ ID NO: 2) would be understood to have undesired activity. Based on the published structural and functional analyses and routine experimentation, it could be readily determined those amino acids within endoH that could be substituted and would retain enzymatic activity and which amino acids could not be substituted.
[0079] In embodiments, the endoH that is surface displayed, e.g., is part of a fusion protein, comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 1 lacks an N-terminal signal peptide that is present in SEQ ID NO: 2. The endoH may be a variant of SEQ ID NO: 1 or SEQ ID NO: 2. The variant may have at least or about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 1 or SEQ ID NO: 2.
Surface Display
[0080] Aspects of the present disclosure include engineered eukaryotic cells comprising a surface displayed catalytic domain of an endoglycosidase.
[0081] In embodiment, surface display occurs by attachment of the catalytic domain to the extracellular surface of the cell via an anchoring domain of a cell surface protein. In the present disclosure, the catalytic domain and anchoring domain are present in a fusion protein, optionally, separated by one or more linkers.
[0082] Surface display is understood as the projection of a protein, e.g., a fusion protein, out from a cell's surface and/or from the cell's membrane and into the extracellular space, e.g., into the growth medium in which the engineered eukaryotic cell is being cultured. By projecting into the extracellular space, a surface displayed fusion protein is positioned to interact with soluble glycoproteins present in the extracellular space. Alternately, a surface displayed fusion protein is positioned to interact with cell-associated proteins on adjacent cells. When the surface displayed fusion protein comprise a catalytic domain of an enzyme, e.g., an endoglycosidase, and especially, endoH, the catalytic domain is positioned to cleave off oligonucleotides from soluble glycoproteins present in the extracellular space or cleave off oligonucleotides from cell-associated glycoproteins on adjacent cells.
[0083] In some cases, the cell that expresses a surface displayed fusion protein also expresses (co-expresses) a secreted glycoprotein. This co-expression simplifies the production of deglycosylated proteins in that only one engineered cell needs to be produced and cultured. Moreover, as the secreted glycoprotein is released by the engineered cell, it is an enhanced likelihood of contacting the fusion protein that is located on the surface of the same cell.
[0084] In alternate case, the cell that expresses the fusion protein is different from the cell that secretes the glycoprotein. An advantage of this configuration is that an engineered cell that optimally expresses a fusion protein can be co-cultured with an engineered cell that optimally expresses a secreted glycoprotein.
[0085] To ensure that a fusion protein is surface displayed and remains attached to the extracellular surface of a cell rather than being secreted and released into the extracellular space, a fusion protein comprises an anchoring domain from a cell surface protein. These anchoring domains either bind to a component of the cell's membrane or its cell wall or the anchoring domain comprises a motif that is used to attach the protein to the cell's membrane, e.g., via a glycosylphosphatidylinositol (GPI) anchor. Thus, the anchoring domain stably attaches the fusion protein to the extracellular surface of the engineered cell.
[0086] In some cases, a fusion protein comprises a portion of the cell surface protein in addition to its anchoring domain. In embodiments, a fusion protein comprises substantially the entire amino acid sequence of the cell surface protein.
[0087] In various embodiments, the cell surface protein is selected from Sed1p, Flo5-2, Flo11, Saccharomyces cerevisiae Flo5, CWP, and PIR.
[0088] Sed1p is a major component of the Saccharomyces cerevisiae cell wall. It is required to stabilize the cell wall and for stress resistance in stationary-phase cells. See, e.g., the world wide web (at) uniprot.org/uniprot/Q01589. It is believed that Asn 318 (with respect to SEQ ID NO: 3) is the most likely candidate for the GPI attachment site in Sed1p. In some embodiments, a fusion protein comprising a Sed1p anchoring domain has a sequence having at least 95% or more sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4. In some cases, the sequence identity may be greater than or about 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the Sed1p anchoring domain of a fusion protein of the present disclosure comprises a GPI attachment site; thus, the anchoring domain may only require a short fragment of SEQ ID NO: 3 or SEQ ID NO: 4, i.e., a fragment that is 5, 10, 25, 50, 100, 200, or 300 or more amino acids in length, as long as it is capable of projecting the catalytic domain of the fusion protein into the extracellular space. In some embodiments, the anchoring domain comprises, at least, Sed1p's GPI attachment site.
[0089] In some cases, the cell surface protein is Sed1p and the endoglycosidase is endoglycosidase H. The fusion protein may comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 or SEQ ID NO: 10. In some cases, the sequence identity may be greater than or about 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 9 or SEQ ID NO: 10.
[0090] Komagataella phaffii Flo5-2 is considered to be an ortholog of both Saccharomyces Flo1 and Flo5. See, e.g., the world wide web (at) uniprot.org/uniprot/F2QXPO. The two Saccharomyces flocculation proteins are highly similar in their amino acid sequence, only significantly differing in the length of the linker portion used to extend the protein past the cell wall. The Saccharomyces flocculation proteins are cell wall proteins that participate directly in adhesive cell-cell interactions during yeast flocculation, a reversible, asexual process in which cells adhere to form aggregates (flocs) consisting of thousands of cells. The lectin-like proteins stick out of the cell wall of flocculent cells and selectively bind mannose residues in the cell walls of adjacent cells. Literature on Saccharomyces Flo1p shows that monomeric mannose added to the media can prevent flocculation, suggesting that flocculation by Flo1p results from binding to mannose in the cell wall and free-floating mannose can compete for the binding spot. Thus, the flocculation family of proteins are useful in the present disclosure, for, at least, two reasons. First, they generally extend relative far from the cell wall, and, second, it is believed that they bind and capture some exopolysaccharides. Notably, Flo5-2 has a GPI anchor site towards its C-terminus which can tether the protein to a cell's membrane. Therefore, a fusion protein comprising an anchoring domain of Flo5-2 may anchor the fusion protein to the extracellular surface of an engineered cell via its GPI anchor or by the domain's interaction with exopolysaccharides located on the extracellular surface of an engineered cell. Moreover, without wishing to be bound by theory, inclusion of an anchoring domain of Flo5-2 may promote capture of a secreted glycoprotein for deglycosylation.
[0091] In some embodiments, a fusion protein comprising a Flo5-2 anchoring domain has a sequence that has 95% or more sequence identity with SEQ ID NO: 5 or SEQ ID NO: 6. In some cases, the sequence identity may be greater than or about 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the Flo5-2 anchoring domain of a fusion protein of the present disclosure comprises a GPI attachment site; thus, the anchoring domain may only require a short fragment of SEQ ID NO: 5 or SEQ ID NO: 6, i.e., a fragment that is 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more amino acids in length, as long as it is capable of projecting the catalytic domain of the fusion protein into the extracellular space. In some embodiments, the anchoring domain comprises, at least, Flo5-2's GPI attachment site. In some embodiments, the anchoring domain lacks Flo5-2's GPI attachment site yet retains the ability to capture exopolysaccharides and retain the fusion protein at the extracellular surface.
[0092] In some cases, the cell surface protein is Flo5-2 and the endoglycosidase is endoglycosidase H. The fusion protein may comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 11 or SEQ ID NO: 12. In some cases, the sequence identity may be greater than or about 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 11 or SEQ ID NO: 12.
[0093] Saccharomyces cerevisiae Flo5 has a GPI anchor site towards its C-terminus which can tether the protein to a cell's membrane. Therefore, a fusion protein comprising an anchoring domain of Flo5 may anchor the fusion protein to the extracellular surface of an engineered cell via its GPI anchor or by the domain's interaction with exopolysaccharides located on the extracellular surface of an engineered cell. Moreover, without wishing to be bound by theory, inclusion of an anchoring domain of Flo5 may promote capture of a secreted glycoprotein for deglycosylation.
[0094] In some embodiments, a fusion protein comprising a Saccharomyces cerevisiae Flo5 anchoring domain has a sequence that has 95% or more sequence identity with SEQ ID NO: 20. In some cases, the sequence identity may be greater than or about 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the Flo5 anchoring domain of a fusion protein of the present disclosure comprises a GPI attachment site; thus, the anchoring domain may only require a short fragment of SEQ ID NO: 20, i.e., a fragment that is 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more amino acids in length, as long as it is capable of projecting the catalytic domain of the fusion protein into the extracellular space. In some embodiments, the anchoring domain comprises, at least, Flo5's GPI attachment site. In some embodiments, the anchoring domain lacks Flo5's GPI attachment site yet retains the ability to capture exopolysaccharides and retain the fusion protein at the extracellular surface.
[0095] In some cases, the cell surface protein is Saccharomyces cerevisiae Flo5 and the endoglycosidase is endoglycosidase H. The fusion protein may comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 293. In some cases, the sequence identity may be greater than or about 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 293.
[0096] Flo11 is another GPI-anchored cell surface glycoprotein (flocculin). See, e.g., the world wide web (at) uniprot.org/uniprot/F2QRD4. Flo11 is believed to be required for pseudohyphal and invasive growth, flocculation, and biofilm formation. It is a major determinant of colony morphology and required for formation of fibrous interconnections between cells. Like the other yeast flocculation proteins, its adhesive activity is inhibited by mannose, but not by glucose, maltose, sucrose or galactose. Thus, use of Flo11 in a fusion protein of the present disclosure may be useful extending the fusion protein relatively far from the cell wall, and for binding and capturing some exopolysaccharides. Like, Flo5-2, Flo11 has a GPI anchor site towards its C-terminus which can tether the protein to a cell's membrane. Therefore, a fusion protein comprising an anchoring domain of Flo11 may anchor the fusion protein to the extracellular surface of an engineered cell via its GPI anchor or by the domain's interaction with exopolysaccharides located on the extracellular surface of an engineered cell. Moreover, without wishing to be bound by theory, inclusion of an anchoring domain of Flo11 may promote capture of a secreted glycoprotein for deglycosylation.
[0097] In some embodiments, a fusion protein comprising a Flo11 anchoring domain has a sequence that has 95% or more sequence identity with SEQ ID NO: 7 or SEQ ID NO: 8. In some cases, the sequence identity may be greater than or about 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In various embodiments, the Flo11 anchoring domain of a fusion protein of the present disclosure comprises a GPI attachment site; thus, the anchoring domain may only require a short fragment of SEQ ID NO: 7 or SEQ ID NO: 8, i.e., a fragment that is 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more amino acids in length, as long as it is capable of projecting the catalytic domain of the fusion protein into the extracellular space. In some embodiments, the anchoring domain comprises, at least, Flo11's GPI attachment site. In some embodiments, the anchoring domain lacks Flo11's GPI attachment site yet retains the ability to capture exopolysaccharides and retain the fusion protein at the extracellular surface.
[0098] In some cases, the cell surface protein is Flo11 and the endoglycosidase is endoglycosidase H. The fusion protein may comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 13 or SEQ ID NO: 14. In some cases, the sequence identity may be greater than or about 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 13 or SEQ ID NO: 14.
Engineered Eukaryotic Cells
[0099] The present disclosure relates to engineered eukaryotic cells. These engineered cells are transfected to express a surface displayed catalytic domain of an endoglycosidase. In various embodiments, the engineered cells are transfected to express a surface displayed fusion protein comprising a catalytic domain of an endoglycosidase and an anchoring domain of a cell surface protein.
[0100] In some cases, the engineered eukaryotic cell is a yeast cell, e.g., yeast cell that is a Pichia species
[0101] A fusion protein may be expressed by the cell by nucleic acid sequence, e.g., an expression cassette, that is stably integrated into a cell's chromosome. Alternately, a fusion protein may be expressed by the cell by an extrachromosomal nucleic acid sequence, e.g., plasmid, vector, or YAC which comprises an expression cassette. Any method for transfecting cells with suitable constructs that express the fusion protein may be used.
[0102] An expression cassette is any nucleic acid sequence that contains a subsequence that codes for a transgene and can confer expression of that subsequence when contained in a microorganism and is heterologous to that microorganism. It may comprise one or more of a coding sequence, a promoter, and a terminator. It may encode a secretory signal. It may further encode a signal sequence. In some embodiments, a nucleic acid sequence, e.g., which is expressed by a recombinant cell, may comprise an expression cassette.
[0103] The expression cassettes useful herein can be obtained using chemical synthesis, molecular cloning or recombinant methods, DNA or gene assembly methods, artificial gene synthesis, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence. For preparing polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable cloning or expression vector, and the cloning or expression vector in turn can be introduced into a suitable host cell for replication and amplification. Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected fvmay vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the expression vector. Methods for obtaining cloning and expression vectors are well-known (see, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, New York (2012)), the contents of which is incorporated herein by reference in its entirety.
[0104] In some cases, it is desirable for a engineered cell to express multiple copies of the fusion protein and/or to control expression of the fusion protein. Thus, a nucleic acid sequence or expression cassette may comprise a constitutive promoter, inducible promoter, and hybrid promoter. A promoter refers to a polynucleotide subsequence of nucleic acid sequence or an expression cassette that is located upstream, or 5, to a coding sequence and is involved in initiating transcription of the coding sequence when the nucleic acid sequence or expression cassette is integrated into a chromosome or located extrachromosomally in a host cell.
[0105] Notably, in some cases, it is undesirable for a cell to excessively express the fusion protein. The main purpose of the recombinant cells of the present disclosure is to produce the recombinant glycoproteins, e.g., for inclusion in composition for human or animal use. Should a cell express excessive amounts of the fusion protein, then the transcriptional and translational machinery dedicated to producing the fusion protein cannot be used to produce the recombinant glycoproteins. If so, the cell may become stressed and produce either less recombinant glycoproteins and/or may produce undesirable byproducts. Thus, in some embodiments, a nucleic acid encoding a fusion protein is fused to a weak promoter or to an intermediate strength promoter rather than a strong promoter.
[0106] In embodiments, the nucleic acid sequence or expression cassette comprises an inducible promoter. The inducible promoter may be an AOX1, DAK2, PEX11, FLD1, FGH1, DAS2, CAT1, MDH3, HAC1, BiP, RAD30, RVS161-2, MPP10, THP3, or GBP2 promoter. In some embodiments, the promoter used may have a sequence that has 95% or more sequence identity with any of SEQ ID NO: 26 to SEQ ID NO: 40. In some cases, the sequence identity may be greater than or about 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 26 to SEQ ID NO: 40.
[0107] Useful promoters may be selected from acu-5, adh1+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, -amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbh1), ccg-1, cDNA1, cellular filament polypeptide (cfp), cpc-2, ctr4+, CUP1, dihydroxyacetone synthase (DAS), enolase (ENO, ENO1), formaldehyde dehydrogenase (FLD1), FMD, formate dehydrogenase (FMDH), G1, G6, GAA, GAL1, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10, GCW14, gdhA, gla-1, -glucoamylase (glaA), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUT1), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, -galactosidase (lac4), LEU2, me10, MET3, methanol oxidase (MOX), nmt1, NSP, pcbC, PETS, phosphoglycerate kinase (PGK, PGK1), pho 1, PH05, PH089, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase (pki1), RPS7, sorbitol dehydrogenase (SDH), 3-phosphoserine aminotransferase (SERI), SSA4, SV40, TEF, translation elongation factor 1 alpha-(TEF1), THI11, homoserine kinase (THR1), tpi, TPS1, triose phosphate isomerase (TPI1), XRP2, YPT1, GCW14, GAP, a sequence or subsequence chosen from SEQ ID NO: 26 to SEQ ID NO: 48, and any combination thereof. In some cases, the sequence identity may be greater than or about 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 26 to SEQ ID NO: 48.
[0108] In embodiments, the nucleic acid sequence or expression cassette comprises a terminator sequence. A terminator is a section of nucleic acid sequence that marks the end of a gene during transcription. In some cases, the terminator is an AOX1, TDH3, RPS25A, or RPL2A terminator. In some embodiments, the terminator used may have a sequence that has 95% or more sequence identity with any of SEQ ID NO: 53 to SEQ ID NO: 56. In some cases, the sequence identity may be greater than or about 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 53 to SEQ ID NO: 56.
[0109] Certain combinations of promoter and terminator may provide more preferred expression of the fusion protein and/or more preferred activity of the fusion protein, e.g., in deglycosylating glycoproteins. It is well-within the skill of an artisan to determine which combinations of promoters and terminators achieve desirability and which combinations do not.
[0110] Moreover, in some cases, the same combination of promoter and terminator may have preferred activity in one strain and have less preferred activity in another strain. Without wishing to be bound by theory, the strain difference may be due to a construct's integration into the host cell's genome or it may be due to epigenetic reasons. It is well-within the skill of an artisan to determine which strains for a certain combination of promoter and terminator achieve desirability and which strains do not.
[0111] Additionally, some combinations of promoters and terminators and certain strains perform better when cells are cultured at higher density (e.g., in bioreactors) versus low density cell cultures, as in a high throughput screen. Thus, a combination or strain may appear to be less desirable when assayed in small scale cultures, but may actually be a preferred combination or strain when cultured at higher cell density, which would be the case for commercial scale production of deglycosylated proteins. It is well-within the skill of an artisan to determine the culturing conditions that ensure certain combination of promoter and terminator and specific strains provided desirable amounts of glycoprotein deglycosylation.
[0112] In some cases, the nucleic acid sequence or expression cassette encodes a signal peptide and/or a secretory signal. A signal peptide, also known as a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide, may support secretion of a protein or polynucleotide. Extracellular secretion (for the purposes of surface display) of a recombinant or heterologously expressed fusion protein is facilitated by having a signal peptide included in the fusion protein. A signal peptide may be derived from a precursor (e.g., prepropeptide, preprotein) of a protein. Signal peptides may be derived from a precursor of a protein including, but not limited to, acid phosphatase (e.g., Pichia pastoris PHO1), albumin (e.g., chicken), alkaline extracellular protease (e.g., Yarrowia lipolytica XRP2), -mating factor (-MF, MAT) (e.g., Saccharomyces cerevisiae), amylase (e.g., -amylase, Rhizopus oryzae, Schizosaccharomyces pombe putative amylase SPCC63.02c (Amy 1)), -casein (e.g., bovine), carbohydrate binding module family 21 (CBM21)-starch binding domain, carboxypeptidase Y (e.g., Schizosaccharomyces pombe Cpy 1), cellobiohydrolase I (e.g., Trichoderma reesei CBH1), dipeptidyl protease (e.g., Schizosaccharomyces pombe putative dipeptidyl protease SPBC1711.12 (Dpp1)), glucoamylase (e.g., Aspergillus awamori), heat shock protein (e.g., bacterial Hsp70), hydrophobin (e.g., Trichoderma reesei HBFI, Trichoderma reesei HBFII), inulase, invertase (e.g., Saccharomyces cerevisiae SUC2), killer protein or killer toxin (e.g., 128 kDa pGKL killer protein, -subunit of the K1 killer toxin (e.g., Kluyveromyces lactis), K1 toxin KILM1, K28 pre-pro-toxin, Pichia acaciae), leucine-rich artificial signal peptide CLY-L8, lysozyme (e.g., chicken CLY), phytohemagglutinin (PHA-E) (e.g., Phaseolus vulgaris), maltose binding protein (MBP) (e.g., Escherichia coli), P-factor (e.g., Schizosaccharomyces pombe P3), Pichia pastoris Dse, Pichia pastoris Exg, Pichia pastoris Pir1, Pichia pastoris Scw, and cell wall protein Pir4 (protein with internal repeats). In some embodiments, the signal peptide used may have a sequence that has 80% or more sequence identity with any of SEQ ID NO: 57 to SEQ ID NO: 156. In some cases, the sequence identity may be greater than or about 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 57 to SEQ ID NO: 156. In some cases, the signal peptide used may have a sequence that has 80% or more sequence identity with any of SEQ ID NO: 57 to SEQ ID NO: 61. In some cases, the sequence identity may be greater than or about 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 57 to SEQ ID NO: 61.
[0113] In various embodiments, a fusion protein comprises an -mating factor (-MF, MAT) (e.g., Saccharomyces cerevisiae) secretion signal. In some cases the alpha mating factor signal peptide and secretion signal has a sequence that has 95% or more sequence identity with SEQ ID NO: 290 or SEQ ID NO: 291. In some cases, the sequence identity may be greater than or about 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of with SEQ ID NO: 290 or SEQ ID NO: 291. The -mating factor secretion signal targets a fusion protein through the secretory pathway and is removed before exiting the cell.
[0114] In some cases, a nucleic acid sequence or expression cassette encodes a selectable marker. The selectable maker may be an antibiotic resistance gene (e.g., zeocin, ampicillin, blasticidin, kanamycin, nourseothricin, chloroamphenicol, tetracycline, triclosan, ganciclovir, and any combination thereof), an auxotrophic marker (e.g., f ade1, arg4, his4, ura3, met2, and any combination thereof).
[0115] In various embodiments, a nucleic acid sequence or expression cassette comprises codons that are optimized for the species of the engineered cell, e.g., a yeast cell including a Pichia cell. As known in the art, codon optimization may improve stability and/or increase expression of a recombinant protein, e.g., a fusion protein of the present disclosure. Surprisingly, codon optimization of a nucleic acid sequence or expression cassette my improve the transfection efficiency of the nucleic acid sequence or expression cassette into the genome of a host cell. Codon utilization tables for various species of host cell are publicly available. See, e.g., the world wide web (at) kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4922&aa=15&style=N.
[0116] Host cells useful for expression fusion proteins of the present disclosure include but are not limited to: Arxula spp., Arxula adeninivorans, Kluyveromyces spp., Kluyveromyces lactis, Pichia spp., Pichia angusta, Pichia pastoris, Saccharomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces spp., Schizosaccharomyces pombe, Yarrowia spp., Yarrowia lipolytica, Agaricus spp., Agaricus bisporus, Aspergillus spp., Aspergillus awamori, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Colletotrichum spp., Colletotrichum gloeosporiodes, Endothia spp., Endothia parasitica, Fusarium spp., Fusarium graminearum, Fusarium solani, Mucor spp., Mucor miehei, Mucor pusillus, Myceliophthora spp., Myceliophthora thermophila, Neurospora spp., Neurospora crassa, Penicillium spp., Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium (Talaromyces) emersonii, Penicillium funiculosum, Penicillium purpurogenum, Penicillium roqueforti, Pleurotus spp., Pleurotus ostreatus, Rhizomucor spp., Rhizomucor miehei, Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus oryzae, Trichoderma spp., Trichoderma altroviride, Trichoderma reesei, Trichoderma vireus, Aspergillus oryzae, Bacillus subtilis, Escherichia coli, Myceliophthora thermophila, Neurospora crassa, Pichia pastoris, Komagataella phaffii and Komagataella pastoris.
[0117] Transfection of a host cell with an expression cassette can exploit the natural ability of a host cell to integrate exogenous DNA into its chromosome. This natural ability is well documented for yeast cells, including Pichia cells. In some embodiments an additional vector and or additional elements may be designed to aide (as deemed necessary by one skilled in the art) for the particular method of transfection (e.g. CAS9 and gRNA vectors for a CRISPR/CAS9 based method).
[0118] In some cases, a host eukaryotic cell that expresses a fusion protein comprises a mutation in its AOX1 gene and/or its AOX2 gene. A deletion in either the AOX1 gene or AOX2 gene generates a methanol-utilization slow (mutS) phenotype that reduces the strain's ability to consume methanol as an energy source. A deletion in both the AOX1 gene and the AOX2 gene generates a methanol-utilization minus (mutM) phenotype that substantially limits the strain's ability to consume methanol as an energy source. Using an AOX1 mutant and/or AOX2 mutant cell is especially useful in the context of a fusion protein encoded by an expression cassette that comprises a methanol-inducible promoter, e.g., OAX1, DAS1, and FDH1. In this configuration, the host cell does not use methanol as an energy source, thus, when the cell is provided methanol, the methanol is primarily used to activate the methanol-inducible promoter, thereby especially activating the promoter and causing increased expression of the fusion protein.
[0119] Another aspect of the present disclosure is a population of engineered eukaryotic cells of any of the herein disclosed aspects or embodiments. The present disclosure further relates to a bioreactor comprising this population of engineered eukaryotic cells.
[0120] Yet another aspect of the present disclosure is a method for expressing a fusion protein comprising an anchoring domain of a cell surface protein and a catalytic domain of an endoglycosidase. The method comprises obtaining any herein disclosed engineered eukaryotic cell and culturing the engineered eukaryotic cell under conditions that promote expression of the fusion protein.
[0121] The conditions that promote expression of the fusion protein may be standard growth conditions. However, when the engineered eukaryotic cell comprises a nucleic acid sequence that encodes the fusion protein and comprises an inducible promoter, culturing the engineered eukaryotic cell under conditions that promote expression of the fusion protein comprises contacting the cell with an agent that activates the inducible promoter. When the inducible promoter is an AOX1, DAK2, PEX11 promoter the agent that activates the inducible promoter is methanol.
Glycoprotein and Sources Thereof
[0122] In some cases, the engineered eukaryotic cell that expresses the surface display fusion protein further comprises a genomic modification that overexpresses a secretory glycoprotein. Here, as a cell secretes the glycoprotein into the extracellular space, it comes in contact with a surface displayed fusion protein, which cleaves the oligosaccharide from the glycoprotein, with both the deglycosylated protein and the liberated oligosaccharide progressing into the extracellular space, e.g., the growth medium in which the eukaryotic cell is being cultured.
[0123] In alternate cases, a first engineered eukaryotic cell expresses the surface display fusion protein and a second engineered eukaryotic cell overexpresses a secretory glycoprotein. Here, the second cell secretes the glycoprotein into the extracellular space and it comes in contact with a surface displayed fusion protein on the first cell. The fusion protein cleaves the oligosaccharide from the glycoprotein, with both the deglycosylated protein and the liberated oligosaccharide progressing into the extracellular space, e.g., the growth medium in which the engineered eukaryotic cell is being cultured.
[0124] In other cases, a first engineered eukaryotic cell expresses the surface display fusion protein and further comprises a genomic modification that overexpresses a secretory glycoprotein, however, the fusion protein cleaves a secretory glycoprotein that was overexpressed by a second engineered eukaryotic cell.
[0125] The genomic modification that overexpresses a secretory glycoprotein may comprise a promoter (constitutive promoter, inducible promoter, and hybrid promoter) as disclosed herein; the genomic modification that overexpresses a secretory glycoprotein may comprise a terminator sequence as disclosed herein; the genomic modification that overexpresses a secretory glycoprotein may encode a secretory signal as disclosed herein; and/or the genomic modification that overexpresses a secretory glycoprotein may encode a signal sequence as disclosed herein.
[0126] A host cell may comprise a first promoter driving the expression of the fusion protein and a second promoter driving the expression secretory glycoprotein. The first and second promoter may be selected from the list of promoters provided herein. In some cases, the first promoter and the second promoter may be the same. Alternatively, the first and the second promoter may be different.
[0127] In various embodiments, the secreted glycoprotein is an animal protein. In some embodiments, the animal protein is an egg protein, e.g., selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
[0128] The glycoprotein may have amino acid sequence of any one of SEQ ID NO: 157 to SEQ ID NO: 290. The glycoprotein may be a variant of any one of SEQ ID NO: 157 to SEQ ID NO: 290. The variant may have at least or about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 157 to SEQ ID NO: 290.
[0129] Another aspect of the present disclosure is a population of engineered eukaryotic cells (that express a surface display fusion protein alone or that express a surface display fusion protein and overexpress a secretory glycoprotein) of any of the herein disclosed aspects or embodiment. The present disclosure further relates to a bioreactor comprising this population of engineered eukaryotic cells.
Compositions
[0130] The present disclosure further relates to composition comprising any herein disclosed engineered eukaryotic cell, a secreted protein that has been deglycosylated, and one or more oligosaccharides cleaved from the secreted protein.
[0131] Also, the present disclosure further relates to a composition comprising a secreted protein that has been deglycosylated and one or more oligosaccharides cleaved from the secreted protein.
[0132] Further, the present disclosure relates to a composition comprising a secreted protein that has been deglycosylated.
[0133] Additionally, the present disclosure relates to a composition comprising one or more oligosaccharides cleaved from a secreted protein.
[0134] In various embodiments, the secreted glycoprotein is an animal protein. In some embodiments, the animal protein is an egg protein, e.g., selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
[0135] The glycoprotein may have amino acid sequence of any one of SEQ ID NO: 157 to SEQ ID NO: 290. The glycoprotein may be a variant of any one of SEQ ID NO: 157 to SEQ ID NO: 290. The variant may have at least or about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 157 to SEQ ID NO: 290.
[0136] These compositions may be liquid or dried. The secreted protein that has been deglycosylated and/or one or more oligosaccharides cleaved from the secreted protein may be lyophilized. In some cases, the secreted protein that has been deglycosylated and/or one or more oligosaccharides cleaved from the secreted protein are isolated, e.g., from each other and/or from a growth medium. The secreted protein that has been deglycosylated and/or one or more oligosaccharides cleaved from the secreted protein may be concentrated.
[0137] Deglycosylated proteins and/or one or more oligosaccharides cleaved from the secreted protein, as disclosed herein, may be used in a consumable composition comprising. Illustrative uses and features of such consumable compositions are described in WO 2016/077457, the contents of which is incorporated herein by reference in its entirety.
[0138] A consumable composition may comprise one or more deglycosylated proteins. As used herein, a consumable composition refers to a composition, which comprises an isolated deglycosylated protein and/or a cleaved oligosaccharide and may be consumed by an animal, including but not limited to humans and other mammals. Consumable food compositions include food products, beverage products, dietary supplements, food additives, and nutraceuticals as non-limiting examples. The consumable composition may comprise one or more components in addition to the deglycosylated protein. The one or more components may include ingredients, solvents used in the formation of foodstuff or beverages. For instance, the deglycosylated protein may be in the form of a powder which can be mixed with solvents to produce a beverage or mixed with other ingredients to form a food product.
[0139] The nutritional content of the deglycosylated protein may be higher than the nutritional content of an identical quantity of a control protein. The control protein may be the same protein produced recombinantly but not treated with a fusion protein of the present disclosure. The control protein may be the same protein produced recombinantly in a host cell which does not express a surface displayed fusion protein. The control protein may be the same protein isolated from a naturally occurring source. For instance, the control protein may be an isolated an egg white protein.
[0140] The nutritional content of a composition comprising the deglycosylated protein can be more than the nutritional content of the composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 1% to 80% more than the protein content of a composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 1% to 5% more than the protein content of a composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 1% to 10% more than the protein content of a composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 1% to 20% more than the protein content of a composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 1% to 50% more than the protein content of a composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 1% to 80% more than the protein content of a composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 5% to 10%, 5-15%, 5-20%, 5-30%, 5-50%, 5-80% more than the protein content of a composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 10% to 80%, 10-20%, 10-30%, 10-50%, 10-70%, 10-80% more than the protein content of a composition comprising a control protein. The protein content of the deglycosylated protein composition may be about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% more than the protein content of a composition comprising a control protein.
[0141] Protein content of a deglycosylated protein composition may be measured using conventional methods. For instance, protein content may be measured using nitrogen quantitation by combustion and then using a conversion factor to estimate quantity of protein in a sample followed by calculating the percentage (w/w) of the dry matter.
[0142] The nitrogen to carbon ratio of a deglycosylated protein be higher than the nitrogen to carbon ratio of a control protein. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.1. The nitrogen to carbon ratio of a deglycosylated protein be higher than the nitrogen to carbon ratio of a control protein. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.25. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.3. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.35. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.4. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.5.
[0143] Solubility of a deglycosylated protein may be greater than the solubility of a control protein. Solubility of a composition comprising a deglycosylated protein may be higher than the solubility of a composition comprising the control protein. Thermal stability of the deglycosylated protein may be greater than the thermal stability of a control protein.
[0144] The degree of glycosylation of the recombinant protein may be dependent on the consumable composition being produced. For instance, a consumable composition may comprise a lower degree of glycosylation to increase the protein content of the composition. Alternatively, the degree of glycosylation may be higher to increase the solubility of the protein in the composition.
Methods for Deglycosylating a Secreted Protein
[0145] Another aspect of the present disclosure is a method for deglycosylating a secreted glycoprotein. The method comprises contacting a secreted protein with a fusion protein anchored to any herein-disclosed engineered eukaryotic cell. By contacting a secreted protein with the fusion protein, the catalytic domain cleaves and releases an oligonucleotide from the secreted glycoprotein.
[0146] In some cases, the secreted glycoprotein is expressed by the engineered eukaryotic cell.
[0147] Notably, a fusion protein anchored to an engineered eukaryotic cell (of the present disclosure) is more effective at deglycosylating the secreted glycoprotein than an intracellular endoglycosidase, e.g., an intracellular endoglycosidase located within a Golgi vesicle. In particular, a fusion protein anchored to the surface of an engineered eukaryotic cell (of the present disclosure) is more effective at deglycosylating the secreted glycoprotein than an intracellular endoglycosidase that is linked to a membrane associating domain, e.g., a membrane associating domain that comprises an amino acid sequence of OCH1. Preferably, the amino acid sequence of OCH1 that is included in a fusion protein of the present disclosure lacks the wild-type OCH1 Golgi retention domain. This retention domain comprises at least a portion of the first 48 residues of Pichia OCH1 protein. If the Golgi retention domain of OCH1 is included in a fusion protein of the present disclosure, then it is unlikely that the fusion protein would be displayed on the exterior of the cell, as needed to be a surface displayed fusion protein of the present disclosure. In embodiments, a fusion protein having an OCH1 anchoring domain lacks the OCH1 Golgi retention domain. In some embodiments, a fusion protein having an OCH1 anchoring domain lacks at least a portion of the first 48 residues of Pichia OCH1 protein. In various embodiments, a fusion protein having an OCH1 anchoring domain lacks the first 48 residues of Pichia OCH1 protein.
[0148] A deglycosylated protein of the present disclosure can have a level of N-linked glycosylation that is reduced by at least about 10 percent (e.g., 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, or 100 percent) as compared to the level of N-linked glycosylation of the same glycoprotein that is not contacted with a fusion protein of the present disclosure, including a glycoprotein contacted with an intracellular endoglycosidase.
[0149] In some cases, the secreted glycoprotein is expressed by a cell other than the engineered eukaryotic cell.
[0150] In some embodiments, the method further comprises a step of isolating the deglycosylated secreted protein, e.g., from a cleaved oligosaccharide and/or from its growth medium. In some embodiments, the method further comprises a step of drying the deglycosylated secreted protein and/or the cleaved oligosaccharides.
[0151] In various embodiments, the secreted glycoprotein is an animal protein. In some embodiments, the animal protein is an egg protein, e.g., selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
[0152] The glycoprotein may have amino acid sequence of any one of SEQ ID NO: 157 to SEQ ID NO: 290. The glycoprotein may be a variant of any one of SEQ ID NO: 157 to SEQ ID NO: 290. The variant may have at least or about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 157 to SEQ ID NO: 290.
[0153] Another aspect of the present disclosure is a method for deglycosylating a plurality of secreted glycoproteins. The method comprises contacting the plurality of secreted glycoproteins with a population of any herein disclosed engineered eukaryotic cells. By contacting the plurality of secreted glycoprotein with the fusion protein, the catalytic domains cleave and release oligonucleotides from the plurality secreted glycoprotein and provide a plurality of deglycosylated secreted proteins.
[0154] In some cases, substantially every secreted glycoprotein in the plurality of secreted glycoproteins is deglycosylated upon contact with the population of engineered eukaryotic cells.
[0155] Notably, the amount of deglycosylation of the secreted glycoproteins is not increased by further contacting the secreted protein with an isolated endoglycosidase.
[0156] Further, the amount of deglycosylation of the secreted glycoproteins is more than the amount obtained from a population of cells that express an intracellular endoglycosidase in addition to expressing the secreted glycoprotein.
[0157] In some embodiments, the method further comprises a step of isolating the plurality of deglycosylated secreted proteins and may further comprise a step of drying the plurality of deglycosylated secreted proteins.
[0158] In various embodiments, the secreted glycoprotein is an animal protein. In some embodiments, the animal protein is an egg protein, e.g., selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, -ovomucin, -ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
[0159] The glycoprotein may have amino acid sequence of any one of SEQ ID NO: 157 to SEQ ID NO: 290. The glycoprotein may be a variant of any one of SEQ ID NO: 157 to SEQ ID NO: 290. The variant may have at least or about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 157 to SEQ ID NO: 290.
Additional Catalytic Domains
[0160] Much of the above disclosure relates to surface displayed fusion proteins comprising a catalytic domain of an endoglycosidase, e.g., endoglycosidase H.
[0161] The engineered cells, nucleic acid sequences, compositions, and method disclosed herein may be adapted to relate to fusion proteins with catalytic domains of enzymes other than endoglycosidases. As used herein, the term catalytic domain comprises a portion of an enzyme that provides catalytic activity.
[0162] Accordingly, another aspect of the present disclosure is an engineered eukaryotic cell which expresses a surface displayed catalytic domain of an enzyme, wherein the catalytic domain is directly or indirectly tethered to the exterior surface of the cell.
[0163] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.
Definitions
[0164] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0165] As used in the specification and claims, the singular forms a, an and the include plural references unless the context clearly dictates otherwise.
[0166] As used herein, the phrases at least one, one or more, and and/or are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions at least one of A, B and C, at least one of A, B, or C, one or more of A, B, and C, one or more of A, B, or C and A, B, and/or C mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.
[0167] As used herein, or may refer to and, or, or and/or and may be used both exclusively and inclusively. For example, the term A or B may refer to A or B, A but not B, B but not A, and A and B. In some cases, context may dictate a particular meaning.
[0168] As used herein, the term about a number refers to that number plus or minus 10% of that number and/or within one standard deviation (plus or minus) from that number. The term about a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value and that range minus one standard deviation its lowest value and plus one standard deviation of its greatest value.
[0169] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[0170] The terms increased, increasing, or increase are used herein to generally mean an increase by a statically significant amount relative to a reference level. In some aspects, the terms increased, or increase, mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level. Other examples of increase include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.
[0171] The terms decreased, decreasing, or decrease are used herein generally to mean a decrease in a value relative to a reference level. In some aspects, decreased or decrease means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level.
[0172] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
INCORPORATION BY REFERENCE
[0173] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
EXAMPLES
[0174] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Construction of a Surface Displayed EndoH-Sed1p Fusion Protein
[0175] A nucleic acid sequence that expressed a surface displayed fusion protein of SEQ ID NO: 10 was constructed and transfected in to Pichia cells. Transfected cells that faithfully expressed and surface displayed the fusion protein were isolated and expanded in culture.
[0176] The fusion protein included the Saccharomyces cerevisiae alpha mating factor signal peptide and secretion signal (89 residues, ending in EAEA; SEQ ID NO: 21), EndoH codon variant 2 (271 residues; SEQ ID NO: 1), a flex linker of 26 residues [GSS].sub.8 (eight repeats of SEQ ID NO: 23), a semi-rigid alpha helix linker of 20 residues [EAAAR].sub.4, (SEQ ID NO: 24) another flex linker of 15 residues [GGGGS].sub.3 (three repeats of SEQ ID NO: 22) and the full Sed1 gene minus the N term 18 amino acid signal peptide (320 residues; SEQ ID NO: 3). Glycine-Serine linkers are commonly used in fusion proteins to space them out with no intervening secondary structure. The ratio of serine to glycine determines the relative stiffness of the linker, but even high serine content GS linkers are still fairly flexible. The entire linker of this fusion protein has an amino acid sequence of SEQ ID NO: 25. The full fusion protein had the amino acid sequence of SEQ ID NO: 10.
[0177] During translation and processing by the engineered cell, the signal peptide (MRFPSIFTAVLFAASSALA; SEQ ID NO: 59) was first cleaved off in the cell's endoplasmic reticulum. When the protein arrives in the late Golgi, the secretion signal (APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV SLDKR; SEQ ID NO: 291) was cleaved off. Around the same time, the propeptide on the C-term (APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV SLDKREAEA; SEQ ID NO: 292) was also cleaved off for the attachment of the GPI anchor. The final resultant fusion protein is as below, and include the full EndoH protein, the mature Sed1 protein, plus various linker elements and having the amino acid sequence of SEQ ID NO: 9.
[0178] The surface displayed fusion protein was incorporated into the cell membrane via a GPI anchor attached to the protein's C-terminus.
[0179] This surface displayed fusion protein was shown to be effective at deglycosylating an illustrative secreted glycoprotein (here, ovomucoid (OVD)). A high-throughput screen of cells engineered cells to express OVD and the surface displayed EndoH-Sed1p fusion protein was performed. In this screen, all engineered cell lines were capable of fully deglycosylating OVD while maintaining OVD titer. As shown in
[0180] To expand production of EndoH-Sed1p fusion protein/glycoprotein secreting P. pastoris cells, a seed strain was removed from cryo-storage and thawed to room temperature. Contents of the thawed seed vials were used to inoculate liquid seed culture media in baffled flasks which were grown at 30 C. in shaking incubators. These seed flasks were then transferred and grown in a series of larger and larger seed fermenters containing a basal salt media, trace metals, and glucose. The temperature in the seed reactors were controlled at 30 C., pH at 5, and dissolved oxygen (DO) at 30%. pH was maintained by feeding ammonia hydroxide which also acted as a nitrogen source. Once sufficient cell mass was reached, the grown EndoH-Sed1p fusion protein/glycoprotein secreting P. pastoris was inoculated in a production-scale reactor containing basal salt media, trace metals, and glucose. Like in the seed tanks, the culture was also controlled at 30 C., pH 5 and 30% DO throughout the process. pH was again maintained by feeding ammonia hydroxide. During the initial batch glucose phase, the culture was left to consume all glucose and subsequently-produced ethanol. Once the target cell density was achieved and glucose and ethanol concentrations were confirmed to be zero, the glucose fed-batch growth phase was initiated. In this phase, glucose was fed until the culture reaches a target cell density. Glucose was fed at a limiting rate to prevent ethanol from building up in the presence of non-zero glucose concentrations. In the final induction phase, the culture was co-fed glucose and methanol which induced the cells to produce EndoH-Sed1p fusion protein via a methanol-inducible promoter included in the construct expressing the fusion protein. Glucose was fed at an amount to produce a desired growth rate, while methanol was fed to maintain the methanol concentration at 1% to ensure that fusion protein expression was consistently induced. Regular samples were taken throughout the fermentation process for analyses of specific process parameters (e.g., cell density, glucose/methanol concentrations, product titer, and quality).
[0181] The bioreactor-expanded cells were assayed for their ability to deglycosylate an illustrative glycoprotein. As shown in
[0182] Another version of the surface displayed fusion protein described above was generated with a shorter linker (i.e., [GGGGS].sub.3) and with a different EndoH codon set. Surprisingly, this other version of the fusion protein has much lower deglycosylation ability.
Example 2: Construction of a Surface Displayed EndoHFlo5-2 Fusion Protein
[0183] A nucleic acid sequence that expressed a surface displayed fusion protein of SEQ ID NO: 12 was constructed and transfected into Pichia cells. Transfected cells that faithfully expressed and surface displayed the fusion protein were isolated and expanded in culture.
[0184] Overexpression results in Pichia cells showed that Flo5-2 strongly flocculates pichia cells. These results were conducted in cells that did not co-express a secreted glycoprotein and had low exopolysaccharides.
[0185] The EndoHFlo5-2 fusion protein was designed to take advantage of Flo5-2's ability to flocculate pichia cells and endoH's ability to cleave off oligosaccharides from glycoproteins. Without wishing to be bound by theory, the endoH on the N terminal end of the fusion protein should shield the Flo5-2 protein and reduce the risk of flocculation while giving enough space (via linkers) for exopolysaccharides present in the extracellular space be captured. Flo proteins naturally extend well into the extracellular space because they need to be able to adhere to cell wall of another cell. Therefore, combining EndoH with Flo5-2 would provide an extended reach for the enzyme to bind to and cleave secreted glycoproteins present in the extracellular space.
[0186] The surface displayed EndoHFlo5-2 fusion protein had the following structure: a Flo5-2 signal peptide (MKFPVPLLFLLQLFFIIATQG; SEQ ID NO: 61), EndoH (SEQ ID NO: 1), a complex linker (SEQ ID NO: 25), and a Flo5-2 mature protein (SEQ ID NO: 5) plus the propeptide that gets cut off for GPI anchoring. The propeptide that's cleaved off within the cell is on Flo5-2's the C-terminal and is likely around the same size as Sed1's propeptide of about 20 amino acids.
[0187] The surface displayed EndoHFlo5-2 fusion protein uses Flo5-2's native signal peptide. Flo5-2 secretes itself without needing another secretion signal. So, this fusion protein did not include an alpha factor secretion signal, as used in the EndoH-Sed1 fusion protein. However, adding an alpha factor secretion signal is considered and may improve secretion of the fusion protein.
[0188] In a high throughput screen, surface displayed EndoH Flo5-2 fusion protein was capable of fully deglycosylating an illustrative co-expressed glycoprotein (here, OVD) and at a fairly high rate.
Example 3: Construction of a Surface Displayed EndoHSaccharomyces cerevisiae Flo5 Fusion Protein
[0189] A nucleic acid sequence that expressed a surface displayed fusion protein of SEQ ID NO: 293 was constructed and transfected into Pichia cells. Transfected cells that faithfully expressed and surface displayed the fusion protein were isolated and expanded in culture.
[0190] A high throughput screen showed that the surface displayed EndoHSaccharomyces cerevisiae Flo5 fusion protein fully deglycosylated an illustrative co-expressed glycoprotein (here, OVD).
Example 4: Construction of a Surface Displayed EndoH-Flo11 Fusion Protein
[0191] A nucleic acid sequence that expressed a surface displayed fusion protein of SEQ ID NO: 14 are constructed and are transfected into Pichia cells. Transfected cells that faithfully express and surface display the fusion protein will be isolated and expanded in culture. And the fusion protein's ability to fully deglycosylated an illustrative co-expressed glycoprotein will be assayed.
[0192] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
TABLE-US-00001 TABLE1 Sequences matureEndoHseq SEQIDNO:1 APAPVKQGPTSVAYVEVNNNSMLNVGKYTLADGGGNAFDV onlywithout AVIFAANINYDTGTKTAYLHFNENVQRVLDNAVTQIRPLQ itsnative QQGIKVLLSVLGNHQGAGFANFPSQQAASAFAKQLSDAVA signalpeptide KYGLDGVDFDDEYAEYGNNGTAQPNDSSFVHLVTALRANM PDKIISLYNIGPAASRLSYGGVDVSDKFDYAWNPYYGTWQ VPGIALPKAQLSPAAVEIGRTSRSTVADLARRTVDEGYGV YLTYNLDGGDRTADVSAFTRELYGSEAVRTP endoH SEQIDNO:2 MFTPVRRRVRTAALALSAAAALVLGSTAASGASATPSPAP (withsignalpeptide APAPAPVKQGPTSVAYVEVNNNSMLNVGKYTLADGGGNAF underlined) DVAVIFAANINYDTGTKTAYLHFNENVQRVLDNAVTQIRP LQQQGIKVLLSVLGNHQGAGFANFPSQQAASAFAKQLSDA VAKYGLDGVDFDDEYAEYGNNGTAQPNDSSFVHLVTALRA NMPDKIISLYNIGPAASRLSYGGVDVSDKFDYAWNPYYGT WQVPGIALPKAQLSPAAVEIGRTSRSTVADLARRTVDEGY GVYLTYNLDGGDRTADVSAFTRELYGSEAVRTP Sed1from SEQIDNO:3 QFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGT Saccharomyces STAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEA cerevisiae PTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGT TSAFPPTTSLPPSNTTTTPPYNPSTDYTTDYTVVTEYTTY CPEPTTFTTNGKTYTVTEPTTLTITDCPCTIEKPTTTSTT EYTVVTEYTTYCPEPTTFTTNGKTYTVTEPTTLTITDCPC TIEKSEAPESSVPVTESKGTTTKETGVTTKQTTANPSLTV STVVPVSSSASSHSVVINSNGANVVVPGALGLAGVAMLFL Sed1from SEQIDNO:4 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSISTS Saccharomyces SGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGT cerevisiae STEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEA (underlined PTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTPPYN issignalpeptide,not PSTDYTTDYTVVTEYTTYCPEPTTFTTNGKTYTVTEPTTL utilizedindesign) TITDCPCTIEKPTTTSTTEYTVVTEYTTYCPEPTTFTTNG KTYTVTEPTTLTITDCPCTIEKSEAPESSVPVTESKGTTT KETGVTTKQTTANPSLTVSTVVPVSSSASSHSVVINSNGA NVVVPGALGLAGVAMLFL Flo5-2from SEQIDNO:5 DESGNGDESDTAYGCDITSNAFDGFDATIYEYNANDLKLI Komagataellaphaffii RDPVFMSTGYLGRNVLNKISGVTVPGFNIWNPRSRTATVY GVQNVNYYNMVLELKGYFKAAVSGDYKLTLSNIDDSSMLF FGKNTAFQCCDTGSIPVDQAPTDYSLFTIKPSNQVNSEVI SSTQYLEAGKYYPVRIVFVNALERALFNFKLTIPSGTVLD DFQDYIYQFGALDENSCYETTVSKITEWTTYTTPWTGTFE TTRTITPTGTEGTVVIETPESYVTTTQPWTGTYETTYTVP PTGTEPGTVIIETPEIIDCEAVCCGPFLTAFSFRKREECQ CENICCPGDTNCETYVTTTQPWTGTYETTYTVPPTGTEPG TVIIETPESYVTTTQPWTGTYETTYTVPPTGTEPGTVIIE TPESYVTTTQPWTGTYETTYTVPPSGTEPGTVVIETPEIV DCEAYCCASVAIKKRELCQCENFCCSWDQSCQTYVTTTQP WTGTYETTYTVPPTGTEPGTVIIETPESYVTTTQPWTGTY ETTYTVPPTGTEPGTVIIETPESYVTTTQPWTGTYETTYT VPPTGTEPGTVIIETPEIIDCEAVCCGPFLTAFSFRKREE CQCENICCPGDTNCETYVTTTQPWTGTYETTYTVPPTGTE PGTVIIETPESYVTTTQPWTGTYETTYTVPPTGTEPGTVI IETPESYVTTTQPWTGTYETTYTVPPTGTEPGTVIIETPE IINCEAVCCGPFLTAFSFRKREECQCENICCPGDTNCETY VTTTQPWTGTYETTYTVPPTGTEPGTVIIETPESYVTTTQ PWTGTYETTYTVPSTGTEPGTVIIETPESYVTTTQPWTGT YETTFTVPPTGTEPGTVVIETPESYVTTTQPWTGTYETTY SVPPSGTEPGTVVIETPESYVTTTQPWTGTYETTYSVPPS GTEPGTVVIETPEASTARTKFTTVTSSWTGVFTTTKTLPA SGTEPATIVIQTPTGYFNTSSLVSTRTKTNVDTVTRVIPC PICTAPKTITVVPEEPNESVSVIISQPQSSSTDTTLSKPD SVRVISQPETASQMDTSLSKTDSAVISTETAGNNIIPLAG SHSYNTIVTTVTDSPQVAQSTTATSSSNVHLTISTQTTTP SLVYSSSLSTVHQVSPSNGGFRSSITVHPLLSVIGAIFGA LFM Flo5-2from SEQIDNO:6 MKFPVPLLFLLQLFFIIATQGDESGNGDESDTAYGCDITS Komagataellaphaffii NAFDGFDATIYEYNANDLKLIRDPVFMSTGYLGRNVLNKI (underlinedissignal SGVTVPGFNIWNPRSRTATVYGVQNVNYYNMVLELKGYFK peptide,usedinsome AAVSGDYKLTLSNIDDSSMLFFGKNTAFQCCDTGSIPVDQ versionsandnot APTDYSLFTIKPSNQVNSEVISSTQYLEAGKYYPVRIVFV others) NALERALFNFKLTIPSGTVLDDFQDYIYQFGALDENSCYE TTVSKITEWTTYTTPWTGTFETTRTITPTGTEGTVVIETP ESYVTTTQPWTGTYETTYTVPPTGTEPGTVIIETPEIIDC EAVCCGPFLTAFSFRKREECQCENICCPGDTNCETYVTTT QPWTGTYETTYTVPPTGTEPGTVIIETPESYVTTTQPWTG TYETTYTVPPTGTEPGTVIIETPESYVTTTQPWTGTYETT YTVPPSGTEPGTVVIETPEIVDCEAYCCASVAIKKRELCQ CENFCCSWDQSCQTYVTTTQPWTGTYETTYTVPPTGTEPG TVIIETPESYVTTTQPWTGTYETTYTVPPTGTEPGTVIIE TPESYVTTTQPWTGTYETTYTVPPTGTEPGTVIIETPEII DCEAVCCGPFLTAFSFRKREECQCENICCPGDTNCETYVT TTQPWTGTYETTYTVPPTGTEPGTVIIETPESYVTTTQPW TGTYETTYTVPPTGTEPGTVIIETPESYVTTTQPWTGTYE TTYTVPPTGTEPGTVIIETPEIINCEAVCCGPFLTAFSFR KREECQCENICCPGDTNCETYVTTTQPWTGTYETTYTVPP TGTEPGTVIIETPESYVTTTQPWTGTYETTYTVPSTGTEP GTVIIETPESYVTTTQPWTGTYETTFTVPPTGTEPGTVVI ETPESYVTTTQPWTGTYETTYSVPPSGTEPGTVVIETPES YVTTTQPWTGTYETTYSVPPSGTEPGTVVIETPEASTART KFTTVTSSWTGVFTTTKTLPASGTEPATIVIQTPTGYFNT SSLVSTRTKTNVDTVTRVIPCPICTAPKTITVVPEEPNES VSVIISQPQSSSTDTTLSKPDSVRVISQPETASQMDTSLS KTDSAVISTETAGNNIIPLAGSHSYNTIVTTVTDSPQVAQ STTATSSSNVHLTISTQTTTPSLVYSSSLSTVHQVSPSNG GFRSSITVHPLLSVIGAIFGALFM Flo11from SEQIDNO:7 SSGKTCPTSEVSPACYANQWETTFPPSDIKITGATWVQDN Komagataellaphaffii IYDVTLSYEAESLELENLTELKIIGLNSPTGGTKLVWSLN (nosignalsequence) SKVYDIDNPAKWTTTLRVYTKSSADDCYVEMYPFQIQVDW CEAGASTDGCSAWKWPKSYDYDIGCDNMQDGVSRKHHPVY KWPKKCSSNCGVEPTTSDEPEEPTTSEEPEEPTTSEEPEE PTSSDEEPTTSEEPEEPTTSDEPEEPTTSEEPEEPTTSEE PEEPTTSEEPTTSEEPEEPTSSDEEPTTSDEPEEPTTSDE PEEPTTSEEPTTSEEPEEPTTSSEEPTPSEEPEGPTCPTS EVSPACYADQWETTFPPSDIKITGATWVEDNIYDVTLSYE AESLELENLTELKIIGLNSPTGGTKVVWSLNSGIYDIDNP AKWTTTLRVYTKSSADDCYVEMYPFQIQVDWCEAGASTDG CSAWKWPKSYDYDIGCDNMQDGVSRKHHPVYKWPKKCSSD CGVEPTTSDEPEEPTTSEEPVEPTSSDEEPTTSEEPTTSE EPEEPTTSDEPEEPTTSEEPEEPTTSEEPEEPTTSEEPTT SEEPEEPTSSDEEPTTSDEPEEPTTSEEPEEPTTSEEPEE PTTSEEPEEPTTSDEPEEPTTSEEPEEPTTSEEPEEPTSS DEEPTTSEEPEEPTTSEEPEEPTTSEEPEEPTTSEEPEEP TSSDEEPTTSEEPEEPTTSDEPEEPTTSEEPEEPTTSEEP EEPTSSDEEPTTSEEPEEPTTSDEPEEPTTSEEPEEPTTS EEPEEPTTSEEPEEPTTSEEPEEPTSSDEEPTTSEEPEEP TTSDEPEEPTTSEEPEEPTTSEEPEEPTTSEEPEEPTTSD EEPGTTEEPLVPTTKTETDVSTTLLTVTDCGTKTCTKSLV ITGVTKETVTTHGKTTVITTYCPLPTETVTPTPVTVTSTI YADESVTKTTVYTTGAVEKTVTVGGSSTVVVVHTPLTTAV VQSQSTDEIKTVVTARPSTTTIVRDVCYNSVCSVATIVTG VTEKTITFSTGSITVVPTYVPLVESEEHQRTASTSETRAT SVVVPTVVGQSSSASATSSIFPSVTIHEGVANTVKNSMIS GAVALLFNALFL Flo11from SEQIDNO:8 MVSLRSIFTSSILAAGLTRAHGSSGKTCPTSEVSPACYAN Komagataellaphaffii QWETTFPPSDIKITGATWVQDNIYDVTLSYEAESLELENL (withsignalsequence) TELKIIGLNSPTGGTKLVWSLNSKVYDIDNPAKWTTTLRV YTKSSADDCYVEMYPFQIQVDWCEAGASTDGCSAWKWPKS YDYDIGCDNMQDGVSRKHHPVYKWPKKCSSNCGVEPTTSD EPEEPTTSEEPEEPTTSEEPEEPTSSDEEPTTSEEPEEPT TSDEPEEPTTSEEPEEPTTSEEPEEPTTSEEPTTSEEPEE PTSSDEEPTTSDEPEEPTTSDEPEEPTTSEEPTTSEEPEE PTTSSEEPTPSEEPEGPTCPTSEVSPACYADQWETTFPPS DIKITGATWVEDNIYDVTLSYEAESLELENLTELKIIGLN SPTGGTKVVWSLNSGIYDIDNPAKWTTTLRVYTKSSADDC YVEMYPFQIQVDWCEAGASTDGCSAWKWPKSYDYDIGCDN MQDGVSRKHHPVYKWPKKCSSDCGVEPTTSDEPEEPTTSE EPVEPTSSDEEPTTSEEPTTSEEPEEPTTSDEPEEPTTSE EPEEPTTSEEPEEPTTSEEPTTSEEPEEPTSSDEEPTTSD EPEEPTTSEEPEEPTTSEEPEEPTTSEEPEEPTTSDEPEE PTTSEEPEEPTTSEEPEEPTSSDEEPTTSEEPEEPTTSEE PEEPTTSEEPEEPTTSEEPEEPTSSDEEPTTSEEPEEPTT SDEPEEPTTSEEPEEPTTSEEPEEPTSSDEEPTTSEEPEE PTTSDEPEEPTTSEEPEEPTTSEEPEEPTTSEEPEEPTTS EEPEEPTSSDEEPTTSEEPEEPTTSDEPEEPTTSEEPEEP TTSEEPEEPTTSEEPEEPTTSDEEPGTTEEPLVPTTKTET DVSTTLLTVTDCGTKTCTKSLVITGVTKETVTTHGKTTVI TTYCPLPTETVTPTPVTVTSTIYADESVTKTTVYTTGAVE KTVTVGGSSTVVVVHTPLTTAVVQSQSTDEIKTVVTARPS TTTIVRDVCYNSVCSVATIVTGVTEKTITFSTGSITVVPT YVPLVESEEHQRTASTSETRATSVVVPTVVGQSSSASATS SIFPSVTIHEGVANTVKNSMISGAVALLFNALFL EndoH-Sed1fusion SEQIDNO:9 EAEAAPAPVKQGPTSVAYVEVNNNSMLNVGKYTLADGGGN (partialORF,without AFDVAVIFAANINYDTGTKTAYLHFNENVQRVLDNAVTQI peptidesthatare RPLQQQGIKVLLSVLGNHQGAGFANFPSQQAASAFAKQLS cleavedoffpost- DAVAKYGLDGVDFDDEYAEYGNNGTAQPNDSSFVHLVTAL translationally) RANMPDKIISLYNIGPAASRLSYGGVDVSDKFDYAWNPYY GTWQVPGIALPKAQLSPAAVEIGRTSRSTVADLARRTVDE GYGVYLTYNLDGGDRTADVSAFTRELYGSEAVRTPGSSGS SGSSGSSGSSGSSGSSGSSEAAAREAAAREAAAREAAARG GGGSGGGGSGGGGSQFSNSTSASSTDVTSSSSISTSSGSV TITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEA PTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDT TTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTPPYNPSTD YTTDYTVVTEYTTYCPEPTTFTTNGKTYTVTEPTTLTITD CPCTIEKPTTTSTTEYTVVTEYTTYCPEPTTFTTNGKTYT VTEPTTLTITDCPCTIEKSEAPESSVPVTESKGTTTKETG VTTKQTTANPSLTVSTVVPVSSSASSHSVVINSN EndoH-Sedlfusion SEQIDNO:10 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG (fullORF,including YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV peptidesthatare SLDKREAEAAPAPVKQGPTSVAYVEVNNNSMLNVGKYTLA cleavedoffpost- DGGGNAFDVAVIFAANINYDTGTKTAYLHFNENVQRVLDN translationally) AVTQIRPLQQQGIKVLLSVLGNHQGAGFANFPSQQAASAF AKQLSDAVAKYGLDGVDFDDEYAEYGNNGTAQPNDSSFVH LVTALRANMPDKIISLYNIGPAASRLSYGGVDVSDKFDYA WNPYYGTWQVPGIALPKAQLSPAAVEIGRTSRSTVADLAR RTVDEGYGVYLTYNLDGGDRTADVSAFTRELYGSEAVRTP GSSGSSGSSGSSGSSGSSGSSGSSEAAAREAAAREAAARE AAARGGGGSGGGGSGGGGSQFSNSTSASSTDVTSSSSIST SSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNG TSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTE APTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTPPY NPSTDYTTDYTVVTEYTTYCPEPTTFTTNGKTYTVTEPTT LTITDCPCTIEKPTTTSTTEYTVVTEYTTYCPEPTTFTTN GKTYTVTEPTTLTITDCPCTIEKSEAPESSVPVTESKGTT TKETGVTTKQTTANPSLTVSTVVPVSSSASSHSVVINSNG ANVVVPGALGLAGVAMLFL EndoH-Flo5-2fusion SEQIDNO:11 APAPVKQGPTSVAYVEVNNNSMLNVGKYTLADGGGNAFDV (partialORF,without AVIFAANINYDTGTKTAYLHFNENVQRVLDNAVTQIRPLQ signalpeptidethatis QQGIKVLLSVLGNHQGAGFANFPSQQAASAFAKQLSDAVA cleavedoffpost- KYGLDGVDFDDEYAEYGNNGTAQPNDSSFVHLVTALRANM translationally) PDKIISLYNIGPAASRLSYGGVDVSDKFDYAWNPYYGTWQ VPGIALPKAQLSPAAVEIGRTSRSTVADLARRTVDEGYGV YLTYNLDGGDRTADVSAFTRELYGSEAVRTPGSSGSSGSS GSSGSSGSSGSSGSSEAAAREAAAREAAAREAAARGGGGS GGGGSGGGGSDESGNGDESDTAYGCDITSNAFDGFDATIY EYNANDLKLIRDPVFMSTGYLGRNVLNKISGVTVPGFNIW NPRSRTATVYGVQNVNYYNMVLELKGYFKAAVSGDYKLTL SNIDDSSMLFFGKNTAFQCCDTGSIPVDQAPTDYSLFTIK PSNQVNSEVISSTQYLEAGKYYPVRIVFVNALERALFNFK LTIPSGTVLDDFQDYIYQFGALDENSCYETTVSKITEWTT YTTPWTGTFETTRTITPTGTEGTVVIETPESYVTTTQPWT GTYETTYTVPPTGTEPGTVIIETPEIIDCEAVCCGPFLTA FSFRKREECQCENICCPGDTNCETYVTTTQPWTGTYETTY TVPPTGTEPGTVIIETPESYVTTTQPWTGTYETTYTVPPT GTEPGTVIIETPESYVTTTQPWTGTYETTYTVPPSGTEPG TVVIETPEIVDCEAYCCASVAIKKRELCQCENFCCSWDQS CQTYVTTTQPWTGTYETTYTVPPTGTEPGTVIIETPESYV TTTQPWTGTYETTYTVPPTGTEPGTVIIETPESYVTTTQP WTGTYETTYTVPPTGTEPGTVIIETPEIIDCEAVCCGPFL TAFSFRKREECQCENICCPGDTNCETYVTTTQPWTGTYET TYTVPPTGTEPGTVIIETPESYVTTTQPWTGTYETTYTVP PTGTEPGTVIIETPESYVTTTQPWTGTYETTYTVPPTGTE PGTVIIETPEIINCEAVCCGPFLTAFSFRKREECQCENIC CPGDTNCETYVTTTQPWTGTYETTYTVPPTGTEPGTVIIE TPESYVTTTQPWTGTYETTYTVPSTGTEPGTVIIETPESY VTTTQPWTGTYETTFTVPPTGTEPGTVVIETPESYVTTTQ PWTGTYETTYSVPPSGTEPGTVVIETPESYVTTTQPWTGT YETTYSVPPSGTEPGTVVIETPEASTARTKFTTVTSSWTG VFTTTKTLPASGTEPATIVIQTPTGYFNTSSLVSTRTKTN VDTVTRVIPCPICTAPKTITVVPEEPNESVSVIISQPQSS STDTTLSKPDSVRVISQPETASQMDTSLSKTDSAVISTET AGNNIIPLAGSHSYNTIVTTVTDSPQVAQSTTATSSSNVH LTISTQTTTPSLVYSSSLSTVHQVSPSNGGFRSSITVHPL LSVIGAIFGALFM EndoH-Flo5-2fusion SEQIDNO:12 MKFPVPLLFLLQLFFIIATQGAPAPVKQGPTSVAYVEVNN (fullORF,including NSMLNVGKYTLADGGGNAFDVAVIFAANINYDTGTKTAYL signalpeptidethatis HFNENVQRVLDNAVTQIRPLQQQGIKVLLSVLGNHQGAGF cleavedoffpost- ANFPSQQAASAFAKQLSDAVAKYGLDGVDFDDEYAEYGNN translationally) GTAQPNDSSFVHLVTALRANMPDKIISLYNIGPAASRLSY GGVDVSDKFDYAWNPYYGTWQVPGIALPKAQLSPAAVEIG RTSRSTVADLARRTVDEGYGVYLTYNLDGGDRTADVSAFT RELYGSEAVRTPGSSGSSGSSGSSGSSGSSGSSGSSEAAA REAAAREAAAREAAARGGGGSGGGGSGGGGSDESGNGDES DTAYGCDITSNAFDGFDATIYEYNANDLKLIRDPVFMSTG YLGRNVLNKISGVTVPGFNIWNPRSRTATVYGVQNVNYYN MVLELKGYFKAAVSGDYKLTLSNIDDSSMLFFGKNTAFQC CDTGSIPVDQAPTDYSLFTIKPSNQVNSEVISSTQYLEAG KYYPVRIVFVNALERALFNFKLTIPSGTVLDDFQDYIYQF GALDENSCYETTVSKITEWTTYTTPWTGTFETTRTITPTG TEGTVVIETPESYVTTTQPWTGTYETTYTVPPTGTEPGTV IIETPEIIDCEAVCCGPFLTAFSFRKREECQCENICCPGD TNCETYVTTTQPWTGTYETTYTVPPTGTEPGTVIIETPES YVTTTQPWTGTYETTYTVPPTGTEPGTVIIETPESYVTTT QPWTGTYETTYTVPPSGTEPGTVVIETPEIVDCEAYCCAS VAIKKRELCQCENFCCSWDQSCQTYVTTTQPWTGTYETTY TVPPTGTEPGTVIIETPESYVTTTQPWTGTYETTYTVPPT GTEPGTVIIETPESYVTTTQPWTGTYETTYTVPPTGTEPG TVIIETPEIIDCEAVCCGPFLTAFSFRKREECQCENICCP GDTNCETYVTTTQPWTGTYETTYTVPPTGTEPGTVIIETP ESYVTTTQPWTGTYETTYTVPPTGTEPGTVIIETPESYVT TTQPWTGTYETTYTVPPTGTEPGTVIIETPEIINCEAVCC GPFLTAFSFRKREECQCENICCPGDTNCETYVTTTQPWTG TYETTYTVPPTGTEPGTVIIETPESYVTTTQPWTGTYETT YTVPSTGTEPGTVIIETPESYVTTTQPWTGTYETTFTVPP TGTEPGTVVIETPESYVTTTQPWTGTYETTYSVPPSGTEP GTVVIETPESYVTTTQPWTGTYETTYSVPPSGTEPGTVVI ETPEASTARTKFTTVTSSWTGVFTTTKTLPASGTEPATIV IQTPTGYFNTSSLVSTRTKTNVDTVTRVIPCPICTAPKTI TVVPEEPNESVSVIISQPQSSSTDTTLSKPDSVRVISQPE TASQMDTSLSKTDSAVISTETAGNNIIPLAGSHSYNTIVT TVTDSPQVAQSTTATSSSNVHLTISTQTTTPSLVYSSSLS TVHQVSPSNGGFRSSITVHPLLSVIGAIFGALFM EndoH-Flo11fusion SEQIDNO:13 APAPVKQGPTSVAYVEVNNNSMLNVGKYTLADGGGNAFDV (partialORF,without AVIFAANINYDTGTKTAYLHFNENVQRVLDNAVTQIRPLQ signalpeptidethatis QQGIKVLLSVLGNHQGAGFANFPSQQAASAFAKQLSDAVA cleavedoffpost- KYGLDGVDFDDEYAEYGNNGTAQPNDSSFVHLVTALRANM translationally) PDKIISLYNIGPAASRLSYGGVDVSDKFDYAWNPYYGTWQ VPGIALPKAQLSPAAVEIGRTSRSTVADLARRTVDEGYGV YLTYNLDGGDRTADVSAFTRELYGSEAVRTPGSSGSSGSS GSSGSSGSSGSSGSSEAAAREAAAREAAAREAAARGGGGS GGGGSGGGGSSSGKTCPTSEVSPACYANQWETTFPPSDIK ITGATWVQDNIYDVTLSYEAESLELENLTELKIIGLNSPT GGTKLVWSLNSKVYDIDNPAKWTTTLRVYTKSSADDCYVE MYPFQIQVDWCEAGASTDGCSAWKWPKSYDYDIGCDNMQD GVSRKHHPVYKWPKKCSSNCGVEPTTSDEPEEPTTSEEPE EPTTSEEPEEPTSSDEEPTTSEEPEEPTTSDEPEEPTTSE EPEEPTTSEEPEEPTTSEEPTTSEEPEEPTSSDEEPTTSD EPEEPTTSDEPEEPTTSEEPTTSEEPEEPTTSSEEPTPSE EPEGPTCPTSEVSPACYADQWETTFPPSDIKITGATWVED NIYDVTLSYEAESLELENLTELKIIGLNSPTGGTKVVWSL NSGIYDIDNPAKWTTTLRVYTKSSADDCYVEMYPFQIQVD WCEAGASTDGCSAWKWPKSYDYDIGCDNMQDGVSRKHHPV YKWPKKCSSDCGVEPTTSDEPEEPTTSEEPVEPTSSDEEP TTSEEPTTSEEPEEPTTSDEPEEPTTSEEPEEPTTSEEPE EPTTSEEPTTSEEPEEPTSSDEEPTTSDEPEEPTTSEEPE EPTTSEEPEEPTTSEEPEEPTTSDEPEEPTTSEEPEEPTT SEEPEEPTSSDEEPTTSEEPEEPTTSEEPEEPTTSEEPEE PTTSEEPEEPTSSDEEPTTSEEPEEPTTSDEPEEPTTSEE PEEPTTSEEPEEPTSSDEEPTTSEEPEEPTTSDEPEEPTT SEEPEEPTTSEEPEEPTTSEEPEEPTTSEEPEEPTSSDEE PTTSEEPEEPTTSDEPEEPTTSEEPEEPTTSEEPEEPTTS EEPEEPTTSDEEPGTTEEPLVPTTKTETDVSTTLLTVTDC GTKTCTKSLVITGVTKETVTTHGKTTVITTYCPLPTETVT PTPVTVTSTIYADESVTKTTVYTTGAVEKTVTVGGSSTVV VVHTPLTTAVVQSQSTDEIKTVVTARPSTTTIVRDVCYNS VCSVATIVTGVTEKTITFSTGSITVVPTYVPLVESEEHQR TASTSETRATSVVVPTVVGQSSSASATSSIFPSVTIHEGV ANTVKNSMISGAVALLFNALFL EndoH-Flo11fusion SEQIDNO:14 MVSLRSIFTSSILAAGLTRAHGAPAPVKQGPTSVAYVEVN (fullORF,including NNSMLNVGKYTLADGGGNAFDVAVIFAANINYDTGTKTAY signalpeptidethatis LHFNENVQRVLDNAVTQIRPLQQQGIKVLLSVLGNHQGAG cleavedoffpost- FANFPSQQAASAFAKQLSDAVAKYGLDGVDFDDEYAEYGN translationally) NGTAQPNDSSFVHLVTALRANMPDKIISLYNIGPAASRLS YGGVDVSDKFDYAWNPYYGTWQVPGIALPKAQLSPAAVEI GRTSRSTVADLARRTVDEGYGVYLTYNLDGGDRTADVSAF TRELYGSEAVRTPGSSGSSGSSGSSGSSGSSGSSGSSEAA AREAAAREAAAREAAARGGGGSGGGGSGGGGSSSGKTCPT SEVSPACYANQWETTFPPSDIKITGATWVQDNIYDVTLSY EAESLELENLTELKIIGLNSPTGGTKLVWSLNSKVYDIDN PAKWTTTLRVYTKSSADDCYVEMYPFQIQVDWCEAGASTD GCSAWKWPKSYDYDIGCDNMQDGVSRKHHPVYKWPKKCSS NCGVEPTTSDEPEEPTTSEEPEEPTTSEEPEEPTSSDEEP TTSEEPEEPTTSDEPEEPTTSEEPEEPTTSEEPEEPTTSE EPTTSEEPEEPTSSDEEPTTSDEPEEPTTSDEPEEPTTSE EPTTSEEPEEPTTSSEEPTPSEEPEGPTCPTSEVSPACYA DQWETTFPPSDIKITGATWVEDNIYDVTLSYEAESLELEN LTELKIIGLNSPTGGTKVVWSLNSGIYDIDNPAKWTTTLR VYTKSSADDCYVEMYPFQIQVDWCEAGASTDGCSAWKWPK SYDYDIGCDNMQDGVSRKHHPVYKWPKKCSSDCGVEPTTS DEPEEPTTSEEPVEPTSSDEEPTTSEEPTTSEEPEEPTTS DEPEEPTTSEEPEEPTTSEEPEEPTTSEEPTTSEEPEEPT SSDEEPTTSDEPEEPTTSEEPEEPTTSEEPEEPTTSEEPE EPTTSDEPEEPTTSEEPEEPTTSEEPEEPTSSDEEPTTSE EPEEPTTSEEPEEPTTSEEPEEPTTSEEPEEPTSSDEEPT TSEEPEEPTTSDEPEEPTTSEEPEEPTTSEEPEEPTSSDE EPTTSEEPEEPTTSDEPEEPTTSEEPEEPTTSEEPEEPTT SEEPEEPTTSEEPEEPTSSDEEPTTSEEPEEPTTSDEPEE PTTSEEPEEPTTSEEPEEPTTSEEPEEPTTSDEEPGTTEE PLVPTTKTETDVSTTLLTVTDCGTKTCTKSLVITGVTKET VTTHGKTTVITTYCPLPTETVTPTPVTVTSTIYADESVTK TTVYTTGAVEKTVTVGGSSTVVVVHTPLTTAVVQSQSTDE IKTVVTARPSTTTIVRDVCYNSVCSVATIVTGVTEKTITF STGSITVVPTYVPLVESEEHQRTASTSETRATSVVVPTVV GQSSSASATSSIFPSVTIHEGVANTVKNSMISGAVALLFN ALFL FLO5Saccharomyces SEQIDNO:20 MTIAHHCIFLVILAFLALINVASGATEACLPAGQRKSGMN cerevisiae INFYQYSLKDSSTYSNAAYMAYGYASKTKLGSVGGQTDIS IDYNIPCVSSSGTFPCPQEDSYGNWGCKGMGACSNSQGIA YWSTDLFGFYTTPTNVTLEMTGYFLPPQTGSYTFSFATVD DSAILSVGGSIAFECCAQEQPPITSTNFTINGIKPWDGSL PDNITGTVYMYAGYYYPLKVVYSNAVSWGTLPISVELPDG TTVSDNFEGYVYSFDDDLSQSNCTIPDPSIHTTSTITTTT EPWTGTFTSTSTEMTTITDTNGQLTDETVIVIRTPTTAST ITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVIVIRTP TSEGLITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVI VIRTPTSEGLITTTTEPWTGTFTSTSTEVTTITGTNGQPT DETVIVIRTPTSEGLITTTTEPWTGTFTSTSTEMTTVTGT NGQPTDETVIVIRTPTSEGLISTTTEPWTGTFTSTSTEVT TITGTNGQPTDETVIVIRTPTSEGLITTTTEPWTGTFTST STEMTTVTGTNGQPTDETVIVIRTPTSEGLITRTTEPWTG TFTSTSTEVTTITGTNGQPTDETVIVIRTPTTAISSSLSS SSGQITSSITSSRPIITPFYPSNGTSVISSSVISSSVTSS LVTSSSFISSSVISSSTTTSTSIFSESSTSSVIPTSSSTS GSSESKTSSASSSSSSSSISSESPKSPTNSSSSLPPVTSA TTGQETASSLPPATTTKTSEQTTLVTVTSCESHVCTESIS SAIVSTATVTVSGVTTEYTTWCPISTTETTKQTKGTTEQT KGTTEQTTETTKQTTVVTISSCESDICSKTASPAIVSTST ATINGVTTEYTTWCPISTTESKQQTTLVTVTSCESGVCSE TTSPAIVSTATATVNDVVTVYPTWRPQTTNEQSVSSKMNS ATSETTTNTGAAETKTAVTSSLSRFNHAETQTASATDVIG HSSSVVSVSETGNTMSLTSSGLSTMSQQPRSTPASSMVGS STASLEISTYAGSANSLLAGSGLSVFIASLLLAII N-terminaladdition SEQIDNO:21 EAEA EAEA GGGSlinker SEQIDNO:22 GGGGS GSSlinker SEQIDNO:23 GSS Arigidlinkerthat SEQIDNO:24 EAAAREAAAREAAAREAAAR forms4turnsofan alphahelix Fulllinker SEQIDNO:25 GSSGSSGSSGSSGSSGSSGSSGSSEAAAREAAAREAAARE AAARGGGGSGGGGSGGGGS AOX1promoter SEQIDNO:26 GATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTT TGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTG CCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTT GCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCA CTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGAT TGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAA CACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGG CGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCC GCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGA GTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAA AACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACA AAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGT TGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAA AAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACG AATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTC TCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGC AAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGT CTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTG CTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTA ACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGC CCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTA CTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGA TTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAA CAACTAATTATTGGATCCCGA DAK2promoter SEQIDNO:27 AAATAAGCATGTTTGTTTCAGATCAAAGATTAGCGTTTCA AAGTTGTGGAAAAGTGACCATGCAACAATATGCAACACAT TCGGATTATCTGATAAGTTTCAAAGCTACTAAGTAAGCCC GTTTCAAGTCTCCAGACCGACATCTGCCATCCAGTGATTT TCTTAGTCCTGAAAAATACGATGTGTAAACATAAACCACA AAGATCGGCCTCCGAGGTTGAACCCTTACGAAAGAGACAT CTGGTAGCGCCAATGCCAAAAAAAAATCACACCAGAAGGA CAATTCCCTTCCCCCCCAGCCCATTAAAGCTTACCATTTC CTATTCCAATACGTTCCATAGAGGGCATCGCTCGGCTCAT TTTCGCGTGGGTCATACTAGAGCGGCTAGCTAGTCGGCTG TTTGAGCTCTCTAATCGAGGGGTAAGGATGTCTAATATGT CATAATGGCTCACTATATAAAGAACCCGCTTGCTCAACCT TCGACTCCTTTCCCGATCCTTTGCTTGTTGCTTCTTCTTT TATAACAGGAAACAAAGGAATTTATACACTTTAAGAATT PEX11promoter SEQIDNO:28 CTTCCCCATTTCACTGACAGTTTGTAGAAATAGGGCAACA ATTGATGCAAATCGATTTTCAACGCATTGGTTTTGATAGC ATTGATGATCTTGGAGCTGTAAAAGTCCGGCTGGATAAGC TCAATGAAATAGGTTGGTTGATCTGGATCTTCTTTTGGGT CATTTTGTTCGCTCTGTATTTCACAAATTGCCAGAATCTC TGCCAACCACAGTGGTAGGTCCAACTTGGTGTTCTGAATC ACAGGCTTCCCCGGGTTGTTCTCTAAATAACCGAGGCCCG GCACAGAAATCGTAAACCGACACGGTATCTTTTGTCCGTC CGCCAGTATCTCATCAAGGTCGTAGTAGCCCATGATGAGT ATCAAAGGGGATTTGGTTATGCGATGCAACGAGAGATTGT TTATCCCAGATGCTGATGTAAAAACCTTAACCAGCGTGAC AGTAGAAATAAGACACGTTAAAATTACCCGCGCTTCCCTA ACAATTGGCTCTGCCTTTCGGCAAGTTTCTAACTGCCCTC CCCTCTCACATGCACCACGAACTTACCGTTCGCTCCTAGC AGAACCACCCCAAAGTTTAATCAGGACCGCATTTTAGCCT ATTGCTGTAGAACCCCACAACATAACCTGGTCCAGAGCCA GCCCTTTATATATGGTAAATCCCGTTTGAACTTCGAAGTG GAATCGGAATTTTTACATCAAAGAAACTGATACTGAAACT TTTGGCTTCGACTTGGACTTTCTCTTAATC FLD1promoter SEQIDNO:29 AAATCAGCCATTAATCTCACCTCAGTTTTTGAATCAGTAG AATTTTCAATGAAACAAACGGTTGGTATATTATTTGATAG GGTAGCCAAATTTCCAAAAATGAACTTTTCATCAGGTAAT ATCTTGAATACCGTAATGTAGTGACTATTGGAAGAAACTG CTATCAAATTATATTTCGGATAGAAATCCAAACCCCAGAC TGATCTCTTGAGTCTCAACTCTAAGTCAGCCGCGACTCTA ATTATCTGTGGATTAGGAGTTAGTGTGGACAAAGCATCAG TATAGTATAACTTTACGGTTCCATTATCAGACGCTATTGC AAGAACTTCCTTTCCATTGATCTCTCCAATTCGACAGTAA TTGATATCATAAGGTAGGTCTGGAAACACACTGGCGCTTG TATCCCATTCTGCAGGAATTTCTGGAACGGTGGTAATGGT AGTTATCCAACGGAGTTGGGGTAGTTGGTATATCTGGATA TGCCGCCTATAGGATAAAAACAGGAGAGAGTGAACCTTGC TTACGGCTACTAGATTGTTCTTGTACTCGGAATTGTCGTT ATCGGAAACTAGACTAATCTCATCTGTGTGTTGCAGTACT ATTGAGTCGTTGTAGTATCTACCAGGAGGGCATTCCATGA ACTAGTGAGACAAATGAGTTGGATTTTCTCAATAGACATA TGCAAGAATGCTACACAACGGATGTCGCACTCTTTTTCTT AGTTGATAATATCATCCAATCAGAAGACACGGGCTAGAAG GACTTGCTCCCGAAGGATAATCCACTGCTACTATCTCCCT TCCTCACATATAGTCTTGCAGGGCTCATGCCCCTTTCTCC TTCGAACTGCCCGATGAGGAAGTCTTTAGCCTATCAAGGA ATTCGGGACCATCATCAATTTTTAGAGCCTTACCTGATCG CAATCAGGATTTCACTACTCATATAAATACATCACTCAAA CTCCAACTTTGCTTGTTCATACAATTCTTGATATTCACAG GATC FGH1promoter SEQIDNO:30 GTGAATTTGTCACGGAATTGACCAAGAGGTCAGACGATCC TGTATCCCATTGAGCCGTTATGCTTTGTGGGGGAAACCCT ATTTCTATCGTACTAAGAAAACCAATGGTGAACTCATATT CGGTATCAATGGCGACGATTCCAGCATAGCCTGTAGACAG TAACAACACTAGGGCAACAGCAACTAACATATCTTCATTG ATGAAACGTTGTGATCGGTGTGACTTTTATAGTAAAAGCT ACAACTGTTTGAAATACCAAGATATCATTGTGAATGGCTC AAAAGGGTAATACATCTGAAAAACCTGAAGTGTGGAAAAT TCCGATGGAGCCAACTCATGATAACGCAGAAGTCCCATTT TGCCATCTTCTCTTGGTATGAAACGGTAGAAAATGATCCG AGTATGCCAATTGATACTCTTGATTCATGCCCTATAGTTT GCGTAGGGTTTAATTGATCTCCTGGTCTATCGATCTGGGA CGCAATGTAGACCCCATTAGTGGAAACACTGAAAGGGATC CAACACTCTAGGCGGACCCGCTCACAGTCATTTCAGGACA ATCACCACAGGAATCAACTACTTCTCCCAGTCTTCCTTGC GTGAAGCTTCAAGCCTACAACATAACACTTCTTACTTAAT CTTTGATTCTCGAATTGTTTACCCAATCTTGACAACTTAG CCTAAGCAATACTCTGGGGTTATATATAGCAATTGCTCTT CCTCGCTGTAGCGTTCATTCCATCTTTCTAGAATTCGT DAS2promoter SEQIDNO:31 CCTGTTGATAAGACGCATTCTAGAGTTGTTTCATGAAAGG GTTACGGGTGTTGATTGGTTTGAGATATGCCAGAGGACAG ATCAATCTGTGGTTTGCTAAACTGGAAGTCTGGTAAGGAC TCTAGCAAGTCCGTTACTCAAAAAGTCATACCAAGTAAGA TTACGTAACACCTGGGCATGACTTTCTAAGTTAGCAAGTC ACCAAGAGGGTCCTATTTAACGTTTGGCGGTATCTGAAAC ACAAGACTTGCCTATCCCATAGTACATCATATTACCTGTC AAGCTATGCTACCCCACAGAAATACCCCAAAAGTTGAAGT GAAAAAATGAAAATTACTGGTAACTTCACCCCATAACAAA CTTAATAATTTCTGTAGCCAATGAAAGTAAACCCCATTCA ATGTTCCGAGATTTAGTATACTTGCCCCTATAAGAAACGA AGGATTTCAGCTTCCTTACCCCATGAACAGAAATCTTCCA TTTACCCCCCACTGGAGAGATCCGCCCAAACGAACAGATA ATAGAAAAAAGAAATTCGGACAAATAGAACACTTTCTCAG CCAATTAAAGTCATTCCATGCACTCCCTTTAGCTGCCGTT CCATCCCTTTGTTGAGCAACACCATCGTTAGCCAGTACGA AAGAGGAAACTTAACCGATACCTTGGAGAAATCTAAGGCG CGAATGAGTTTAGCCTAGATATCCTTAGTGAAGGGTTGTT CCGATACTTCTCCACATTCAGTCATAGATGGGCAGCTTTG TTATCATGAAGAGACGGAAACGGGCATTAAGGGTTAACCG CCAAATTATATAAAGACAACATGTCCCCAGTTTAAAGTTT TTCTTTCCTATTCTTGTATCCTGAGTGACCGTTGTGTTTA ATATAACAAGTTCGTTTTAACTTAAGACCAAAACCAGTTA CAACAAATTATAACCCCTCTAAACACTAAAGTTCACTCTT ATCAAACTATCAAACATCAAAAGAATTCGCG CAT1promoter SEQIDNO:32 TAATCGAACTCCGAATGCGGTTCTCCTGTAACCTTAATTG TAGCATAGATCACTTAAATAAACTCATGGCCTGACATCTG TACACGTTCTTATTGGTCTTTTAGCAATCTTGAAGTCTTT CTATTGTTCCGGTCGGCATTACCTAATAAATTCGAATCGA GATTGCTAGTACCTGATATCATATGAAGTAATCATCACAT GCAAGTTCCATGATACCCTCTACTAATGGAATTGAACAAA GTTTAAGCTTCTCGCACGAGACCGAATCCATACTATGCAC CCCTCAAAGTTGGGATTAGTCAGGAAAGCTGAGCAATTAA CTTCCCTCGATTGGCCTGGACTTTTCGCTTAGCCTGCCGC AATCGGTAAGTTTCATTATCCCAGCGGGGTGATAGCCTCT GTTGCTCATCAGGCCAAAATCATATATAAGCTGTAGACCC AGCACTTCAATTACTTGAAATTCACCATAACACTTGCTCT AGTCAAGACTTACAATTAAA MDH3promoter SEQIDNO:33 TAGCTTGGGTAGGACTTGACAAGTACGGCTTCCGTGGTCA TACCAAACGCCTTTGTTACCGTTGGCTATACCTAATGACC AAGGCATTTGTGGATTATAACGGTATCGTAGTTGAAAAAT ATGACGTAACCACTGGTACTAGCCCCCACAAGGTTGATGC TGAATACGGGAATCAAGGTGCCGATTTTAAAGGAGTAGCC ACTGAAGGGTTTGGCTGGGTCAATGCCTCTTTTATTTTGG GATTAACCTACTTAGATGTCCAAGGCATCCGTGCGATAGG CGCCGTTACGTCCCCTGATGTATTTTTCAGGAAGCTCAAA CCTTGGGAACGCGCAAGTTATGGCCTAAGGCCATGTAACG AGATAGTCAAGTCAAACTAGAAGTATACGGTTTCCCCGCA GAAATAGCAGAAATAGGCGACAAATACATACAACATTTTC ATTGTGATAGGGGGCGGCGGTTCCTAGGAGGGACAACCCC CAGAAACCTTGTAGACTACGTTTTCACGACGATGGGTTAT TACTGTAAAGGAAGAATATACTACCCACCAGTTGAATGTT TGAACGGATCAAAGGTCGAAGGGAGTACACGGCCCAACCA ACGTAGCTACCGGAGAAAGCAAGACTTTCCCAAACCAAAT AGCTCCGGGTTTCTTCTCCGGCAACCCGTCAGTTTTTGTG TGGCCGGACAAAAATTCGCACCCTCAGTCTAATTGAAAGG TCGGGCTCCGAGCTCTAGGCGTTTGCGCATGTAATATTGC ATCCCCTCCCATAGATAATACTGCGCGAACACAGGGTGCA AATTATGATGACCACACATGCCAGTGACCAAAACAGTTTT TTAGTCTTTAAAAACCCTCGGAACTTCTGAGTATATAAAG GCTTCTCATTTCCTACAAGCAAACAAAGAAGAAACTTCCA CTTTCTAACTTTTTATCTATAGACTTTAGAGTTACAACCA ACGAACAATAACAAA HAC1promoter SEQIDNO:34 TGAAGCTTATCTGCTGAGCAAGTTGTTTGACCAAACTTGA GTCAACAGTGGTTAACTATATCCTCTATTATTTTAGATGG GAGCACATCAAGTGTACGGGAACAATGCAATCGACAACCT GTAGCCTGACATACATAGCCATCTTGAATTGACAAAACTT AGAATGTCTTGAATGTGATAGATATGAGTTCCCAAAAATC TCTTTTACGATTTCCCAGTTGCGGTGTACTATTACACAGA GGATATCATAGCAGACTTACAATCCTCAGGCATAAAACGA GCTTTCTTATCAAAGTGTATTCAAATGGACCATTTGATTG CACCAAGGCATTAGCCCCAAACCATACCACACAGTAACTT GATATTCTCAGCATGCATGGAAATTCCACTCATAACGCGC TATTCACCGCGAATACTTATCTATGAAACTGGGTTCTTTA GTATTCTTTGCCAAATTTCACCGATTAGAAATTATTAGGT AATATAATTTCTTTGGGGAACCCCTTCCCGTTACGCCCGC TGCGGCTTTGTGGTTCTTTTCCAGTCTTGAGCAAATTACA TCTGGTCTAGACAGTTCTTCCGTGCCCCAGTATGCGAGCG CAAACTTTCAATCAAACCTCGTAGCAAATTGGTACTTGAA CTTCGTATTTAACCGCTATTAAATGTACTGACTCTTACAT TATGAAAAATTTTGATAAAGATTTTATATTTCATCTCAGT TAATCTCCTAATAATAATAGTCTGCATAACTCAAACGGTA CTTCCTTTTCGGAACGCGAAGAGTAGTCTCTATGTCATTC TCACACTATCCGCAGCGCAATAGAGAACGAGCATGTTACC CGACTCATCCCTTGTCGATTCGGAAACGATTTATAAATAC AATTAGATCGCCACCGATCTTCTTTTGTCAATATTATAAA AATAGTACAGATTTTCCTTAGTCGAATCAGATCGCAGAAA BiPpromoter SEQIDNO:35 AGATCTGAGGGTGTATACGATGTATCGTGCCGAACACATG CACTTGACGGCACAGCAAATGGTATTCAAGAAGACCACTT TAGAATGGGAGTTAATAGGGATGGTTTCATGGAGGTTAAA ACACTTCAAGGAGGCATCTGAAGCATTCAAGTATGCACTA GGTCTGAGGTTTTCGGTCAAGGCATGCAAGAAATTAATTG TATTCTATCTGAACGAACGCTCCAGAATGAACCAGCCAGA AACCTCAATTGCCCTCAACAACTTAAATCAATCCACATTA TCCATCCAAGAGATTCTCAAGTATCGTTCGTTCCTCGATA TCAACCTAATTTCAAACTTGGTCAAACTAGGAGTTTGGAA TCACCGCTGGTATGCTGAGTTTTCTCCAAAACTCATAGAA AGCCTTGCGGTTGTTGTGGAGAACGGAGGGCTTATCAAGG TAGAAAACGAGGTTAAGGCTACCTATTTCGATTCACAAGA TGGAGTTTACGACTTGATGAACGAGGTATTCAAGTTCATG AAGCATTACGATTATCCTGGGACTGACAACTAAGAGCTCC TAGTGAAGACTTGAGATGGACATGATAAACAATTATAGTG AAAATAGAAACCATAATACAATATTCTAATAGAGGAACCG TTTACCTGTGGTTCCTATTGTGGCCTACTGTTACTAGCTA GTGTAATACACCCTTGCCTCAGCTTTGCAAGTTGACAACT CAGCCAAATGATCTTTGAATGCGCGAAACCTCAAGGTCCA TCGAATTTTCTCGAATTTTCAGTGTTTTCATACAGCGTGT CATCTTCTTTCGCGTACTTATTAAAATCGTACCCAGATCC CTTCTTCTTCCTTAATTTCAATTCCAACACTCAAGA RAD30promoter SEQIDNO:36 AGATCTTGCAAAATACCTTTCCAGCTTTCCAGCTTCCTAG CACTCATCTTGAAGATATCAAATATTCTCCATTCAAACCA ACATCAAAAAATAGAATAATTATAATCAGTTTGAAGAGCA AGAGTAATTTTAAAGGAAACACATTCATGGTCAGCTAGAA GGTTGACTGAAGAGTCGCAAGATATCTGAGAATAAAAAAG AGCATAGCTAACAAGATGAGTAAACACGGCAAACAGATTT AGGAACAGGTGAAGGGTTTCTGGCTCTTCAATGTATATCC TGCTAGCCACCCATTCAGAAATAACACAAAGTAGGACCCT ACTGAAAAATAAATTTAATACATCTTCATCCTCTCATTAA ACCACCGACCACTCAAACCATACCAGCCTTGTCCAATTCC ATGCATCGTGCTATCCGTCAGAATTTTCAGTGTTAATCGA ATCGGTCATTATAGCTCCGTCTGGGGCGACAACTTGTCAT CACAGAATAGCACAATTATGCGTTGGAATCGTCAAAAAAT CACCTCCAGGTCTGTATACATACAGAACTGGTTGTAACGA CAACCTTGTTTGATTGAGGTGACTGGAAGGTGGAAAGAAA GGGAGGAAATAAATATTGCAAGGAAAGAAAAAAAAATTGT TCACAGTCACCTCTTCACCTTCGCGATTTCATGTTTCTTT CATGTGCTAACTGATCCCAGGGCTTCTCCAGCGCCCTTAT CTGTTAG RVS161-2promoter SEQIDNO:37 CTGCCCATCTATGACTGAATGTGGAGAAGTATCGGAACAA CCCTTCACTAAGGATATCTAGGCTAAACTCATTCGCGCCT TAGATTTCTCCAAGGTATCGGTTAAGTTTCCTCTTTCGTA CTGGCTAACGATGGTGTTGCTCAACAAAGGGATGGAACGG CAGCTAAAGGGAGTGCATGGAATGACTTTAATTGGCTGAG AAAGTGTTCTATTTGTCCGAATTTCTTTTTTCTATTATCT GTTCGTTTGGGCGGATCTCTCCAGTGGGGGGTAAATGGAA GATTTCTGTTCATGGGGTAAGGAAGCTGAAATCCTTCGTT TCTTATAGGGGCAAGTATACTAAATCTCGGAACATTGAAT GGGGTTTACTTTCATTGGCTACAGAAATTATTAAGTTTGT TATGGGGTGAAGTTACCAGTAATTTTCATTTTTTCACTTC AACTTTTGGGGTATTTCTGTGGGGTAGCATAGCTTGACAG GTAATATGATGTACTATGGGATAGGCAAGTCTTGTGTTTC AGATACCGCCAAACGTTAAATAGGACCCTCTTGGTGACTT GCTAACTTAGAAAGTCATGCCCAGGTGTTACGTAATCTTA CTTGGTATGACTTTTTGAGTAACGGACTTGCTAGAGTCCT TACCAGACTTCCAGTTTAGCAAACCACAGATTGATCTGTC CTCTGGCATATCTCAAACCAATCAACACCCGTAACCCTTT CATGAAACAACTCTAGAATGCGTCTTATCAACAGGATTGC CCAAAACAGTAATTGGGGCGGTGGAATCTACATGGGAGTT CCATCGTTGTCTCGGTTTTTCTCCCTATAAGCTACTCTGG AGACGAAGTAACTAACACCCTCAAATATCATT MPP10promoter SEQIDNO:38 TCTGAATCCGACCTCCTCTAATCTACCACTGAAGAGAAGC AGTGTATTGTTCGTCTACGTAAATTTGAATGTGTAAATGG CAAACATGGCTTCGGGGATGATTTGGCATATATATTATTG TAGCATCGTCTGTGGCTCTATGAGTTGTGTGGCGGATGAT GAAAAGTTTCGTGCTGATCCCACAATGCGGCATTTACCAA ATGGGGAAAGACCAGATTTCTTCGCTGCGCCAGCTAGGGA CAGCATAATGTTCCAAGAAGAAGCGATTACAGGTGGATTA CAAAGCGTTCGTCTGCAGTTGATGTTCTACGTGATGGGTA TGAGTTGTAGTGCTACGCTCCATGAATACTTCTAATTTGT CGTTGACAATCCATGAATAATTTAAGTTTGCTTCCCAAGA GTCTATTGCGAAGGGTGAGCCGAATCTCTTGGCGTATGCA CCCGACTCGTCGGCTTTTGTGCGTTCCTTGCAAAGCTCGG TAGCAATCCGTTGGTGGGAGAAATTTGTCTCACGAATTTC AGTTGGGAGTAGCTGTTCCTGGTAGCAAGTTCGAGGGGAT CTGTGCTCATAAAACGTGCTCACGCCAAAAATATTCTTAC AAAATCTTCGCGGGGTGTTTGTCTTACATAATCGATTGGA TATTTTCTTCAAATTTTTTTTTCTTACTGAAGTCCCCTAT AGAG THP3promoter SEQIDNO:39 TCTTGCCAGTTGTCTCCTAAGATGTCATCGGAGTAGGCTC GGCTAAAGAGTAGTAATGCATCAAGACCAACCAAAACACC TTCCACGAGTTCAGATGAACC TTTTAATAACTTCAGGTCACTTTGATGCCGGCACAACTGG GCGAGTTTCGTATAGTTAACTCTGATCTTGCACTCCAGAA CGGGAATAGGATTGACTTTTTGCTTCCGAGAAACGATTTG CTCTCTCTTCGTCTGGCTTTTCACTTTATATCGCACGGAA TCAATGGATGGAACTCCTAAAGCTCCTAACTTCGATGATT TGCTAGCCATGACTCTGTGGGACATTTTCTTGCATCTCGT TTGTAACCTGTCTGTTCCTACACTAAGTTTATGAGAGGCT ACTTTGGATTCTAGCCTCGGTGGTAAAGTGGGAGATAACA ACGGCATAAGGCAAGAACCAGAAGTACCATAACGGTCTGG TAAAGTTGGTGATAACTTAATTGGAAGAGTGTAAGTAAGA CGTGGCTTGTAATAAGGCTTTCCATCAAAAAGGTTCTCCG GGTTGGAGTTTGTGAGGCTCACATCTTTGATCAGTCTTTC AATATAAATTGGTAACGTTGATGACAATGCCGGAGGTAAT TTCTGTAGTTGTTGATATACGCAGATAACAGATTCAAATC TCCATTGGTTTTCATCATTGTGGCTTAAATTAGATCAGAA CATGGTAGTATTTAAAAATGGATCTCTTTGCAGATTTACT CAATATAGCGAAAAAAGGAGACATTCGTTACAAAATATGA AGATAATTCGCCTCATAACTCGATTAATCAAAACAGACGG TCCAGTTCTTCTTTTGGTAGT GBP2promoter SEQIDNO:40 ATCTGTACTGGTACTGACAAAGGTTATCCAGAATCCGAGA CATTTCAACAACAGAGATTCCAGGCTTCAAAACATCCATT TTATCACCAATATCTAGTAATGCTTGCAACAATTCTGGAT ACTTCTTCTGTGTAACCAAATCTCTTATAAACTGAACAGC TTTCTGTACGTTGTCGTCAGTAGTTGGATCAACCTCAGTG GTGACCTGGCCTATCGGTTTTCCAAAAGACTTGTTTATCA CGTCCGAAAGCTCCCATTTTTGCAGATGCGCAACTTTAAA AGGCCTGGCTTGAACATTTGCATCTCTTGTTGTGTGTTCT TTGAGAAAATATTCATCGATCTGGGTGCTTCCAACGACAG AAGATACTCTTCTGAGACCAGAAAGTCCCCAGCCATGCTT CCTAATTACAAAATATTTGTAGGAAGATCCCTGATTAGGA CAAAGTTGTCTTCTCATGAGTTCAACTGAAACTGGGGCTC AAACGGATTATGAAAGGGGTGATTAAAGGTTTTCCTAGCC TTACTTTCCAAATGTCGACCGAGACGAACATTTAAAATCC TAACATCAGAAATTTCTATCCTTAATCTCATTGATGGTTA GTACACTTCGCAGAGTCTCCACATTTGCAGACCCTCCTGG ATAACCAAAGCTTATCTAACAGCGGCATTGGACCTTTGAA AAGACCCTC DAS1promoter SEQIDNO:41 AAATCTGAACACGATGAAACCTCCCCGTAGATTCCACCGC CCCGTTACTTTTTTGGGCAATCCCGTTGATAAGATCCATT TTAGAGTTGTTTCTGAAAGGATTACAGGCGTTGAAGGGTC AGAGAGATGCCAGAGAACAGACCAATTGGTAGTTTGCTAA AGTGGACGTCTGGCAGGTGCTCTATCGTGTTCTTTATTTA GGGCGTTACACTTAGTAGGATTACGTAACAATTTGGCTTA ACCTTCTAAGTTAGAAAGAAACCAAGAGGGGTCCTCTTTA ACGTTCAGCAGTATCTAAAACACAAAACCTGCCCTCATAA TACATCATTCTATCTGTCAAGCTGTGCTACCCCACAGAAA TACCCCCAAGAGTTAAAGTGAAAAGAAAAGCTAAATCTGT TAGACTTCACCCCATAACAAACTTGATAGTTCCTGTAGCC AATGAAAGTTAACCCCATTCAATGTTCCGAGATCTAGTAT GCTTGCTCCTATAAGGAACGAAGGGTTCCAGCTTCCTTAC CCCATCAATGGAAATCTCCTATTTACCCCCCACTGGAA AGATCCGTCCGAACGAACGGATAATAGAAAAAAGAAATTC GGACAAAATAGAACACTTATTTAGCCAATGAAATCCATTT CCAGCATCTCCTTCAACTGCCGTTCCATCCCCTTTGTTGA GCTACACCATCGTCAGCCAGTACCGAATAGGAAACTTAAC CGATATCTTGGAGAATTCTAATGCGCGAATGAGTTTAGCC TAGATATCCTTAGTGAAGGGTTGTTCCGATACTTCTCCAC ATTCAGTCATTTCAGATGGGCAGCATTGTTATCATGAAGA AACGGAAACGGGCAGTAAGGGTTAACCGCCAAATTATATA AAGACAACATGTCCCCAGTTTAAAGTTTTTCTTTCCTATT CTTGTATCCTGAGTGACCGTTGTGTTTAAAATAACAAGTT CGTTTTAACTTAAGACCAAAACCAGTTACAACAAATTATT CCCCAACTAAACACTAAAGTTCACTCTTATCAAACTATCA AACATCAAAG Methanolinducible SEQIDNO:42 CTTCCCCATTTCACTGACAGTTTGTAGAAA promoter TAGGGCAACAATTGATGCAAATCGATTTTCAACGCATTGG TTTTGATAGCATTGATGATCTTGGAGCTGTAAAAGTCCGG CTGGATAAGCTCAATGAAATAGGTTGGTTGATCTGGATCT TCTTTTGGGTCATTTTGTTCGCTCTGTATTTCACAAATTG CCAGAATCTCTGCCAACCACAGTGGTAGGTCCAACTTGGT GTTCTGAATCACAGGCTTCCCCGGGTTGTTCTCTAAATAA CCGAGGCCCGGCACAGAAATCGTAAACCGACACGGTATCT TTTGTCCGTCCGCCAGTATCTCATCAAGGTCGTAGTAGCC CATGATGAGTATCAAAGGGGATTTGGTTATGCGATGCAAC GAGAGATTGTTTATCCCAGATGCTGATGTAAAAACCTTAA CCAGCGTGACAGTAGAAATAAGACACGTTAAAATTACCCG CGCTTCCCTAACAATTGGCTCTGCCTTTCGGCAAGTTTCT AACTGCCCTCCCCTCTCACATGCACCACGAACTTACCGTT CGCTCCTAGCAGAACCACCCCAAAGTTTAATCAGGACCGC ATTTTAGCCTATTGCTGTAGAACCCCACAACATAACCTGG TCCAGAGCCAGCCCTTTATATATGGTAAATCCCGTTTGAA CTTCGAAGTGGAATCGGAATTTTTACATCAAAGAAACTGA TACTGAAACTTTTGGCTTCGACTTGGACTTTCTCTTAATC GAATTCGT GCW14promoter SEQIDNO:43 CAGGTGAACCCACCTAACTATTTTTAACTGGCATCCAGTG AGCTCGCTGGGTGAAAGCCAACCATCTTTTGTTTCGGGGA ACCGTGCTCGCCCCGTAAAGTTAATTTTTTTTTCCCGCGC AGCTTTAATCTTTCGGCAGAGAAGGCGTTTTCATCGTAGC GTGGGAACAGAATAATCAGTTCATGTGCTATACAGGCACA TGGCAGCAGTCACTATTTTGCTTTTTAACCTTAAAGTCGT TCATCAATCATTAACTGACCAATCAGATTTTTTGCATTTG CCACTTATCTAAAAATACTTTTGTATCTCGCAGATACGTT CAGTGGTTTCCAGGACAACACCCAAAAAAAGGTATCAATG CCACTAGGCAGTCGGTTTTATTTTTGGTCACCCACGCAAA GAAGCACCCACCTCTTTTAGGTTTTAAGTTGTGGGAACAG TAACACCGCCTAGAGCTTCAGGAAAAACCAGTACCTGTGA CCGCAATTCACCATGATGCAGAATGTTAATTTAAACGAGT GCCAAATCAAGATTTCAACAGACAAATCAATCGATCCATA GTTACCCATTCCAGCCTTTTCGTCGTCGAGCCTGCTTCAT TCCTGCCTCAGGTGCATAACTTTGCATGAAAAGTCCAGAT TAGGGCAGATTTTGAGTTTAAAATAGGAAATATAAACAAA TATACCGCGAAAAAGGTTTGTTTATAGCTTTTCGCCTGGT GCCGTACGGTATAAATACATACTCTCCTCCCCCCCCTGGT TCTCTTTTTCTTTTGTTACTTACATTTTACCGTTCCGT FDH1promoter SEQIDNO:44 AAATAAATGGCAGAAGGATCAGCCTGGACGAAGCAACCAG TTCCAACTGCTAAGTAAAGAAGATGCTAGACGAAGGAGAC TTCAGAGGTGAAAAGTTTGCAAGAAGAGAGCTGCGGGAAA TAAATTTTCAATTTAAGGACTTGAGTGCGTCCATATTCGT GTACGTGTCCAACTGTTTTCCATTACCTAAGAAAAACATA AAGATTAAAAAGATAAACCCAATCGGGAAACTTTAGCGTG CCGTTTCGGATTCCGAAAAACTTTTGGAGCGCCAGATGAC TATGGAAAGAGGAGTGTACCAAAATGGCAAGTCGGGGGCT ACTCACCGGATAGCCAATACATTCTCTAGGAACCAGGGAT GAATCCAGGTTTTTGTTGTCACGGTAGGTCAAGCATTCAC TTCTTAGGAATATCTCGTTGAAAGCTACTTGAAATCCCAT TGGGTGCGGAACCAGCTTCTAATTAAATAGTTCGATGATG TTCTCTAAGTGGGACTCTACGGCTCAAACTTCTACACAGC ATCATCTTAGTAGTCCCTTCCCAAAACACCATTCTAGGTT TCGGAACGTAACGAAACAATGTTCCTCTCTTCACATTGGG CCGTTACTCTAGCCTTCCGAAGAACCAATAAAAGGGACCG GCTGAAACGGGTGTGGAAACTCCTGTCCAGTTTATGGCAA AGGCTACAGAAATCCCAATCTTGTCGGGATGTTGCTCCTC CCAAACGCCATATTGTACTGCAGTTGGTGCGCATTTTAGG GAAAATTTACCCCAGATGTCCTGATTTTCGAGGGCTACCC CCAACTCCCTGTGCTTATACTTAGTCTAATTCTATTCAGT GTGCTGACCTACACGTAATGATGTCGTAACCCAGTTAAAT GGCCGAAAAACTATTTAAGTAAGTTTATTTCTCCTCCAGA TGAGACTCTCCTTCTTTTCTCCGCTAGTTATCAAACTATA AACCTATTTTACCTCAAATACCTCCAACATCACCCACTTA AACAGAATT FBA1promoter SEQIDNO:45 TGCTTAAGTAATTGAAAACAGTGTTGTGATTATATAAGCA TGGTATTTGAATAGAACTACTGGGGTTAACTTATCTAGTA GGATGGAAGTTGAGGGAGATCAAGATGCTTAAAGAAAAGG ATTGGCCAATATGAAAGCCATAATTAGCAATACTTATTTA ATCAGATAATTGTGGGGCATTGTGACTTGACTTTTACCAG GACTTCAAACCTCAACCATTTAAACAGTTATAGAAGACGT ACCGTCACTTTTGCTTTTAATGTGATCTAAATGTGATCAC ATGAACTCAAACTAAAATGATATCTTTTACTGGACAAAAA TGTTATCCTGCAAACAGAAAGCTTTCTTCTATTCTAAGAA GAACATTTACATTGGTGGGAAACCTGAAAACAGAAAATAA ATACTCCCCAGTGACCCTATGAGCAGGATTTTTGCATCCC TATTGTAGGCCTTTCAAACTCACACCTAATATTTCCCGCC ACTCACACTATCAATGATCACTTCCCAGTTCTCTTCTTCC CCTATTCGTACCATGCAACCCTTACACGCCTTTTCCATTT CGGTTCGGATGCGACTTCCAGTCTGTGGGGTACGTAGCCT ATTCTCTTAGCCGGTATTTAAACATACAAATTCACCCAAA TTCTACCTTGATAAGGTAATTGATTAATTTCATAAATGAA TTCGCG GAPpromoter SEQIDNO:46 TTTTTGTAGAAATGTCTTGGTGTCCTCGTCCAATCAGGTA GCCATCTCTGAAATATCTGGCTCCGTTGCAACTCCGAACG ACCTGCTGGCAACGTAAAATTCTCCGGGGTAAAACTTAAA TGTGGAGTAATGGAACCAGAAACGTCTCTTCCCTTCTCTC TCCTTCCACCGCCCGTTACCGTCCCTAGGAAATTTTACTC TGCTGGAGAGCTTCTTCTACGGCCCCCTTGCAGCAATGCT CTTCCCAGCATTACGTTGCGGGTAAAACGGAGGTCGTGTA CCCGACCTAGCAGCCCAGGGATGGAAAAGTCCCGGCCGTC GCTGGCAATAATAGCGGGCGGACGCATGTCATGAGATTAT TGGAAACCACCAGAATCGAATATAAAAGGCGAACACCTTT CCCAATTTTGGTTTCTCCTGACCCAAAGACTTTAAATTTA ATTTATTTGTCCCTATTTCAATCAATTGAACAACTAT PGKpromoter SEQIDNO:47 AAATAGCAGTTTGCGGTTTCTTGATTTCATGGGGGGAACA AACAATAGTGTTGCCTTAATTCTAATTGGCATTGTTGCTT GGAATCGAAATTGGGGGATAACGTCATATCTGAAAAGTAA ACAACTTCGGGAAATCAGGCTGTTTGAATGGCTTGGAAGC GAGATAGAAAGGGGATAGCGAGATAGAGGGGGCGGAGTAG ACGAAGGGTGTTAAACTGCTGAAATCTCTCAATCTGGAAG AAACGGAATAAATTAACTCCTTGCGATAATAAAATCCGAG TCCGTTATGACCCCACACCGTGTTGACCACGGCATACCCC ATGGAATCTGGTACAAAGCGTCAGTCTTGAAGACACCATC ACGTGTAGGAGACTGATTGTCTGACCGTCCAGCAAAAAGG GCATTATAAATCTTGCTGTTAAAGGGGTGAGGGGAGATGC AGGTTGTTCTTTTATTCGCCTTGAACTTTTTAATTTTCCC GGGGTTGCGGAGCGTGAACAGTTAGCCCGATCTGATAGCT TGCAAGATTCAACAGTTTATCCACTACAGGTCAGAGAGAT CGCCGCAGAAGAAATGCTCGTCTCGTGTTCCAGCACACAT ACTGGTGAAGTCGTTATTTTGCCGAAGGGGGGGTAATAAG GTTATGCACCCCCTCTCCACACCCCAGAATCATTTTTTAG CTGGGTTCAAGGCATTAGACTTTGCACATTTTTCCCTTAA ACACCCTTGAAACGCGGATAAACAGTTGCATGTGCATCCT AAAACTAGGTGAGATGCGTACTCCGTGCTCCGATAATAAC AGTGGTGTTGGGGTTGCTGCTAGCTCACGCACTCCGTTCT TTTTTTTCAACCAGCAAAATTCGATGGGGAGAAACTTGGG GTACTTTGCCGACTCCTCCACCATGCTGGTATATAAATAA TACTCGCCCACTTTTCGTTTGCTGCTTTTATATTTCATAG ACTGAAAAAGACTCTTCTTCTACTTTTTCATAATATATCT CAGATATCACTACTATAG TEFg_promoter SEQIDNO:48 GCGATTTAAATTCGCGAAAGAACAGCCTAATAAACTCCGA AGCATGATGGCCTCTATCCGGAAAACGTTAAGAGATGTGG CAACAGGAGGGCACATAGAATTTTTAAAGACGCTGAAGAA TGCTATCATAGTCCGTAAAAATGTGATAGTACTTTGTTTA GTGCGTACGCCACTTATTCGGGGCCAATAGCTAAACCCAG GTTTGCTGGCAGCAAATTCAACTGTAGATTGAATCTCTCT AACAATAATGGTGTTCAATCCCCTGGCTGGTCACGGGGAG GACTATCTTGCGTGATCCGCTTGGAAAATGTTGTGTATCC CTTTCTCAATTGCGGAAAGCATCTGCTACTTCCCATAGGC ACCAGTTACCCAATTGATATTTCCAAAAAAGATTACCATA TGTTCATCTAGAAGTATAAATACAAGTGGACATTCAATGA ATATTTCATTCAATTAGTCATTGACACTTTCATCAACTTA CTACGTCTTATTCAACAATGAATTCGCG AOX1terminator SEQIDNO:53 TCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAG GCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGT ATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGC TTGCTCCTGATCAGCCTATCTCGCAGCAGATGAATATCTT GTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTT TCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTA AGTGAAACCTTCGTTTGTGCG TDH3terminator SEQIDNO:54 TCGATTTGTATGTGAAATAGCTGAAATTCGAAAATTTCAT TATGGCTGTATCTACTTTAGCGTATTAGGCATTTGAGCAT TGGCTTGAACAATGCGGGCTGTAGTGTGTCACCAAAGAAA CCATTCGGGTTCGGATCTGGAAGTCCTCATCACGTGATGC CGATCTCGTGTATTTTATTTTCAGATAACACCTGAAGACT TT RPS25Aterminator SEQIDNO:55 ATTAGTGTACATCTGATAATATAGTACTACCACGTATGAT AATGTAGAGAATAGTCTTCCTTGTCGAGTGTGTTTGCAGT TTTCTTGAGTTTCAAGGTTTAAATGCTGGTATATTAGTTC ATCGAAGGTTTCAGCCAATAGCACCTTAAATCAATCAAAC TAATTCGACTCTTACGAAAGAGCCTACTGTGTTTAGTATC GAAGTCGTTTACCTTTCATGTTGAATAGCTTCCTCTCTGA CCCTAACATTTCAAGATCCTCCTAAAGTTACCCGGATTGT GAAATTCTAATGATCCACCTGCCCAATGCATTTTTTCTTT ATTCAGTTTACCTTTTTTACCTAATATACGAGCTTGTTAA AGTAAGTGGCACTGCAATACTAGGCTTATTGTTGATATTA TGATGAATCGTTTTCACAAACTTGATTTCCTGTGAACTCA CCATGTACTAAGGAAAAAAACATGCATCACCATCTGAATA TTTGAC RPL2Aterminator SEQIDNO:56 ACTATGTAACTAACGAAACAGCATGTACTAATAGAACCGT ATCGAGAATATTTATTTAGGTGAGTAGTAGGAGTGAACCA GACAGTCAATTTAGTGAGCTGTCCCAGCTTTTGTGCATTC CAGAATTGCCGGTCAAATTGGTTATGGGTTATGGGGCTTT TCCGATTGAGGTTCAGTTTCTGCGGTTATCTCTTTCTTGA CCTGGTCTTTTACAGGCTGTTCTTTCTCCCCATGATTATT CTTTAGCTGAAGATACCGCTTAGCCTGATAATGTCGTCGT TTTGTAATCAAAATCTTTAGTTGGGCATCGTCTGAGGTTT CCTTTGGCTTCTGGGGTTGTTAGTAGGAACGTAGGAACCA TAGTAACTTTTACACATACATTCTTATGATTGCGAAGTAA GCTGAGTCTGCTGCTTGGCTCCCGAAGTACTTTCTCTTTC TCTACCGGTTGATTCTCCTTCTGGTGCTCCTAAACGATTG TGTTAGAAGGGATTGAC SignalPeptide SEQIDNO:57 MFTPVRRRVRTAALALSAAAALVLGSTAASGASATPSPAP AP SignalPeptide SEQIDNO:58 MKLSTVLLSAGLASTTLA SignalPeptide SEQIDNO:59 MRFPSIFTAVLFAASSALA SignalPeptide SEQIDNO:60 MVSLRSIFTSSILAAGLTRAHG SignalPeptide SEQIDNO:61 MKFPVPLLFLLQLFFIIATQG SignalPeptide SEQIDNO:62 MQVKSIVNLLLACSLAVA SignalPeptide SEQIDNO:63 MQFNWNIKTVASILSALTLAQA SignalPeptide SEQIDNO:64 MYRNLIIATALTCGAYSAYVPSEPWSTLTPDASLESALKD YSQTFGIAIKSLDADKIKR SignalPeptide SEQIDNO:65 MNLYLITLLFASLCSAITLPKR SignalPeptide SEQIDNO:66 MFEKSKFVVSFLLLLQLFCVLGVHG SignalPeptide SEQIDNO:67 MQFNSVVISQLLLTLASVSMG SignalPeptide SEQIDNO:68 MKSQLIFMALASLVASAPLEHQQQHHKHEKR SignalPeptide SEQIDNO:69 MKFAISTLLIILQAAAVFA SignalPeptide SEQIDNO:70 MKLLNFLLSFVTLFGLLSGSVFA SignalPeptide SEQIDNO:71 MIFNLKTLAAVAISISQVSA SignalPeptide SEQIDNO:72 MKISALTACAVTLAGLAIAAPAPKPEDCTTTVQKRHQHKR SignalPeptide SEQIDNO:73 MSYLKISALLSVLSVALA SignalPeptide SEQIDNO:74 MLSTILNIFILLLFIQASLQ SignalPeptide SEQIDNO:75 MKLSTNLILAIAAASAVVSAAPVAPAEEAANHLHKR SignalPeptide SEQIDNO:76 MFKSLCMLIGSCLLSSVLA SignalPeptide SEQIDNO:77 MKLAALSTIALTILPVALA SignalPeptide SEQIDNO:78 MSFSSNVPQLFLLLVLLTNIVSG SignalPeptide SEQIDNO:79 MQLQYLAVLCALLLNVQSKNVVDFSRFGDAKISPDDTDLE SRERKR SignalPeptide SEQIDNO:80 MKIHSLLLWNLFFIPSILG SignalPeptide SEQIDNO:81 MSTLTLLAVLLSLQNSALA SignalPeptide SEQIDNO:82 MINLNSFLILTVTLLSPALALPKNVLEEQQAKDDLAKR SignalPeptide SEQIDNO:83 MFSLAVGALLLTQAFG SignalPeptide SEQIDNO:84 MKILSALLLLFTLAFA SignalPeptide SEQIDNO:85 MKVSTTKFLAVFLLVRLVCA SignalPeptide SEQIDNO:86 MQFGKVLFAISALAVTALG SignalPeptide SEQIDNO:87 MWSLFISGLLIFYPLVLG SignalPeptide SEQIDNO:88 MRNHLNDLVVLFLLLTVAAQA SignalPeptide SEQIDNO:89 MFLKSLLSFASILTLCKA SignalPeptide SEQIDNO:90 MFVFEPVLLAVLVASTCVTA SignalPeptide SEQIDNO:91 MFSPILSLEIILALATLQSVFA SignalPeptide SEQIDNO:92 MIINHLVLTALSIALA SignalPeptide SEQIDNO:93 MLALVRISTLLLLALTASA SignalPeptide SEQIDNO:94 MRPVLSLLLLLASSVLA SignalPeptide SEQIDNO:95 MVLIQNFLPLFAYTLFFNQRAALA SignalPeptide SEQIDNO:96 MVSLTRLLITGIATALQVNA SignalPeptide SEQIDNO:97 MIFDGTTMSIAIGLLSTLGIGAEA SignalPeptide SEQIDNO:98 MVLVGLLTRLVPLVLLAGTVLLLVFVVLSGG SignalPeptide SEQIDNO:99 MLSILSALTLLGLSCA SignalPeptide SEQIDNO:100 MRLLHISLLSIISVLTKANA SignalPeptide SEQIDNO:101 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG YLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV SLDKREAEA SignalPeptide SEQIDNO:102 MFKSVVYSILAASLANA SignalPeptide SEQIDNO:103 MLLQAFLFLLAGFAAKISA SignalPeptide SEQIDNO:104 MASSNLLSLALFLVLLTHANS SignalPeptide SEQIDNO:105 MNIFYIFLFLLSFVQGLEHTHRRGSLVKR SignalPeptide SEQIDNO:106 MLIIVLLFLATLANSLDCSGDVFFGYTRGDKTDVHKSQAL TAVKNIKR SignalPeptide SEQIDNO:107 MESVSSLFNIFSTIMVNYKSLVLALLSVSNLKYARGMPTS ERQQGLEER SignalPeptide SEQIDNO:108 MFAFYFLTACISLKGVFG SignalPeptide SEQIDNO:109 MRFSTTLATAATALFFTASQVSA SignalPeptide SEQIDNO:110 MKFAYSLLLPLAGVSASVINYKR SignalPeptide SEQIDNO:111 MKFFAIAALFAAAAVAQPLEDR SignalPeptide SEQIDNO:112 MQFFAVALFATSALA SignalPeptide SEQIDNO:113 MKWVTFISLLFLFSSAYSRGVFRR SignalPeptide SEQIDNO:114 MRSLLILVLCFLPLAALG SignalPeptide SEQIDNO:115 MKVLILACLVALALA SignalPeptide SEQIDNO:116 MFNLKTILISTLASIAVA SignalPeptide SEQIDNO:117 MYRKLAVISAFLATARAQSA WT SEQIDNO:118 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG YLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV QLDKR App3 SEQIDNO:119 MRFPPIFTAALFAASSALAAPANTTTEDETAQIPAEAVIG YLDSEGDSDVAVLPFSNSTNNGLSFINTTIASIAAKEEGV QLDKR App8 SEQIDNO:120 MRFPSIFTAVLFAASSALAAPANTTTEDETAQIPAEAVIS YSDLEGDFDAAALPLSNSTNNGLSSTNTTIASIAAKEEGV QLDKR App9 SEQIDNO:121 MRPPSIFTAVLFAASSALAAPANTTTEDETTQIPAEAVAT YLDLEGDVDVAVLPFSSSTNNGLSFINTTIASIAAKEEGV QLDKR App10 SEQIDNO:122 MRFPSIFTAALFAASSALAAPANTTTEGETAQTPAEAVIG YRDLEGDFDVAVLPFPNSTNNGLLFTNTTTASIAAKEEGV QLDKR appS1 SEQIDNO:123 MRFPSIFTAVLLAAPSALAAPANATTEDEAAQIPAEAVIG YLDLEGDFDAAVLPFSNSTNNGLLSINTTIASIAAKEEGV QLDKR appS4 SEQIDNO:124 MRFPSIFTAVVFAASSALAAPANTTAEDETAQIPAEAVIG YLGLEGDSDVAALPLSDSTNNGSLSTNTTIASIAAKEEGV QLDKR appS6 SEQIDNO:125 MRLPSIFTAAVFAASSALAAPANTTTEDETAQIPAEAAIG YLDLEGDSDVAVLPLSNSTNNGLLFINTTIASIAAKEEGV QLDKR appS8 SEQIDNO:126 MRFPSIFTAVLFAASSALAAPANTTTEDETAQIPAEAVIG YLDLEGDFDVAVLPFSNSTNDGLSFINTTTASIAAKEEGV QLDKR a-Factor SEQIDNO:127 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPA PpScw11p SEQIDNO:128 MLSTILNIFILLLFIQASLQAPIPVVTKYVTEGIAVV PpDse4p SEQIDNO:129 MSFSSNVPQLFLLLVLLTNIVSGAVISVWSTSKVTK PpExg1p SEQIDNO:130 MNLYLITLLFASLCSAITLPKRDIIWDYSSEKIMG a-EGFP SEQIDNO:131 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPA S-EGFP SEQIDNO:132 MLSTILNIFILLLFIQASLQEFDYKDDDDKMVSKG D-EGFP SEQIDNO:133 MSFSSNVPQLFLLLVLLTNIVSGEFDYKDDDDKMV E-EGFP SEQIDNO:134 MNLYLITLLFASLCSAEFDYKDDDDKMVSKGEELF a-CALB SEQIDNO:135 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPA S-CALB SEQIDNO:136 MLSTILNIFILLLFIQASLQEFLPSGSDPAFSQPK D-CALB SEQIDNO:137 MSFSSNVPQLFLLLVLLTNIVSGEFLPSGSDPAFS E-CALB SEQIDNO:138 MNLYLITLLFASLCSAEFLPSGSDPAFSQPKSVLD Amylase(AA) SEQIDNO:139 MVAWWSLFLYGLQVAAPALAAEVDCSRFPNATDKEGKDVL VCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDG ECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGV TYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSE YPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTL SHFGKC AlphaK(AK) SEQIDNO:140 MRFPSIFTAVLFAASSALAAPVNTTTEDELEGDFDVAVLP FSASIAAKEEGVSLEKRAEVDCSRFPNATDKEGKDVLVCN KDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECK ETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYD NECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPK PDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHF GKC AlphaT(AT) SEQIDNO:141 MRFPSIFTAVLFAASSALAAEVDCSRFPNATDKEGKDVLV CNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGE CKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVT YDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEY PKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLS HFGKC Lysozyme(LZ) SEQIDNO:142 MLGKNDPMCLVLVLLGLTALLGICQGAEVDCSRFPNATDK EGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNI SKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPV CGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAV SVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVES NGTLTLSHFGKC KillerProtein(KP) SEQIDNO:143 MTKPTQVLVRSVSILFFITLLHLVVAAEVDCSRFPNATDK EGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNI SKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPV CGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAV SVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVES NGTLTLSHFGKC Invertase(IV) SEQIDNO:144 MLLQAFLFLLAGFAAKISAAEVDCSRFPNATDKEGKDVLV CNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGE CKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVT YDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEY PKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLS HFGKC SerumAlbumin(SA) SEQIDNO:145 MKWVTFISLLFLFSSAYSAEVDCSRFPNATDKEGKDVLVC NKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGEC KETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTY DNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYP KPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSH FGKC Glucoamyl(GA) SEQIDNO:146 MSFRSLLALSGLVCSGLAAEVDCSRFPNATDKEGKDVLVC NKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGEC KETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTY DNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYP KPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSH FGKC Inulase(IN)-IC SEQIDNO:147 MKLAYSLLLPLAGVSAAEVDCSRFPNATDKEGKDVLVCNK DLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKE TVPMNCSSYANTTSEDGKVMVLCN RAFNPVCGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCR KELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFC NAVVESNGTLTLSHFGKC AlphaKS(AKS) SEQIDNO:148 MRFPSIFTAVLFAASSALAAPVNTTTEDELEGDFDVAVLP FSASIAAKEEGVSLEKREAEAAEVDCSRFPNATDKEGKDV LVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHD GECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDG VTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCS EYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLT LSHFGKC Ovomucoidsignal SEQIDNO:149 MAMAGVFVLFSFVLCGFLPDAAFG peptide Lysozymesignal SEQIDNO:150 MRSLLILVLCFLPLAALG peptide OvalbuminSignal SEQIDNO:151 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG Peptide YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV SLDKREAEA OvotransferrinSignal SEQIDNO:152 MKLILCTVLSLGIAAVCFA Peptide BovineLactoferrin SEQIDNO:153 MKLFVPALLSLGALGLCLA SignalPeptide PorcineLactoferrin SEQIDNO:154 MKLFIPALLFLGTLGLCLA SignalPeptide KidLipaseSignal SEQIDNO:155 MESKALLLLALSVWLQSLTVSHG Peptide PorcineLipase SEQIDNO:156 MLLIWTLSLLLGAVLG SignalPeptide Ovomucoid SEQIDNO:157 AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTND (canonical) CLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSED GKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVD KRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKT YGNKCNFCNAVVESNGTLTLSHFGKC* Ovomucoid SEQIDNO:158 AEVDCSRFPNATDMEGKDVLVCNKDLRPICGTDGVTYTND CLLCAYSVEFGTNISKEHDGECKETVPMNCSSYANTTSED GKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVD KRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKT YGNKCNFCNAVVESNGTLTLSHFGKC* Ovomucoid SEQIDNO:159 AEVDCSRFPNATDMEGKDVLVCNKDLRPICGTDGVTYTND G162MF167A CLLCAYSVEFGTNISKEHDGECKETVPMNCSSYANTTSED GKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVD KRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKT YMNKCNACNAVVESNGTLTLSHFGKC* Ovomucoidisoform1 SEQIDNO:160 MAMAGVFVLFSFVLCGFLPDAAFGAEVDCSRFPNATDKEG precursorfulllength KDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISK EHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCG TDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSV DCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNG TLTLSHFGKC Ovomucoid[Gallus SEQIDNO:161 MAMAGVFVLFSFVLCGFLPDAVFGAEVDCSRFPNATDMEG gallus] KDVLVCNKDLRPICGTDGVTYTNDCLLCAYSVEFGTNISK EHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCG TDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSV DCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNG TLTLSHFGKC Ovomucoidisoform2 SEQIDNO:162 MAMAGVFVLFSFVLCGFLPDAAFGAEVDCSRFPNATDKEG precursor[Gallus KDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISK gallus] EHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCG TDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVDC SEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTL TLSHFGKC Ovomucoid[Gallus SEQIDNO:163 AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYNNE gallus] CLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSED GKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVD KRHDGECRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKT YGNKCNFCNAVVESNGTLTLSHFGKC Ovomucoid[Numida SEQIDNO:164 MAMAGVFVLFSFALCGFLPDAAFGVEVDCSRFPNATNEEG meleagris] KDVLVCTEDLRPICGTDGVTYSNDCLLCAYNIEYGTNISK EHDGECREAVPVDCSRYPNMTSEEGKVLILCNKAFNPVCG TDGVTYDNECLLCAHNVEQGTSVGKKHDGECRKELAAVDC SEYPKPACTMEYRPLCGSDNKTYDNKCNFCNAVVESNGTL TLSHFGKC PREDICTED: SEQIDNO:165 MQTITWRQPQGDHLRSRAPAATCRAGQYLTMAMAGIFVLF Ovomucoidisoform SFALCGFLPDAAFGVEVDCSRFPNTTNEEGKDVLVCTEDL X1[Meleagris RPICGTDGVTHSECLLCAYNIEYGTNISKEHDGECREAVP gallopavo] MDCSRYPNTTNEEGKVMILCNKALNPVCGTDGVTYDNECV LCAHNLEQGTSVGKKHDGGCRKELAAVSVDCSEYPKPACT LEYRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC Ovomucoid SEQIDNO:166 VEVDCSRFPNTTNEEGKDVLVCTEDLRPICGTDGVTHSEC [Meleagrisgallopavo] LLCAYNIEYGTNISKEHDGECREAVPMDCSRYPNTTSEEG KVMILCNKALNPVCGTDGVTYDNECVLCAHNLEQGTSVGK KHDGECRKELAAVSVDCSEYPKPACTLEYRPLCGSDNKTY GNKCNFCNAVVESNGTLTLSHFGKC PREDICTED: SEQIDNO:167 MQTITWRQPQGDHLRSRAPAATCRAGQYLTMAMAGIFVLF Ovomucoidisoform SFALCGFLPDAAFGVEVDCSRFPNTTNEEGKDVLVCTEDL X2[Meleagris RPICGTDGVTHSECLLCAYNIEYGTNISKEHDGECREAVP gallopavo] MDCSRYPNTTNEEGKVMILCNKALNPVCGTDGVTYDNECV LCAHNLEQGTSVGKKHDGGCRKELAAVDCSEYPKPACTLE YRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC Ovomucoid SEQIDNO:168 EYGTNISIKHNGECKETVPMDCSRYANMTNEEGKVMMPCD [Bambusicola RTYNPVCGTDGVTYDNECQLCAHNVEQGTSVDKKHDGVCG thoracicus] KELAAVSVDCSEYPKPECTAEERPICGSDNKTYGNKCNFC NAVVYVQP Ovomucoid SEQIDNO:169 VDCSRFPNTTNEEGKDVLACTKELHPICGTDGVTYSNECL [Callipeplasquamata] LCYYNIEYGTNISKEHDGECTEAVPVDCSRYPNTTSEEGK VLIPCNRDFNPVCGSDGVTYENECLLCAHNVEQGTSVGKK HDGGCRKEFAAVSVDCSEYPKPDCTLEYRPLCGSDNKTYA SKCNFCNAVVIWEQEKNTRHHASHSVFFISARLVC Ovomucoid[Colinus SEQIDNO:170 MLPLGLREYGTNTSKEHDGECTEAVPVDCSRYPNTTSEEG virginianus] KVRILCKKDINPVCGTDGVTYDNECLLCSHSVGQGASIDK KHDGGCRKEFAAVSVDCSEYPKPACMSEYRPLCGSDNKTY VNKCNFCNAVVYVQPWLHSRCRLPPTGTSFLGSEGRETSL LTSRATDLQVAGCTAISAMEATRAAALLGLVLLSSFCELS HLCFSQASCDVYRLSGSRNLACPRIFQPVCGTDNVTYPNE CSLCRQMLRSRAVYKKHDGRCVKVDCTGYMRATGGLGTAC SQQYSPLYATNGVIYSNKCTFCSAVANGEDIDLLAVKYPE EESWISVSPTPWRMLSAGA Ovomucoid-like SEQIDNO:171 MSWWGIKPALERPSQEQSTSGQPVDSGSTSTTTMAGIFVL isoformX2[Anser LSLVLCCFPDAAFGVEVDCSRFPNTTNEEGKEVLLCTKDL cygnoidesdomesticus] SPICGTDGVTYSNECLLCAYNIEYGTNISKDHDGECKEAV PVDCSTYPNMTNEEGKVMLVCNKMFSPVCGTDGVTYDNEC MLCAHNVEQGTSVGKKYDGKCKKEVATVDCSDYPKPACTV EYMPLCGSDNKTYDNKCNFCNAVVDSNGTLTLSHFGKC Ovomucoid-like SEQIDNO:172 MSSQNQLHRRRRPLPGGQDLNKYYWPHCTSDRFSWLLHVT isoformX1[Anser AEQFRHCVCIYLQPALERPSQEQSTSGQPVDSGSTSTTTM cygnoidesdomesticus] AGIFVLLSLVLCCFPDAAFGVEVDCSRFPNTTNEEGKEVL LCTKDLSPICGTDGVTYSNECLLCAYNIEYGTNISKDHDG ECKEAVPVDCSTYPNMTNEEGKVMLVCNKMFSPVCGTDGV TYDNECMLCAHNVEQGTSVGKKYDGKCKKEVATVDCSDYP KPACTVEYMPLCGSDNKTYDNKCNFCNAVVDSNGTLTLSH FGKC Ovomucoid[Coturnix SEQIDNO:173 VEVDCSRFPNTTNEEGKDEVVCPDELRLICGTDGVTYNHE japonica] CMLCFYNKEYGTNISKEQDGECGETVPMDCSRYPNTTSED GKVTILCTKDFSFVCGTDGVTYDNECMLCAHNVVQGTSVG KKHDGECRKELAAVSVDCSEYPKPACPKDYRPVCGSDNKT YSNKCNFCNAVVESNGTLTLNHFGKC Ovomucoid[Coturnix SEQIDNO:174 MAMAGVFLLFSFALCGFLPDAAFGVEVDCSRFPNTTNEEG japonica] KDEVVCPDELRLICGTDGVTYNHECMLCFYNKEYGTNISK EQDGECGETVPMDCSRYPNTTSEDGKVTILCTKDFSFVCG TDGVTYDNECMLCAHNIVQGTSVGKKHDGECRKELAAVSV DCSEYPKPACPKDYRPVCGSDNKTYSNKCNFCNAVVESNG TLTLNHFGKC Ovomucoid[Anas SEQIDNO:175 MAGVFVLLSLVLCCFPDAAFGVEVDCSRFPNTTNEEGKDV platyrhynchos] LLCTKELSPVCGTDGVTYSNECLLCAYNIEYGTNISKDHD GECKEAVPADCSMYPNMTNEEGKMTLLCNKMFSPVCGTDG VTYDNECMLCAHNVEQGTSVGKKYDGKCKKEVATVDCSGY PKPACTMEYMPLCGSDNKTYGNKCNFCNAVVDSNGTLTLS HFGEC Ovomucoid,partial SEQIDNO:176 QVDCSRFPNTTNEEGKEVLLCTKELSPVCGTDGVTYSNEC [Anasplatyrhynchos] LLCAYNIEYGTNISKDHDGECKEAVPADCSMYPNMTNEEG KMTLLCNKMFSPVCGTDGVTYDNECMLCAHNVEQGTSVGK KYDGKCKKEVATVSVDCSGYPKPACTMEYMPLCGSDNKTY GNKCNFCNAVV Ovomucoid-like[Tyto SEQIDNO:177 MTMPGAFVVLSFVLCCFPDATFGVEVDCSTYPNTTNEEGK alba] EVLVCSKILSPICGTDGVTYSNECLLCANNIEYGTNISKY HDGECKEFVPVNCSRYPNTTNEEGKVMLICNKDLSPVCGT DGVTYDNECLLCAHNLEPGTSVGKKYDGECKKEIATVDCS DYPKPVCSLESMPLCGSDNKTYSNKCNFCNAVVDSNETLT LSHFGKC Ovomucoid[Balearica SEQIDNO:178 MTMAGVFVLLSFALCCFPDAAFGVEVDCSTYPNTTNEEGK regulorum EVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKD gibbericeps] HDGECKEVVPVDCSRYPNSTNEEGKVVMLCSKDLNPVCGT DGVTYDNECVLCAHNVESGTSVGKKYDGECKKETATVDCS DYPKPACTLEYMPFCGSDSKTYSNKCNFCNAVVDSNGTLT LSHFGKC Turkeyvulture SEQIDNO:179 MTTAGVFVLLSFALCSFPDAAFGVEVDCSTYPNTTNEEGK [Cathartesaura]OVD EVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKD (nativesequence) HDGECKEFVPVDCSRYPNTTNEDGKVVLLCNKDLSPICGT boldedisnativesignal DGVTYDNECLLCARNLEPGTSVGKKYDGECKKEIATVDCS sequence DYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLT LSHFGKC Ovomucoid-like SEQIDNO:180 MTTAGVFVLLSFTLCSFPDAAFGVEVDCSPYPNTTNEEGK [Cuculuscanorus] EVLVCNKILSPICGTDGVTYSNECLLCAYNLEYGTNISKD YDGECKEVAPVDCSRHPNTTNEEGKVELLCNKDLNPICGT NGVTYDNECLLCARNLESGTSIGKKYDGECKKEIATVDCS DYPKPVCTLEEMPLCGSDNKTYGNKCNFCNAVVDSNGTLT LSHFGKC Ovomucoid SEQIDNO:181 MTTAVVFVLLSFALCCFPDAAFGVEVDCSTYPNSTNEEGK [Antrostomus DVLVCPKILGPICGTDGVTYSNECLLCAYNIQYGTNVSKD carolinensis] HDGECKEIVPVDCSRYPNTTNEEGKVVFLCNKNFDPVCGT DGDTYDNECMLCARSLEPGTTVGKKHDGECKREIATVDCS DYPKPTCSAEDMPLCGSDSKTYSNKCNFCNAVVDSNGTLT LSRFGKC Ovomucoid[Cariama SEQIDNO:182 MTMTGVFVLLSFAICCFPDAAFGVEVDCSTYPNTTNEEGK cristata] EVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKD HDGECKEVVPVDCSKYPNTTNEEGKVVLLCSKDLSPVCGT DGVTYDNECLLCARNLEPGSSVGKKYDGECKKEIATIDCS DYPKPVCSLEYMPLCGSDSKTYDNKCNFCNAVVDSNGTLT LSHFGKC Ovomucoid-like SEQIDNO:183 MTTAGVFVLLSFVLCCFPDAVFGVEVDCSTYPNTTNEEGK isoformX2 EVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKD [Pygoscelisadeliae] HDGECKEVVPVNCSRYPNTTNEEGKVVLRCSKDLSPVCGT DGVTYDNECLMCARNLEPGAVVGKNYDGECKKEIATVDCS DYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLT LSHFGKC Ovomucoid-like SEQIDNO:184 MTTAGVFVLLSIALCCFPDAAFGVEVDCSAYSNTTSEEGK [Nipponianippon] EVLSCTKILSPICGTDGVTYSNECLLCAYNIEYGTNISKD HDGECKEVVSVDCSRYPNTTNEEGKAVLLCNKDLSPVCGT DGVTYDNECLLCAHNLEPGTSVGKKYDGACKKEIATVDCS DYPKPVCTLEYLPLCGSDSKTYSNKCDFCNAVVDSNGTLT LSHFGKC Ovomucoid-like SEQIDNO:185 MTTAGVFVLLSFALCCFPDAAFGVEVDCSTYPNTTNEEGK [Phaethonlepturus] EVLVCTKILSPICGTDGTTYSNECLLCAYNIEYGTNVSKD HDGECKVVPVDCSKYPNTTNEDGKVVLLCNKALSPICGTD RVTYDNECLMCAHNLEPGTSVGKKHDGECQKEVATVDCSD YPKPVCSLEYMPLCGSDGKTYSNKCNFCNAVVNSNGTLTL SHFEKC Ovomucoid-like SEQIDNO:186 MTTAGVFVLLSFVLCCFFPDAAFGVEVDCSTYPNTTNEEG isoformX1 KEVLVCAKILSPVCGTDGVTYSNECLLCAHNIENGTNVGK [Melopsittacus DHDGKCKEAVPVDCSRYPNTTDEEGKVVLLCNKDVSPVCG undulatus] TDGVTYDNECLLCAHNLEAGTSVDKKNDSECKTEDTTLAA VSVDCSDYPKPVCTLEYLPLCGSDNKTYSNKCRFCNAVVD SNGTLTLSRFGKC Ovomucoid[Podiceps SEQIDNO:187 MTTAGVFVLLSFALCCSPDAAFGVEVDCSTYPNTTNEEGK cristatus] EVLACTKILSPICGTDGVTYSNECLLCAYNMEYGTNVSKD HDGKCKEVVPVDCSRYPNTTNEEGK VVLLCNKDLSPVCGTDGVTYDNECLLCARNLEPGASVGKK YDGECKKEIATVDCSDYPKPVCSLEHMPLCGSDSKTYSNK CTFCNAVVDSNGTLTLSHFGKC Ovomucoid-like SEQIDNO:188 MTTAGVFVLLSFALCCFPDAAFGVEVDCSTYPNTTNEEGR [Fulmarusglacialis] EVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKD HDGECKEVAPVGCSRYPNTTNEEGKVVLLCNKDLSPVCGT DGVTYDNECLLCARHLEPGTSVGKKYDGECKKEIATVDCS DYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVLDSNGTLT LSHFGKC Ovomucoid SEQIDNO:189 MTTAGVFVLLSFALCCFPDAVFGVEVDCSTYPNTTNEEGK [Aptenodytesforsteri] EVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKD HDGECKEVVPVDCSRYPNTTNEEGKVVLRCNKDLSPVCGT DGVTYDNECLMCARNLEPGAIVGKKYDGECKKEIATVDCS DYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLI LSHFGKC Ovomucoid-like SEQIDNO:190 MTTAGVFVLLSFVLCCFPDAVFGVEVDCSTYPNTTNEEGK isoformX1 EVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKD [Pygoscelisadeliae] HDGECKEVVPVDCSRYPNTTNEEGKVVLRCSKDLSPVCGT DGVTYDNECLMCARNLEPGAVVGKNYDGECKKEIATVDCS DYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLT LSHFGKC Ovomucoidisoform SEQIDNO:191 MSSQNQLPSRCRPLPGSQDLNKYYQPHCTGDRFCWLFYVT X1[Aptenodytes VEQFRHCICIYLQLALERPSHEQSGQPADSRNTSTMTTAG forsteri] VFVLLSFALCCFPDAVFGVEVDCSTYPNTTNEEGKEVLVC TKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKDHDGEC KEVVPVDCSRYPNTTNEEGKVVLRCNKDLSPVCGTDGVTY DNECLMCARNLEPGAIVGKKYDGECKKEIATVDCSDYPKP VCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLILSHFG KC Ovomucoid,partial SEQIDNO:192 MTTAVVFVLLSFALCCFPDAAFGVEVDCSTYPNSTNEEGK [Antrostomus DVLVCPKILGPICGTDGVTYSNECLLCAYNIQYGTNVSKD carolinensis] HDGECKEIVPVDCSRYPNTTNEEGKVVFLCNKNFDPVCGT DGDTYDNECMLCARSLEPGTTVGKKHDGECKREIATVDCS DYPKPTCSAEDMPLCGSDSKTYSNKCNFCNAVV rOVDasexpressedin SEQIDNO:193 EAEAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVT pichia YTNDCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANT secretedform1 TSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQG ASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGS DNKTYGNKCNFCNAVVESNGTLTLSHFGKC rOVDasexpressedin SEQIDNO:194 EEGVSLEKREAEAAEVDCSRFPNATDKEGKDVLVCNKDLR pichiasecretedform2 PICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVP MNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECL LCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCT AEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC rOVD[gallus]coding SEQIDNO:195 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG sequencecontaining YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV analphamatingfactor SLEKREAEAAEVDCSRFPNATDKEGKDVLVCNKDLRPICG signalsequence TDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMNCS (bolded)asexpressed SYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAH inpichia KVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDR PLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC TurkeyvultureOVD SEQIDNO:196 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG codingsequence YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV containingsecretion SLEKREAEAVEVDCSTYPNTTNEEGKEV signalsasexpressedin LVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKDHD pichia GECKEFVPVDCSRYPNTTNEDGKVVLLCNKDLSPICGTDG boldedisanalpha VTYDNECLLCARNLEPGTSVGKKYDGECKKEIATVDCSDY matingfactorsignal PKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLTLS sequence HFGKC TurkeyvultureOVD SEQIDNO:197 EAEAVEVDCSTYPNTTNEEGKEVLVCTKILSPICGTDGVT insecretedform YSNECLLCAYNIEYGTNVSKDHDGECKEFVPVDCSRYPNT expressedinPichia TNEDGKVVLLCNKDLSPICGTDGVTYDNECLLCARNLEPG TSVGKKYDGECKKEIATVDCSDYPKPVCSLEYMPLCGSDS KTYSNKCNFCNAVVDSNGTLTLSHFGKC Hummingbird SEQIDNO:198 MTMAGVFVLLSFILCCFPDTAFGVEVDCSIYPNTTSEEGK OVD(native EVLVCTETLSPICGSDGVTYNNECQLCAYNVEYGTNVSKD sequence) HDGECKEIVPVDCSRYPNTTEEGRVVMLCNKALSPVCGTD boldedisthenative GVTYDNECLLCARNLESGTSVGKKFDGECKKEIATVDCTD signalsequence YPKPVCSLDYMPLCGSDSKTYSNKCNFCNAVMDSNGTLTL NHFGKC HummingbirdOVD SEQIDNO:199 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG codingsequenceas YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV expressedinPichia SLDKREAEAVEVDCSIYPNTTSEEGKEVLVCTETLSPICG boldedisanalpha SDGVTYNNECQLCAYNVEYGTNVSKDHDGECKEIVPVDCS matingfactorsignal RYPNTTEEGRVVMLCNKALSPVCGTDGVTYDNECLLCARN sequence LESGTSVGKKFDGECKKEIATVDCTDYPKPVCSLDYMPLC GSDSKTYSNKCNFCNAVMDSNGTLTLNHFGKC HummingbirdOVD SEQIDNO:200 EAEAVEVDCSIYPNTTSEEGKEVLVCTETLSPICGSDGVT insecretedformfrom YNNECQLCAYNVEYGTNVSKDHDGECKEIVPVDCSRYPNT Pichia TEEGRVVMLCNKALSPVCGTDGVTYDNECLLCARNLESGT SVGKKFDGECKKEIATVDCTDYPKPVCSLDYMPLCGSDSK TYSNKCNFCNAVMDSNGTLTLNHFGKC Ovalbuminrelated SEQIDNO:201 MFFYNTDFRMGSISAANAEFCFDVFNELKVQHTNENILYS proteinX PLSIIVALAMVYMGARGNTEYQMEKALHFDSIAGLGGSTQ TKVQKPKCGKSVNIHLLFKELLSDITASKANYSLRIANRL YAEKSRPILPIYLKCVKKLYRAGLETVNFKTASDQARQLI NSWVEKQTEGQIKDLLVSSSTDLDTTLVLVNAIYFKGMWK TAFNAEDTREMPFHVTKEESKPVQMMCMNNSFNVATLPAE KMKILELPFASGDLSMLVLLPDEVSGLERIEKTINFEKLT EWTNPNTMEKRRVKVYLPQMKIEEKYNLTSVLMALGMTDL FIPSANLTGISSAESLKISQAVHGAFMELSEDGIEMAGST GVIEDIKHSPELEQFRADHPFLFLIKHNPTNTIVYFGRYW SP* Ovalbuminrelated SEQIDNO:202 MDSISVTNAKFCFDVFNEMKVHHVNENILYCPLSILTALA proteinY MVYLGARGNTESQMKKVLHFDSITGAGSTTDSQCGSSEYV HNLFKELLSEITRPNATYSLEIADKLYVDKTFSVLPEYLS CARKFYTGGVEEVNFKTAAEEARQLINSWVEKETNGQIKD LLVSSSIDFGTTMVFINTIYFKGIWKIAFNTEDTREMPFS MTKEESKPVQMMCMNNSFNVATLPAEKMKILELPYASGDL SMLVLLPDEVSGLERIEKTINFDKLREWTSTNAMAKKSMK VYLPRMKIEEKYNLTSILMALGMTDLFSRSANLTGIS SVDNLMISDAVHGVFMEVNEEGTEATGSTGAIGNIKHSLE LEEFRADHPFLFFIRYNPTNAILFFGRYWSP* Ovalbumin SEQIDNO:203 MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALA MVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNV HSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQ CVKELYRGGLEPINFQTAADQARELINSWVESQINGIIRN VLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFR VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTM SMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIK VYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAE SLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFR ADHPFLFCIKHIATNAVLFFGRCVSP* ChickenOvalbumin SEQIDNO:204 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG withboldedsignal YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV sequence SLDKREAEAGSIGAASMEFCFDVFKELKVHHANENIFYCP IAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEA QCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERY PILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVES QTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDE DTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILE LPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSN VMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSAN LSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDA ASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP ChickenOVA SEQIDNO:205 EAEAGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMS sequenceassecreted ALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTS frompichia VNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPE YLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGI IRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAM PFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFAS GTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEER KIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGIS SAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSE EFRADHPFLFCIKHIATNAVLFFGRCVSP PredictedOvalbumin SEQIDNO:206 MRVPAQLLGLLLLWLPGARCGSIGAASMEFCFDVFKELKV [Achromobacter HHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFD denitrificans] KLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFS LASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQ ARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVF KGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVA SMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIIN FEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAM GITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGRE VVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFG RCVSPLEIKRAAAHHHHHH OLLASepitope- SEQIDNO:207 MTSGFANELGPRLMGKLTMGSIGAASMEFCFDVFKELKVH taggedovalbumin HANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDK LPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSL ASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQA RELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFK GLWEKTFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVAS MASEKMKILELPFASGTMSMLVLLP DEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMK MEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQA VHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLF CIKHIATNAVLFFGRCVSPSR Serpinfamilyprotein SEQIDNO:208 MGGRRVRWEVYISRAGYVNRQIAWRRHHRSLTMRVPAQLL [Achromobacter GLLLLWLPGARCGSIGAASMEFCFDVFKELKVHHANENIF denitrificans] YCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDS IEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAE ERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSW VESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAF KDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMK ILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWT SSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSS SANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAG VDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPLEI KRAAAHHHHHH PREDICTED: SEQIDNO:209 MGSIGAVSMEFCFDVFKELKVHHANENIFYSPFTIISALA ovalbuminisoformX1 MVYLGAKDSTRTQINKVVRFDKLPGFGDSVEAQCGTSVNV [Meleagrisgallopavo] HSSLRDILNQITKPNDVYSFSLASRLYAEETYPILPEYLQ CVKELYRGGLESINFQTAADQARGLINSWVESQTNGMIKN VLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAIPFR VTEQESKPVQMMYQIGLFKVASMASEKMKILELPFASGTM SMWVLLPDEVSGLEQLETTISFEKMTEWISSNIMEERRIK VYLPRMKMEEKYNLTSVLMAMGITDLFSSSANLSGISSAG SLKISQAVHAAYAEIYEAGREVIGSAEAGADATSVSEEFR VDHPFLYCIKHNLTNSILFFGRCISP Ovalbuminprecursor SEQIDNO:210 MGSIGAVSMEFCFDVFKELKVHHANENIFYSPFTIISALA [Meleagrisgallopavo] MVYLGAKDSTRTQINKVVRFDKLPGFGDSVEAQCGTSVNV HSSLRDILNQITKPNDVYSFSLASRLYAEETYPILPEYLQ CVKELYRGGLESINFQTAADQARGLINSWVESQTNGMIKN VLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAIPFR VTEQESKPVQMMYQIGLFKVASMASEKMKILELPFASGTM SMWVLLPDEVSGLEQLETTISFEKMTEWISSNIMEERRIK VYLPRMKMEEKYNLTSVLMAMGITDLFSSSANLSGISSAG SLKISQAAHAAYAEIYEAGREVIGSAEAGADATSVSEEFR VDHPFLYCIKHNLTNSILFFGRCISP Hypotheticalprotein SEQIDNO:211 YYRVPCMVLCTAFHPYIFIVLLFALDNSEFTMGSIGAVSM [Bambusicola EFCFDVFKELRVHHPNENIFFCPFAIMSAMAMVYLGAKDS thoracicus] TRTQINKVIRFDKLPGFGDSTEAQCGKSANVHSSLKDILN QITKPNDVYSFSLASRLYADETYSIQSEYLQCVNELYRGG LESINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDS QTAMVLVNAIVFRGLWEKAFKDEDTQTMPFRVTEQESKPV QMMYQIGSFKVASMASEKMKILELPLASGTMSMLVLLPDE VSGLEQLETTISFEKLTEWTSSNVMEERKIKVYLPRMKME EKYNLTSVLMAMGITDLFRSSANLSGISLAGNLKISQAVH AAHAEINEAGRKAVSSAEAGVDATSVSEEFRADRPFLFCI KHIATKVVFFFGRYTSP Eggalbumin SEQIDNO:212 MGSIGAASMEFCFDVFKELKVHHANDNMLYSPFAILSTLA MVFLGAKDSTRTQINKVVHFDKLPGFGDSIEAQCGTSVNV HSSLRDILNQITKQNDAYSFSLASRLYAQETYTVVPEYLQ CVKELYRGGLESVNFQTAADQARGLINAWVESQTNGIIRN ILQPSSVDSQTAMVLVNAIAFKGLWEKAFKAEDTQTIPFR VTEQESKPVQM MYQIGSFKVASMASEKMKILELPFASGTMSMLVLLPDDVS GLEQLESIISFEKLTEWTSSSIMEERKVKVYLPRMKMEEK YNLTSLLMAMGITDLFSSSANLSGISSVGSLKISQAVHAA HAEINEAGRDVVGSAEAGVDATEEFRADHPFLFCVKHIET NAILLFGRCVSP Ovalbuminisoform SEQIDNO:213 MASIGAVSTEFCVDVYKELRVHHANENIFYSPFTIISTLA X2[Numida MVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNV meleagris] HSSLRDILNQITKPNDVYSFSLASRLYAEETYPILPEYLQ CVKELYRGGLESINFQTAADQARELINSWVESQTSGIIKN VLQPSSVNSQTAMVLVNAIYFKGLWERAFKDEDTQAIPFR VTEQESKPVQMMSQIGSFKVASVASEKVKILELPFVSGTM SMLVLLPDEVSGLEQLESTISTEKLTEWTSSSIMEERKIK VFLPRMRMEEKYNLTSVLMAMGMTDLFSSSANLSGISSAE SLKISQAVHAAYAEIYEAGREVVSSAEAGVDATSVSEEFR VDHPFLLCIKHNPTNSILFFGRCISP Ovalbuminisoform SEQIDNO:214 MALCKAFHPYIFIVLLFDVDNSAFTMASIGAVSTEFCVDV X1[Numida YKELRVHHANENIFYSPFTIISTLAMVYLGAKDSTRTQIN meleagris] KVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPN DVYSFSLASRLYAEETYPILPEYLQCVKELYRGGLESINF QTAADQARELINSWVESQTSGIIKNVLQPSSVNSQTAMVL VNAIYFKGLWERAFKDEDTQAIPFRVTEQESKPVQMMSQI GSFKVASVASEKVKILELPFVSGTMSMLVLLPDEVSGLEQ LESTISTEKLTEWTSSSIMEERKIKVFLPRMRMEEKYNLT SVLMAMGMTDLFSSSANLSGISSAESLKISQAVHAAYAEI YEAGREVVSSAEAGVDATSVSEEFRVDHPFLLCIKHNPTN SILFFGRCISP PREDICTED: SEQIDNO:215 MGSIGAASMEFCFDVFKELKVHHANDNMLYSPFAILSTLA Ovalbuminisoform MVFLGAKDSTRTQINKVVHFDKLPGFGDSIEAQCGTSANV X2[Coturnix HSSLRDILNQITKQNDAYSFSLASRLYAQETYTVVPEYLQ japonica] CVKELYRGGLESVNFQTAADQARGLINAWVESQTNGIIRN ILQPSSVDSQTAMVLVNAIAFKGLWEKAFKAEDTQTIPFR VTEQESKPVQMMHQIGSFKVASMASEKMKILELPFASGTM SMLVLLPDDVSGLEQLESTISFEKLTEWTSSSIMEERKVK VYLPRMKMEEKYNLTSLLMAMGITDLFSSSANLSGISSVG SLKISQAVHAAYAEINEAGRDVVGSAEAGVDATEEFRADH PFLFCVKHIETNAILLFGRCVSP PREDICTED: SEQIDNO:216 MGLCTAFHPYIFIVLLFALDNSEFTMGSIGAASMEFCFDV ovalbuminisoformX1 FKELKVHHANDNMLYSPFAILSTLAMVFLGAKDSTRTQIN [Coturnixjaponica] KVVHFDKLPGFGDSIEAQCGTSANVHSSLRDILNQITKQN DAYSFSLASRLYAQETYTVVPEYLQCVKELYRGGLESVNF QTAADQARGLINAWVESQTNGIIRNILQPSSVDSQTAMVL VNAIAFKGLWEKAFKAEDTQTIPFRVTEQESKPVQMMHQI GSFKVASMASEKMKILELPFASGTMSMLVLLPDDVSGLEQ LESTISFEKLTEWTSSSIMEERKVKVYLPRMKMEEKYNLT SLLMAMGITDLFSSSANLSGISSVGSLKISQAVHAAYAEI NEAGRDVVGSAEAGVDATEEFRADHPFLFCVKHIETNAIL LFGRCVSP Eggalbumin SEQIDNO:217 MGSIGAASMEFCFDVFKELKVHHANDNMLYSPFAILSTLA MVFLGAKDSTRTQINKVVHFDKLPGFGDSIEAQCGTSANV HSSLRDILNQITKQNDAYSFSLASRLYAQETYTVVPEYLQ CVKELYRGGLESVNFQTAADQARGLINAWVESQINGIIRN ILQPSSVDSQTAMVLVNAIAFKGLWEKAFKAEDTQTIPFR VTEQESKPVQM MHQIGSFKVASMASEKMKILELPFASGTMSMLVLLPDDVS GLEQLESTISFEKLTEWTSSSIMEERKVKVYLPRMKMEEK YNLTSLLMAMGITDLFSSSANLSGISSVGSLKIPQAVHAA YAEINEAGRDVVGSAEAGVDATEEFRADHPFLFCVKHIET NAILLFGRCVSP ovalbumin[Anas SEQIDNO:218 MGSIGAASTEFCFDVFRELRVQHVNENIFYSPFSIISALA platyrhynchos] MVYLGARDNTRTQIDKVVHFDKLPGFGESMEAQCGTSVSV HSSLRDILTQITKPSDNFSLSFASRLYAEETYAILPEYLQ CVKELYKGGLESISFQTAADQARELINSWVESQTNGIIKN ILQPSSVDSQTTMVLVNAIYFKGMWEKAFKDEDTQAMPFR MTEQESKPVQMMYQVGSFKVAMVTSEKMKILELPFASGMM SMFVLLPDEVSGLEQLESTISFEKLTEWTSSTMMEERRMK VYLPRMKMEEKYNLTSVFMALGMTDLFSSSANMSGISSTV SLKMSEAVHAACVEIFEAGRDVVGSAEAGMDVTSVSEEFR ADHPFLFFIKHNPTNSILFFGRWMSP PREDICTED: SEQIDNO:219 MGSIGAASTEFCFDVFRELKVQHVNENIFYSPLSIISALA ovalbumin-like[Anser MVYLGARDNTRTQIDQVVHFDKIPGFGESMEAQCGTSVSV cygnoidesdomesticus] HSSLRDILTEITKPSDNFSLSFASRLYAEETYTILPEYLQ CVKELYKGGLESISFQTAADQARELINSWVESQTNGIIKN ILQPSSVDSQTTMVLVNAIYFKGMWEKAFKDEDTQTMPFR MTEQESKPVQMMYQVGSFKLATVTSEKVKILELPFASGMM SMCVLLPDEVSGLEQLETTISFEKLTEWTSSTMMEERRMK VYLPRMKMEEKYNLTSVFMALGMTDLFSSSANMSGISSTV SLKMSEAVHAACVEIFEAGRDVVGSAEAGMDVTSVSEEFR ADHPFLFFIKHNPSNSILFFGRWISP PREDICTED: SEQIDNO:220 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALS Ovalbumin-like MVYLGARENTRAQIDKVLHFDKMPGFGDTIESQCGTSVSI [Aquilachrysaetos HTSLKDMFTQITKPSDNYSLSFASRLYAEETYPILPEYLQ canadensis] CVKELYKGGLETISFQTAAEQARELINSWVESQTNGMIKN ILQPSSVDPQTKMVLVNAIYFKGVWEKAFKDEDTQEVPFR VTEQESKPVQMMYQIGSFKVAVMASEKMKILELPYASGQL SMLVLLPDDVSGLEQLESAITFEKLMAWTSSTTMEERKMK VYLPRMKIEEKYNLTSVLMALGVTDLFSSSANLSGISSAE SLKISKAVHEAFVEIYEAGSEVVGSTEAGMEVTSVSEEFR ADHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:221 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALS Ovalbumin-like MVYLGARENTRTQIDKVLHFDKMTGFGDTVESQCGTSVSI [Haliaeetusalbicilla] HTSLKDIFTQITKPSDNYSLSLASRLYAEETYPILPEYLQ CVKELYKGGLETVSFQTAAEQARELINSWVESQTNGMIKN ILQPSSVDPQTKMVLVNAIYFKGVWEKAFKDEDTQEVPFR VTEQESKPVQMMYQIGSFKVAVMASEKMKILELPYASGQL SMLVLLPDDVSGLEQLESAITSEKLMEWTSSTTMEERKMK VYLPRMKIEEKYNLTSVLMALGVTDLFSSSADLSGISSAE SLKISKAVHEAFVEIYEAGSEVVGSTEGGMEVTSVSEEFR ADHPFLFLIKHKPTNSILFFGRCFSP PREDICTED: SEQIDNO:222 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALS Ovalbumin-like MVYLGARENTRTQIDKVLHFDKMTGFGDTVESQCGTSVSI [Haliaeetus HTSLKDIFTQITKPSDNYSLSLASRLYAEETYPILPEYLQ leucocephalus] CVKELYKGGLETVSFQTAAEQARELINSWVESQTNGMIKN ILQPSSVDPQTKMVLVNAIYFKGVWEKAFKDEDTQEVPFR VTEQESKPVQMMY QIGSFKVAVMASEKMKILELPYASGQLSMLVLLPDDVSGL EQLESAITSEKLMEWTSSTTMEERKMKVYLPRMKIEEKYN LTSVLMALGVTDLFSSSADLSGISSAESLKISKAVHEAFV EIYEAGSEVVGSTEGGMEVTSFSEEFRADHPFLFLIKHKP TNSILFFGRCFSP PREDICTED: SEQIDNO:223 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin[Fulmarus MVYLGARENTRAQIDKVVHFDKITGFGETIESQCGTSVSV glacialis] HTSLKDMFTQITKPSDNYSLSFASRLYAEETYPILPEYLQ CVKELYKGGLETTSFQTAADQARELINSWVESQTNGMIKN ILQPGSVDPQTEMVLVNAIYFKGMWEKAFKDEDTQAVPFR MTEQESKTVQMMYQIGSFKVAVMASEKMKILELPYASGEL SMLVMLPDDVSGLEQLETAITFEKLMEWTSSNMMEERKMK VYLPRMKMEEKYNLTSVLMALGVTDLFSSSANLSGISSAE SLKMSEAVHEAFVEIYEAGSEVVGSTGAGMEVTSVSEEFR ADHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:224 MGSIGAASTEFCFDVFKELRVQHVNENVCYSPLIIISALS Ovalbumin-like LVYLGARENTRAQIDKVVHFDKITGFGESIESQCGTSVSV [Chlamydotis HTSLKDMFNQITKPSDNYSLSVASRLYAEERYPILPEYLQ macqueenii] CVKELYKGGLESISFQTAADQAREAINSWVESQTNGMIKN ILQPSSVDPQTEMVLVNAIYFKGMWQKAFKDEDTQAVPFR ISEQESKPVQMMYQIGSFKVAVMAAEKMKILELPYASGEL SMLVLLPDEVSGLEQLENAITVEKLMEWTSSSPMEERIMK VYLPRMKIEEKYNLTSVLMALGITDLFSSSANLSGISAEE SLKMSEAVHQAFAEISEAGSEVVGSSEAGIDATSVSEEFR ADHPFLFLIKHNATNSILFFGRCFSP PREDICTED: SEQIDNO:225 MGSISAASTEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbuminlike MVYLGARENTRAQIEKVVHFDKITGFGESIESQCSTSVSV [Nipponianippon] HTSLKDMFTQITKPSDNYSLSFASRFYAEETYPILPEYLQ CVKELYKGGLETINFRTAADQARELINSWVESQTNGMIKN ILQPGSVDPQTDMVLVNAIYFKGMWEKAFKDEDTQALPFR VTEQESKPVQMMYQIGSFKVAVLASEKVKILELPYASGQL SMLVLLPDDVSGLEQLETAITVEKLMEWTSSNNMEERKIK VYLPRIKIEEKYNLTSVLMALGITDLFSSSANLSGISSAE SLKVSEAIHEAFVEIYEAGSEVAGSTEAGIEVTSVSEEFR ADHPFLFLIKHNATNSILFFGRCFSP PREDICTED: SEQIDNO:226 MVSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDKVVHFDKITGFEETIESQCSTSVSV isoformX2[Gavia HTSLKDMFTQITKPSDNYSLSFASRLYAEETYPILPEYLQ stellata] CVKELYKGGLETISFQTAADQARELINSWVESQTDGMIKN ILQPGSVDPQTEMVLVNAIYFKGMWEKAFKDEDTQAVPFR MTEQESKPVQMMYQIGSFKVAVMASEKMKILELPYASGGM SMLVMLPDDVSGLEQLETAITFEKLMEWTSSNMMEERKMK VYLPRMKMEEKYNLTSVLMALGMTDLFSSSANLSGISSAE SLKMSEAVHEAFVEIYEAGSEAVGSTGAGMEVTSVSEEFR ADHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:227 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin[Pelecanus MVYLGARENTRAQIDKVVHFDKITGFGEPIESQCGISVSV crispus] HTSLKDMITQITKPSDNYSLSFASRLYAEETYPILPEYLQ CVKELYKGGLETISFQTAADQARELINSWVENQTNGMIKN ILQPGSVDPQTEMVLVNAVYFKGMWEKAFKDEDTQAVPFR MTEQESKPVQMMYQIGSFKVAVMASEKIKILELPYASGEL SMLVLLPDDVSGLEQLETAITLDKLTEWTSSNAMEERKMK VYLPRMKIEKKYNLTSVLIALGMTDLFSSSANLSGISSAE SLKMSEAIHEAFLEIYEAGSEVVGSTEAGMEVTSVSEEFR ADHPFLFLIKHNPTNSILFFGRCLSP PREDICTED: SEQIDNO:228 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALS Ovalbumin-like MVYLGARENTRAQIDKVVHFDKIPGFGDTTESQCGTSVSV [Charadriusvociferus] HTSLKDMFTQITKPSDNYSVSFASRLYAEETYPILPEFLE CVKELYKGGLESISFQTAADQARELINSWVESQTNGMIKN ILQPGSVDSQTEMVLVNAIYFKGMWEKAFKDEDTQTVPFR MTEQETKPVQMMYQIGTFKVAVMPSEKMKILELPYASGEL CMLVMLPDDVSGLEELESSITVEKLMEWTSSNMMEERKMK VFLPRMKIEEKYNLTSVLMALGMTDLFSSSANLSGISSAE PLKMSEAVHEAFIEIYEAGSEVVGSTGAGMEITSVSEEFR ADHPFLFLIKHNPTNSILFFGRCVSP PREDICTED: SEQIDNO:229 MGSIGAVSTEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDKVVHFDKITGSGETIEAQCGTSVSV [Eurypygahelias] HTSLKDMFTQITKPSENYSVGFASRLYADETYPIIPEYLQ CVKELYKGGLEMISFQTAADQARELINSWVESQTNGMIKN ILQPGSVDPQTEMILVNAIYFKGVWEKAFKDEDTQAVPFR MTEQESKPVQMMYQFGSFKVAAMAAEKMKILELPYASGAL SMLVLLPDDVSGLEQLESAITFEKLMEWTSSNMMEEKKIK VYLPRMKMEEKYNFTSVLMALGMTDLFSSSANLSGISSAD SLKMSEVVHEAFVEIYEAGSEVVGSTGSGMEAASVSEEFR ADHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:230 MVSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDKVVHFDKITGFEETIESQVQKKQCS isoformX1[Gavia TSVSVHTSLKDMFTQITKPSDNYSLSFASRLYAEETYPIL stellata] PEYLQCVKELYKGGLETISFQTAADQARELINSWVESQTD GMIKNILQPGSVDPQTEMVLVNAIYFKGMWEKAFKDEDTQ AVPFRMTEQESKPVQMMYQIGSFKVAVMASEKMKILELPY ASGGMSMLVMLPDDVSGLEQLETAITFEKLMEWTSSNMME ERKMKVYLPRMKMEEKYNLTSVLMALGMTDLFSSSANLSG ISSAESLKMSEAVHEAFVEIYEAGSEAVGSTGAGMEVTSV SEEFRADHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:231 MGSIGAASGEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDKVVHFDKIIGFGESIESQCGTSVSV [Egrettagarzetta] HTSLKDMFAQITKPSDNYSLSFASRLYAEETFPILPEYLQ CVKELYKGGLETLSFQTAADQARELINSWVESQTNGMIKD ILQPGSVDPQTEMVLVNAIYFKGVWEKAFKDEDTQTVPFR MTEQESKPVQMMYQIGSFKVAVVAAEKIKILELPYASGAL SMLVLLPDDVSSLEQLETAITFEKLTEWTSSNIMEERKIK VYLPRMKIEEKYNLTSVLMDLGITDLFSSSANLSGISSAE SLKVSEAIHEAIVDIYEAGSEVVGSSGAGLEGTSVSEEFR ADHPFLFLIKHNPTSSILFFGRCFSP PREDICTED: SEQIDNO:232 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDKVVHFDKITGSGEAIESQCGTSVSV [Balearicaregulorum HISLKDMFTQITKPSDNYSLSFASRLYAEETYPILPEYLQ gibbericeps] CVKELYKEGLATISFQTAADQAREFINSWVESQTNGMIKN ILQPGSVDPQTQMVLVNAIYFKGVWEKAFKDEDTQAVPFR MTKQESKPVQMMYQIGSFKVAVMASEKMKILELPYASGQL SMLVMLPDDVSGLEQIENAITFEKLMEWTNPNMMEERKMK VYLPRMKMEEKYNLTSVLMALGMTDLFSSSANLSGISSAE SLKMSEAVHEAFVEIYEAGSEVVGSTGAGIEVTSVSEEFR ADHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:233 MGSIGEASTEFCIDVFRELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDQVVHFDKITGFGDTVESQCGSSLSV [Nestornotabilis] HSSLKDIFAQITQPKDNYSLNFASRLYAEETYPILPEYLQ CVKELYKGGLETISFQTAADQARELINSWVESQTNGMIKN ILQPSSVDPQTEMVLVNAIYFKGVWEKAFKDEETQAVPFR ITEQENRPVQIMYQFGSFKVAVVASEKIKILELPYASGQL SMLVLLPDEVSGLEQLENAITFEKLTEWTSSDIMEEKKIK VFLPRMKIEEKYNLTSVLVALGIADLFSSSANLSGISSAE SLKMSEAVHEAFVEIYEAGSEVVGSSGAGIEAASDSEEFR ADHPFLFLIKHKPTNSILFFGRCFSP PREDICTED: SEQIDNO:234 MGSIGAASTEFCFDIFNELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTKAQIDKVVHFDKITGFGESIESQCSTSASV [Pygoscelisadeliae] HTSFKDMFTQITKPSDNYSLSFASRLYAEETYPILPEYSQ CVKELYKGGLESISFQTAADQARELINSWVESQTNGMIKN ILQPGSVDPQTELVLVNAIYFKGTWEKAFKDKDTQAVPFR VTEQESKPVQMMYQIGSYKVAVIASEKMKILELPYASGEL SMLVLLPDDVSGLEQLETAITFEKLMEWTSSNMMEERKVK VYLPRMKIEEKYNLTSVLMALGMTDLFSPSANLSGISSAE SLKMSEAIHEAFVEIYEAGSEVVGSTEAGMEVTSVSEEFR ADHPFLFLIKCNLTNSILFFGRCFSP Ovalbumin-like SEQIDNO:235 MGSISTASTEFCFDVFKELKVQHVNENIFYSPLSIISALS [Athenecunicularia] MVYLGARENTRAQIEKVVHFDKITGFGESIESQCGTSVSV HTSLKDMLIQISKPSDNYSLSFASKLYAEETYPILPEYLQ CVKELYKGGLESINFQTAADQARQLINSWVESQTNGMIKD ILQPSSVDPQTEMVLVNAIYFKGIWEKAFKDEDTQEVPFR ITEQESKPVQMMYQIGSFKVAVIASEKIKILELPYASGEL SMLIVLPDDVSGLEQLETAITFEKLIEWTSPSIMEERKTK VYLPRMKIEEKYNLTSVLMALGMTDLFSPSANLSGISSAE SLKMSEAIHEAFVEIYEAGSEVVGSAEAGMEATSVSEFRV DHPFLFLIKHNPANIILFFGRCVSP PREDICTED: SEQIDNO:236 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALS Ovalbumin-like LVYLGARENTRAQIDKVFHFDKISGFGETTESQCGTSVSV [Calidrispugnax] HTSLKEMFTQITKPSDNYSVSFASRLYAEDTYPILPEYLQ CVKELYKGGLETISFQTAADQAREVINSWVESQTNGMIKN ILQPGSVDSQTEMVLVNAIYFKGMWEKAFKDEDTQTMPFR ITEQERKPVQMMYQAGSFKVAVMASEKMKILELPYASGEF CMLIMLPDDVSGLEQLENSFSFEKLMEWTTSNMMEERKMK VYIPRMKMEEKYNLTSVLMALGMTDLFSSSANLSGISSAE TLKMSEAVHEAFMEIYEAGSEVVGSTGSGAEVTGVYEEFR ADHPFLFLVKHKPTNSILFFGRCVSP PREDICTED: SEQIDNO:237 MGSIGAASTEFCFDIFNELKVQHVNENIFYSPLSIISALS Ovalbumin MVYLGARENTKAQIDKVVHFDKITGFGETIESQCSTSVSV [Aptenodytesforsteri] HTSLKDTFTQITKPSDNYSLSFASRLYAEETYPILPEYSQ CVKELYKGGLETISFQTAADQARELINSWVESQTNGMIKN ILQPGSVDPQTELVLVNAIYFKGTWEKAFKDKDTQAVPFR VTEQESKPVQMMYQIGSYKVAVIASEKMKILELPYASREL SMLVLLPDDVSGLEQLETAITFEKLMEWTSSNMMEERKVK VYLPRMKIEEKYNLTSVLMALGMTDLFSPSANLSGISSAE SLKMSEAVHEAFVEIYEAGSEVVGSTGAGMEVTSVSEEFR ADHPFLFLIKCNPTNSILFFGRCFSP PREDICTED: SEQIDNO:238 MGSISAASAEFCLDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDKVVHFDKITGSGETIEFQCGTSANI [Pteroclesgutturalis] HPSLKDMFTQITRLSDNYSLSFASRLYAEERYPILPEYLQ CVKELYKGGLETISFQTAADQARELINSWVESQTNGMIKN ILQPGSVNPQTEMVLVNAIYFKGLWEKAFKDEDTQTVPFR MTEQESKPVQMMYQVGSFKVAVMASDKIKILELPYASGEL SMLVLLPDDVTGLEQLETSITFEKLMEWTSSNVMEERTMK VYLPHMRMEEKYNLTSVLMALGVTDLFSSSANLSGISSAE SLKMSEAVHEAFVEIYESGSQVVGSTGAGTEVTSVSEEFR VDHPFLFLIKHNPTNSILFFGRCFSP Ovalbumin-like[Falco SEQIDNO:239 MGSIGAASVEFCFDVFKELKVQHVNENIFYSPLSIISALS peregrinus] MVYLGARENTKAQIDKVVHFDKIAGFGEAIESQCVTSASI HSLKDMFTQITKPSDNYSLSFASRLYAEEAYSILPEYLQC VKELYKGGLETISFQTAADQARDLINSWVESQTNGMIKNI LQPGAVDLETEMVLVNAIYFKGMWEKAFKDEDTQTVPFRM TEQESKPVQMMYQVGSFKVAVMASDKIKILELPYASGQLS MVVVLPDDVSGLEQLEASITSEKLMEWTSSSIMEEKKIKV YFPHMKIEEKYNLTSVLMALGMTDLFSSSANLSGISSAEK LKVSEAVHEAFVEISEAGSEVVGSTEAGTEVTSVSEEFKA DHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:240 MGSIGAASSEFCFDIFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDKVVPFDKITASGESIESQCSTSVSV isoformX2 HTSLKDIFTQITKSSDNHSLSFASRLYAEETYPILPEYLQ [Phalacrocoraxcarbo] CVKELYEGGLETISFQTAADQARELINSWIESQTNGRIKN ILQPGSVDPQTEMVLVNAIYFKGMWEKAFKDEDTQAVPFR MTEQESKPVQVMHQIGSFKVAVLASEKIKILELPYASGEL SMLVLLPDDVSGLEQLETAITFEKLMEWTSPNIMEERKIK VFLPRMKIEEKYNLTSVLMALGITDLFSPLANLSGISSAE SLKMSEAIHEAFVEISEAGSEVIGSTEAEVEVTNDPEEFR ADHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:241 MGSIGAASTEFCFDVFKELKAQYVNENIFYSPMTIITALS Ovalbumin-like MVYLGSKENTRAQIAKVAHFDKITGFGESIESQCGASASI [Meropsnubicus] QFSLKDLFTQITKPSGNHSLSVASRIYAEETYPILPEYLE CMKELYKGGLETINFQTAANQARELINSWVERQTSGMIKN ILQPSSVDSQTEMVLVNAIYFRGLWEKAFKVEDTQATPFR ITEQESKPVQMMHQIGSFKVAVVASEKIKILELPYASGRL TMLVVLPDDVSGLKQLETTITFEKLMEWTTSNIMEERKIK VYLPRMKIEEKYNLTSVLMALGLTDLFSSSANLSGISSAE SLKMSEAVHEAFVEIYEAGSEVVASAEAGMDATSVSEEFR ADHPFLFLIKDNTSNSILFFGRCFSP PREDICTED: SEQIDNO:242 MGSIGAASTEFCFDVFKELKGQHVNENIFFCPLSIVSALS Ovalbumin-like MVYLGARENTRAQIVKVAHFDKIAGFAESIESQCGTSVSI [Tauraco HTSLKDMFTQITKPSDNYSLNFASRLYAEETYPIIPEYLQ erythrolophus] CVKELYKGGLETISFQTAADQAREIINSWVESQTNGMIKN ILRPSSVHPQTELVLVNAVYFKGTWEKAFKDEDTQAVPFR ITEQESKPVQMMYQI GSFKVAAVTSEKMKILEVPYASGELSMLVLLPDDVSGLEQ LETAITAEKLIEWTSSTVMEERKLKVYLPRMKIEEKYNLT TVLTALGVTDLFSSSANLSGISSAQGLKMSNAVHEAFVEI YEAGSEVVGSKGEGTEVSSVSDEFKADHPFLFLIKHNPTN SIVFFGRCFSP PREDICTED: SEQIDNO:243 MGSIGAASTEFCFDVFKELKVHHVNENILYSPLAIISALS Ovalbumin-like MVYLGAKENTRDQIDKVVHFDKITGIGESIESQCSTAVSV [Cuculuscanorus] HTSLKDVFDQITRPSDNYSLAFASRLYAEKTYPILPEYLQ CVKELYKGGLETIDFQTAADQARQLINSWVEDETNGMIKN ILRPSSVNPQTKIILVNAIYFKGMWEKAFKDEDTQEVPFR ITEQETKSVQMMYQIGSFKVAEVVSDKMKILELPYASGKL SMLVLLPDDVYGLEQLETVITVEKLKEWTSSIVMEERITK VYLPRMKIMEKYNLTSVLTAFGITDLFSPSANLSGISSTE SLKVSEAVHEAFVEIHEAGSEVVGSAGAGIEATSVSEEFK ADHPFLFLIKHNPTNSILFFGRCFSP Ovalbumin SEQIDNO:244 MGSIGAASTEFCLDVFKELKVQHVNENIFYSPLSIISALS [Antrostomus MVYLGARENTRAQIDKVVHFDKITGFEDSIESQCGTSVSV carolinensis] HTSLKDMFTQITKPSDNYSVGFASRLYAAETYQILPEYSQ CVKELYKGGLETINFQKAADQATELINSWVESQTNGMIKN ILQPSSVDPQTQIFLVNAIYFKGMWQRAFKEEDTQAVPFR ISEKESKPVQMMYQIGSFKVAVIPSEKIKILELPYASGLL SMLVILPDDVSGLEQLENAITLEKLMQWTSSNMMEERKIK VYLPRMRMEEKYNLTSVFMALGITDLFSSSANLSGISSAE SLKMSDAVHEASVEIHEAGSEVVGSTGSGTEASSVSEEFR ADHPYLFLIKHNPTDSIVFFGRCFSP PREDICTED: SEQIDNO:245 MGSIGAASTEFCFDVFKELKFQHVDENIFYSPLTIISALS Ovalbumin-like MVYLGARENTRAQIDKVVHFDKIAGFEETVESQCGTSVSV [Opisthocomus HTSLKDMFAQITKPSDNYSLSFASRLYAEETYPILPEYLQ hoazin] CVKELYKGGLETISFQTAADQARDLINSWVESQTNGMIKN ILQPSSVGPQTELILVNAIYFKGMWQKAFKDEDTQEVPFR MTEQQSKPVQMMYQTGSFKVAVVASEKMKILALPYASGQL SLLVMLPDDVSGLKQLESAITSEKLIEWTSPSMMEERKIK VYLPRMKIEEKYNLTSVLMALGITDLFSPSANLSGISSAE SLKMSQAVHEAFVEIYEAGSEVVGSTGAGMEDSSDSEEFR VDHPFLFFIKHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:246 MGSIGPLSVEFCCDVFKELRIQHPRENIFYSPVTIISALS Ovalbumin-like MVYLGARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSI [Lepidothrixcoronata] HTSLKDIFTQITKPSDNYTVGIASRLYAEEKYPILPEYLQ CIKELYKGGLEPINFQTAAEQARELINSWVESQTNGMIKN ILQPSSVNPETDMVLVNAIYFKGLWEKAFKDEDIQTVPFR ITEQESKPVQMMFQIGSFRVAEITSEKIRILELPYASGQL SLWVLLPDDISGLEQLETAITFENLKEWTSSTKMEERKIK VYLPRMKIEEKYNLTSVLTSLGITDLFSSSANLSGISSAE SLKVSSAFHEASVEIYEAGSKVVGSTGAEVEDTSVSEEFR ADHPFLFLIKHNPSNSIFFFGRCFSP PREDICTED: SEQIDNO:247 MGSIGTASAEFCFDVFKELKVHHVNENIFYSPLSIISALS Ovalbumin[Struthio MVYLGARENTKTQMEKVIHFDKITGLGESMESQCGTGVSI camelusaustralis] HTALKDMLSEITKPSDNYSLSLASRLYAEQTYAILPEYLQ CIKELYKESLETVSFQTAADQARELINSWIESQTNGVIKN FLQPGSVDSQTELVLVNAIYFKGMWEKAFKDEDTQEVPFR ITEQESRPVQMMYQ AGSFKVATVAAEKIKILELPYASGELSMLVLLPDDISGLE QLETTISFEKLTEWTSSNMMEDRNMKVYLPRMKIEEKYNL TSVLIALGMTDLFSPAANLSGISAAESLKMSEAIHAAYVE IYEADSEIVSSAGVQVEVTSDSEEFRVDHPFLFLIKHNPT NSVLFFGRCISP PREDICTED: SEQIDNO:248 MGSIGAVSTEFSCDVFKELRIHHVQENIFYSPVTIISALS Ovalbumin-like MIYLGARDSTKAQIEKAVHFDKIPGFGESIESQCGTSLSI [Acanthisittachloris] HTSIKDMFTKITKASDNYSIGIASRLYAEEKYPILPEYLQ CVKELYKGGLESISFQTAAEQAREIINSWVESQTNGMIKN ILQPSSVDPQTDIVLVNAIYFKGLWEKAFRDEDTQTVPFK ITEQESKPVQMMYQIGSFKVAEITSEKIKILEVPYASGQL SLWVLLPDDISGLEKLETAITFENLKEWTSSTKMEERKIK VYLPRMKIEEKYNLTSVLTALGITDLFSSSANLSGISSAE SLKVSEAFHEAIVEISEAGSKVVGSVGAGVDDTSVSEEFR ADHPFLFLIKHNPTSSIFFFGRCFSP PREDICTED: SEQIDNO:249 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like[Tyto MVYLGARENTRAQIDKVVHFDKIAGFGESTESQCGTSVSA alba] HTSLKDMSNQITKLSDNYSLSFASRLYAEETYPILPEYSQ CVKELYKGGLESISFQTAAYQARELINAWVESQTNGMIKD ILQPGSVDSQTKMVLVNAIYFKGIWEKAFKDEDTQEVPFR MTEQETKPVQMMYQIGSFKVAVIAAEKIKILELPYASGQL SMLVILPDDVSGLEQLETAITFEKLTEWTSASVMEERKIK VYLPRMSIEEKYNLTSVLIALGVTDLFSSSANLSGISSAE SLRMSEAIHEAFVETYEAGSTESGTEVTSASEEFRVDHPF LFLIKHKPTNSILFFGRCFSP PREDICTED: SEQIDNO:250 MGSIGAASSEFCFDIFKELKVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDKVVPFDKITASGESIESQVQKIQCS isoformX1 TSVSVHTSLKDIFTQITKSSDNHSLSFASRLYAEETYPIL [Phalacrocoraxcarbo] PEYLQCVKELYEGGLETISFQTAADQARELINSWIESQTN GRIKNILQPGSVDPQTEMVLVNAIYFKGMWEKAFKDEDTQ AVPFRMTEQESKPVQVMHQIGSFKVAVLASEKIKILELPY ASGELSMLVLLPDDVSGLEQLETAITFEKLMEWTSPNIME ERKIKVFLPRMKIEEKYNLTSVLMALGITDLFSPLANLSG ISSAESLKMSEAIHEAFVEISEAGSEVIGSTEAEVEVIND PEEFRADHPFLFLIKHNPTNSILFFGRCFSP Ovalbumin-like[Pipra SEQIDNO:251 MGSIGPLSVEFCCDVFKELRIQHARENIFYSPVTIISALS filicauda] MVYLGARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSI HTSLKDIFTQITKPSDNYTVGIASRLYAEEKYPILPEYLQ CIKELYKGGLEPISFQTAAEQARELINSWVESQTNGIIKN ILQPSSVNPETDMVLVNAIYFKGLWEKAFKDEGTQTVPFR ITEQESKPVQMMFQIGSFRVAEIASEKIRILELPYASGQL SLWVLLPDDISGLEQLETAITFENLKEWTSSTKMEERKIK VYLPRMKIEEKYNLTSVLTSLGITDLFSSSANLSGISSAE RLKVSSAFHEASMEINEAGSKVVGAGVDDTSVSEEFRVDR PFLFLIKHNPSNSIFFFGRCFSP Ovalbumin[Dromaius SEQIDNO:252 MGSIGAASTEFCFDMFKELKVHHVNENIIYSPLSIISILS novaehollandiae] MVFLGARENTKTQMEKVIHFDKITGFGESLESQCGTSVSV HASLKDILSEITKPSDNYSLSLASKLYAEETYPVLPEYLQ CIKELYKGSLETVSFQTAADQARELINSWVETQTNGVIKN FLQPGSVDPQTEMVLVDAIYFKGTWEKAFKDEDTQEVPFR ITEQESKPVQMMYQAGSFKVATVAAEKMKILELPYASGEL SMFVLLPDDISGLEQLETTISIEKLSEWTSSNMMEDRKMK VYLPHMKIEEKYNLTSVLVALGMTDLFSPSANLSGISTAQ TLKMSEAIHGAYVEIYEAGSEMATSTGVLVEAASVSEEFR VDHPFLFLIKHNPSNSILFFGRCIFP ChainA,Ovalbumin SEQIDNO:253 MGSIGAASTEFCFDMFKELKVHHVNENIIYSPLSIISILS MVFLGARENTKTQMEKVIHFDKITGFGESLESQCGTSVSV HASLKDILSEITKPSDNYSLSLASKLYAEETYPVLPEYLQ CIKELYKGSLETVSFQTAADQARELINSWVETQTNGVIKN FLQPGSVDPQTEMVLVDAIYFKGTWEKAFKDEDTQEVPFR ITEQESKPVQMMYQAGSFKVATVAAEKMKILELPYASGEL SMFVLLPDDISGLEQLETTISIEKLSEWTSSNMMEDRKMK VYLPHMKIEEKYNLTSVLVALGMTDLFSPSANLSGISTAQ TLKMSEAIHGAYVEIYEAGSEMATSTGVLVEAASVSEEFR VDHPFLFLIKHNPSNSILFFGRCIFPHHHHHH Ovalbumin-like SEQIDNO:254 MGSIGPLSVEFCCDVFKELRIQHARENIFYSPVTIISALS [Corapipoaltera] MVYLGARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSI HTSLKDIFTQITKPSDNYTVGIASRLYAEEKYPILPEYLQ CIKELYKGGLEPISFQTAAEQARELINSWVESQTNGMIKN ILQPSAVNPETDMVLVNAIYFKGLWEKAFKDEGTQTVPFR ITEQESKPVQMMFQIGSFRVAEITSEKIRILELPYASGQL SLWVLLPDDISGLEQLETAITFENLKEWTSSTKMEERKIK VYLPRMKIEEKYNLTSVLTSLGITDLFSSSANLSGISSAE RLKVSSAFHEASMEIYEAGSKVVGSTGAGVDDTSVSEEFR VDRPFLFLIKHNPSNSIFFFGRCFSP Ovalbumin-like SEQIDNO:255 MEDQRGNTGFTMGSIGAASTEFCIDVFRELRVQHVNENIF protein[Amazona YSPLTIISALSMVYLGARENTRAQIDQVVHFDKIAGFGDT aestiva] VESQCGSSPSVHNSLKTVXAQITQPRDNYSLNLASRLYAE ESYPILPEYLQCVKELYNGGLETVSFQTAADQARELINSW VESQTNGIIKNILQPSSVDPQTEMVLVNAIYFKGLWEKAF KDEETQAVPFRITEQENRPVQMMYQFGSFKVAXVASEKIK ILELPYASGQLSMLVLLPDEVSGLEQNAITFEKLTEWTSS DLMEERKIKVFFPRVKIEEKYNLTAVLVSLGITDLFSSSA NLSGISSAENLKMSEAVHEAXVEIYEAGSEVAGSSGAGIE VASDSEEFRVDHPFLFLIXHNPTNSILFFGRCFSP PREDICTED: SEQIDNO:256 MGSIGAASTEFCIDVFRELRVQHVNENIFYSPLSIISALS Ovalbumin-like MVYLGARENTRAQIDEVFHFDKIAGFGDTVDPQCGASLSV [Melopsittacus HKSLQNVFAQITQPKDNYSLNLASRLYAEESYPILPEYLQ undulatus] CVKELYNEGLETVSFQTGADQARELINSWVENQTNGVIKN ILQPSSVDPQTEMVLVNAIYFKGLWQKAFKDEETQAVPFR ITEQENRPVQMMYQFGSFKVAVVASEKVKILELPYASGQL SMWVLLPDEVSGLEQLENAITFEKLTEWTSSDLTEERKIK VFLPRVKIEEKYNLTAVLMALGVTDLFSSSANFSGISAAE NLKMSEAVHEAFVEIYEAGSEVVGSSGAGIEAPSDSEEFR ADHPFLFLIKHNPTNSILFFGRCFSP Ovalbumin-like SEQIDNO:257 MGSIGPLSVEFCCDVFKELRIQHARDNIFYSPVTIISALS [Neopelma MVYLGARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSV chrysocephalum] HTSLKDIFTQITKPRENYTVGIASRLYAEEKYPILPEYLQ CIKELYKGGLEPISFQTAAEQARELINSWVESQTNGMIKN ILQPSSVNPETDMVLVNAIYFKGLWKKAFKDEGTQTVPFR ITEQESKPVQMMFQIGSFRVAEITSEKIRILELPYASGQL SLWVLLPDDISGLEQLESAITFENLKEWTSSTKMEERKIK VYLPRMKIEEKYNLTSVLTSLGITDLFSSSANLSGISSAE KLKVSSAFHEASMEIYEAGNKVVGSTGAGVDDTSVSEEFR VDRPFLFLIKHNPSNSIFFFGRCFSP PREDICTED: SEQIDNO:258 MGSIGAASAEFCVDVFKELKDQHVNNIVFSPLMIISALSM Ovalbumin-like VNIGAREDTRAQIDKVVHFDKITGYGESIESQCGTSIGIY [Bucerosrhinoceros FSLKDAFTQITKPSDNYSLSFASKLYAEETYPILPEYLKC silvestris] VKELYKGGLETISFQTAADQARELINSWVESQTNGMIKNI LQPSSVDPQTEMVLVNAIYFKGLWEKAFKDEDTQAVPFRI TEQESKPVQMMYQIGSFKVAVIASEKIKILELPYASGQLS LLVLLPDDVSGLEQLESAITSEKLLEWTNPNIMEERKTKV YLPRMKIEEKYNLTSVLVALGITDLFSSSANLSGISSAEG LKLSDAVHEAFVEIYEAGREVVGSSEAGVEDSSVSEEFKA DRPFIFLIKHNPTNGILYFGRYISP PREDICTED: SEQIDNO:259 MGSIGAANTDFCFDVFKELKVHHANENIFYSPLSIVSALA Ovalbumin-like MVYLGARENTRAQIDKALHFDKILGFGETVESQCDTSVSV [Cariamacristata] HTSLKDMLIQITKPSDNYSFSFASKIYTEETYPILPEYLQ CVKELYKGGVETISFQTAADQAREVINSWVESHTNGMIKN ILQPGSVDPQTKMVLVNAVYFKGIWEKAFKEEDTQEMPFR INEQESKPVQMMYQIGSFKLTVAASENLKILEFPYASGQL SMMVILPDEVSGLKQLETSITSEKLIKWTSSNTMEERKIR VYLPRMKIEEKYNLKSVLMALGITDLFSSSANLSGISSAE SLKMSEAVHEAFVEIYEAGSEVTSSTGTEMEAENVSEEFK ADHPFLFLIKHNPTDSIVFFGRCMSP Ovalbumin[Manacus SEQIDNO:260 MGSIGPLSVEFCCDVFKELRIQHARENIFYSPVTIISALS vitellinus] MVYLGARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSI HTSLKDIFTQITKPSDNYTVGIASRLYAEEKYPILPEYLQ CIKELYKGGLEPISFQTAAEQARELINSWVESQTNGMIKN ILQPSSVNPETDMVLVNAIYFKGLWEKAFKDESTQTVPFR ITEQESKPVQMMFQIGSFRVAEIASEKIRILELPYASGQL SLWVLLPDDISGLEQLETAITFENLKEWTSSTKMEERKIK VYLPRMKIEEKYNLTSVLTSLGITDLFSSSANLSGISSAE RLKVSSAFHEASMEIYEAGSRVVEAGVDDTSVSEEFRVDR PFLFLIKHNPSNSIFFFGRCFSP Ovalbumin-like SEQIDNO:261 MGSIGPVSTEFCCDIFKELRIQHARENIIYSPVTIISALS [Empidonaxtraillii] MVYLGARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSI HTSLKDILTQITKPSDNYTVGIASRLYAEEKYPILSEYLQ CIKELYKGGLEPISFQTAAEQARELINSWVESQTNGMIKN ILQPSSVNPETDMVLVNAIYFKGLWEKAFKDEGTQTVPFR ITEQESKPVQMMFQIGSFKVAEITSEKIRILELPYASGKL SLWVLLPDDISGLEQLETAITFENLKEWTSSTRMEERKIK VYLPRMKIEEKYNLTSVLTSLGITDLFSSSANLSGISSAE RLKVSSAFHEVFVEIYEAGSKVEGSTGAGVDDTSVSEEFR ADHPFLFLVKHNPSNSIIFFGRCYLP PREDICTED: SEQIDNO:262 MGSTGAASMEFCFALFRELKVQHVNENIFFSPVTIISALS Ovalbumin-like MVYLGARENTRAQLDKVAPFDKITGFGETIGSQCSTSASS [Leptosomusdiscolor] HTSLKDVFTQITKASDNYSLSFASRLYAEETYPILPEYLQ CVKELYKGGLESISFQTAADQARELINSWVESQTNGMIKD ILRPSSVDPQTKIILITAIYFKGMWEKAFKEEDTQAVPFR MTEQESKPVQMMYQIGSFKVAVIPSEKLKILELPYASGQL SMLVILPDDVSGLEQLETAITTEKLKEWTSPSMMKERKMK VYFPRMRIEEKYNLTSVLMALGITDLFSPSANLSGISSAE SLKVSEAVHEASVDIDEAGSEVIGSTGVGTEVTSVSEEIR ADHPFLFLIKHKPTNSILFFGRCFSP Hypotheticalprotein SEQIDNO:263 MEHAQLTQLVNSNMTSNTCHEADEFENIDFRMDSISVTNT H355_008077 KFCFDVFNEMKVHHVNENILYSPLSILTALAMVYLGARGN [Colinusvirginianus] TESQMKKALHFDSITGAGSTTDSQCGSSEYIHNLFKEFLT EITRTNATYSLEIADKLYVDKTFTVLPEYINCARKFYTGG VEEVNFKTAAEEARQLINSWVEKETNGQIKDLLVPSSVDF GTMMVFINTIYFKGIWKTAFNTEDTREMPFSMTKQESKPV QMMCLNDTFNMATLPAEKMRILELPYASGELSMLVLLPDE VSGLEQIEKAINFEKLREWTSTNAMEKKSMKVYLPRMKIE EKYNLTSTLMALGMTDLFSRSANLTGISSVENLMISDAVH GAFMEVNEEGTEAAGSTGAIGNIKHSVEFEEFRADHPFLF LIRYNPTNVILFFDNSEFTMGSIGAVSTEFCFDVFKELRV HHANENIFYSPFTVISALAMVYLGAKDSTRTQINKVVRFD KLPGFGDSIEAQCGTSANVHSSLRDILNQITKPNDIYSFS LASRLYADETYTILPEYLQCVKELYRGGLESINFQTAADQ ARELINSWVESQTSGIIRNVLQPSSVDSQTAMVLVNAIYF KGLWEKGFKDEDTQAMPFRVTEQENKSVQMMYQIGTFKVA SVASEKMKILELPFASGTMSMWVLLPDEVSGLEQLETTIS IEKLTEWTSSSVMEERKIKVFLPRMKMEEKYNLTSVLMAM GMTDLFSSSANLSGISSTLQKKGFRSQELGDKYAKPMLES PALTPQVTAWDNSWIVAHPAAIEPDLCYQIMEQKWKPFDW PDFRLPMRVSCRFRTMEALNKANTSFALDFFKHECQEDDD ENILFSPFSISSALATVYLGAKGNTADQMAKTEIGKSGNI HAGFKALDLEINQPTKNYLLNSVNQLYGEKSLPFSKEYLQ LAKKYYSAEPQSVDFLGKANEIRREINSRVEHQTEGKIKN LLPPGSIDSLTRLVLVNALYFKGNWATKFEAEDTRHRPFR INMHTTKQVPMMYLRDKFNWTYVESVQTDVLELPYVNNDL SMFILLPRDITGLQKLINELTFEKLSAWTSPELMEKMKME VYLPRFTVEKKYDMKSTLSKMGIEDAFTKVDSCGVTNVDE ITTHIVSSKCLELKHIQINKKLKCNKAVAMEQVSASIGNF TIDLFNKLNETSRDKNIFFSPWSVSSALALTSLAAKGNTA REMAEDPENEQAENIHSGFKELMTALNKPRNTYSLKSANR IYVEKNYPLLPTYIQLSKKYYKAEPYKVNFKTAPEQSRKE INNWVEKQTERKIKNFLSSDDVKNSTKSILVNAIYFKAEW EEKFQAGNTDMQPFRMSKNKSKLVKMMYMRHTFPVLIMEK LNFKMIELPYVKRELSMFILLPDDIKDSTTGLEQLERELT YEKLSEWADSKKMSVTLVDLHLPKFSMEDRYDLKDALKSM GMASAFNSNADFSGMTGFQAVPMESLSASTNSFTLDLYKK LDETSKGQNIFFASWSIATALAMVHLGAKGDTATQVAKGP EYEETENIHSGFKELLSAINKPRNTYLMKSANRLFGDKTY PLLPKFLELVARYYQAKPQAVNFKTDAEQARAQINSWVEN ETESKIQNLLPAGSIDSHTVLVLVNAIYFKGNWEKRFLEK DTSKMPFRLSKTETKPVQMMFLKDTFLIHHERTMKFKIIE LPYVGNELSAFVLLPDDISDNTTGLELVERELTYEKLAEW SNSASMMKAKVELYLPKLKMEENYDLKSVLSDMGIRSAFD PAQADFTRMSEKKDLFISKVIHKAFVEVNEEDRIVQLASG RLTGRCRTLANKELSEKNRTKNLFFSPFSISSALSMILLG SKGNTEAQIAKVLSLSKAEDAHNGYQSLLSEINNPDTKYI LRTANRLYGEKTFEFLSSFIDSSQKFYHAGLEQTDFKNAS EDSRKQINGWVEEKTEGKIQKLLSEGIINSMTKLVLVNAI YFKGNWQEKFDKETTKEMPFKINKNETKPVQMMFRKGKYN MTYIGDL ETTVLEIPYVDNELSMIILLPDSIQDESTGLEKLERELTY EKLMDWINPNMMDSTEVRVSLPRFKLEENYELKPTLSTMG MPDAFDLRTADFSGISSGNELVLSEVVHKSFVEVNEEGTE AAAATAGIMLLRCAMIVANFTADHPFLFFIRHNKTNSILF CGRFCSP PREDICTED: SEQIDNO:264 MGSIGTASTEFCFDMFKEMKVQHANQNIIFSPLTIISALS Ovalbuminisoform MVYLGARDNTKAQMEKVIHFDKITGFGESVESQCGTSVSI X2[Apteryxaustralis HTSLKDMLSEITKPSDNYSLSLASRLYAEETYPILPEYLQ mantelli] CMKELYKGGLETVSFQTAADQARELINSWVESQTNGVIKN FLQPGSVDPQTEMVLVNAIYFKGMWEKAFKDEDTQEVPFR ITEQESKPVQMMYQVGSFKVATVAAEKMKILEIPYTHREL SMFVLLPDDISGLEQLETTISFEKLTEWTSSNMMEERKVK VYLPHMKIEEKYNLTSVLMALGMTDLFSPSANLSGISTAQ TLMMSEAIHGAYVEIYEAGREMASSTGVQVEVTSVLEEVR ADKPFLFFIRHNPTNSMVVFGRYMSP Hypotheticalprotein SEQIDNO:265 MTSNTCHEADEFEN ASZ78_006007 IDFRMDSISVTNTKFCFDVFNEMKVHHVNENILYSPLSIL [Callipeplasquamata] TALAMVYLGARGNTESQMKKALHFDSITGGGSTTDSQCGS SEYIHNLFKEFLTEITRTNATYSLEIADKLYVDKTFTVLP EYINCARKFYTGGVEEVNFKTAAEEARQLMNSWVEKETNG QIKDLLVPSSVDFGTMMVFINTIYFKGIWKTAFNTEDTRE MPFSMTKQESKPVQMMCLNDTFNMVTLPAEKMRILELPYA SGELSMLVLLPDEVSGLERIEKAINFEKLREWTSTNAMEK KSMKVYLPRMKIEEKYNLTSTLMALGMTDLFSRSANLTGI SSVDNLMISDAVHGAFMEVNEEGTEAAGSTGAIGNIKHSV EFEEFRADHPFLFLIRYNPTNVILFFDNSEFTMGSIGAVS TEFCFDVFKELRVHHANENIFYSPFTIISALAMVYLGAKD STRTQINKVVRFDKLPGFGDSIEAQCGTSANVHSSLRDIL NQITKPNDIYSFSLASRLYADETYTILPEYLQCVKELYRG GLESINFQTAADQARELINSWVESQTSGIIRNVLQPSSVD SQTAMVLVNAIYFKGLWEKGFKDEDTQAIPFRVTEQENKS VQMMYQIGTFKVASVASEKMKILELPFASGTMSMWVLLPD EVSGLEQLETTISIEKLTEWTSSSVMEERKIKVFLPRMKM EEKYNLTSVLMAMGMTDLFSSSANLSGISSTLQKKGFRSQ ELGDKYAKPMLESPALTPQATAWDNSWIVAHPPAIEPDLY YQIMEQKWKPFDWPDFRLPMRVSCRFRTMEALNKANTSFA LDFFKHECQEDDSENILFSPFSISSALATVYLGAKGNTAD QMAKVLHFNEAEGARNVTTTIRMQVYSRTDQQRLNRRACF QKTEIGKSGNIHAGFKGLNLEINQPTKNYLLNSVNQLYGE KSLPFSKEYLQLAKKYYSAEPQSVDFVGTANEIRREINSR VEHQTEGKIKNLLPPGSIDSLTRLVLVNALYFKGNWATKF EAEDTRHRPFRINTHTTKQVPMMYLSDKFNWTYVESVQTD VLELPYVNNDLSMFILLPRDITGLQKLINELTFEKLSAWT SPELMEKMKMEVYLPRFTVEKKYDMKSTLSKMGIEDAFTK VDNCGVTNVDEITIHVVPSKCLELKHIQINKELKCNKAVA MEQVSASIGNFTIDLFNKLNETSRDKNIFFSPWSVSSALA LTSLAAKGNTAREMAEDPENEQAENIHSGFNELLTALNKP RNTYSLKSANRIYVEKNYPLLPTYIQLSKKYYKAEPHKVN FKTAPEQSRKEINNWVEKQTERKIKNFLSSDDVKNSTKLI LVNAIYFKAEWEEKFQAGNTDMQPFRMSKNKSKLVKMMYM RHTFPVLIMEKLNFKMIELPYVKRELSMFILLPDDIKDST TGLEQLERELTYEKLSEWADSKKMSVTLVDLHLPKFSMED RYDLKDALRSMGMASAFNSNADFSG MTGERDLVISKVCHQSFVAVDEKGTEAAAATAVIAEAVPM ESLSASTNSFTLDLYKKLDETSKGQNIFFASWSIATALTM VHLGAKGDTATQVAKGPEYEETENIHSGFKELLSALNKPR NTYSMKSANRLFGDKTYPLLPTKTKPVQMMFLKDTFLIHH ERTMKFKIIELPYMGNELSAFVLLPDDISDNTTGLELVER ELTYEKLAEWSNSASMMKVKVELYLPKLKMEENYDLKSAL SDMGIRSAFDPAQADFTRMSEKKDLFISKVIHKAFVEVNE EDRIVQLASGRLTGNTEAQIAKVLSLSKAEDAHNGYQSLL SEINNPDTKYILRTANRLYGEKTFEFLSSFIDSSQKFYHA GLEQTDFKNASEDSRKQINGWVEEKTEGKIQKLLSEGIIN SMTKLVLVNAIYFKGNWQEKFDKETTKEMPFKINKNETKP VQMMFRKGKYNMTYIGDLETTVLEIPYVDNELSMIILLPD SIQDESTGLEKLERELTYEKLMDWINPNMMDSTEVRVSLP RFKLEENYELKPTLSTMGMPDAFDLRTADFSGISSGNELV LSEVVHKSFVEVNEEGTEAAAATAGIMLLRCAMIVANFTA DHPFLFFIRHNKTNSILFCGRFCSP PREDICTED: SEQIDNO:266 MASIGAASTEFCFDVFKELKTQHVKENIFYSPMAIISALS Ovalbumin-like MVYIGARENTRAEIDKVVHFDKITGFGNAVESQCGPSVSV [Mesitornisunicolor] HSSLKDLITQISKRSDNYSLSYASRIYAEETYPILPEYLQ CVKEVYKGGLESISFQTAADQARENINAWVESQTNGMIKN ILQPSSVNPQTEMVLVNAIYLKGMWEKAFKDEDTQTMPFR VTQQESKPVQMMYQIGSFKVAVIASEKMKILELPYTSGQL SMLVLLPDDVSGLEQVESAITAEKLMEWTSPSIMEERTMK VYLPRMKMVEKYNLTSVLMALGMTDLFTSVANLSGISSAQ GLKMSQAIHEAFVEIYEAGSEAVGSTGVGMEITSVSEEFK ADLSFLFLIRHNPTNSIIFFGRCISP Ovalbumin,partial SEQIDNO:267 MGSIGAASTEFCFDVFRELRVQHVNENIFYSPFSIISALA [Anasplatyrhynchos] MVYLGARDNTRTQIDKISQFQALSDEHLVLCIQQLGEFFV CTNRERREVTRYSEQTEDKTQDQNTGQIHKIVDTCMLRQD ILTQITKPSDNFSLSFASRLYAEETYAILPEYLQCVKELY KGGLESISFQTAADQARELINSWVESQTNGIIKNILQPSS VDSQTTMVLVNAIYFKGMWEKAFKDEDTQAMPFRMTEQES KPVQMMYQVGSFKVAMVTSEKMKILELPFASGMMSMFVLL PDEVSGLEQLESTISFEKLTEWTSSTMMEERRMKVYLPRM KMEEKYNLTSVFMALGMTDLFSSSANMSGISSTVSLKMSE AVHAACVEIFEAGRDVVGSAEAGMDVTSVSEEFRADHPFL FFIKHNPTNSILFFGRWMSP PREDICTED: SEQIDNO:268 MGSIGAASAEFCLDIFKELKVQHVNENIIFSPMTIISALS Ovalbumin-like LVYLGAKEDTRAQIEKVVPFDKIPGFGEIVESQCPKSASV [Chaeturapelagica] HSSIQDIFNQIIKRSDNYSLSLASRLYAEESYPIRPEYLQ CVKELDKEGLETISFQTAADQARQLINSWVESQTNGMIKN ILQPSSVNSQTEMVLVNAIYFRGLWQKAFKDEDTQAVPFR ITEQESKPVQMMQQIGSFKVAEIASEKMKILELPYASGQL SMLVLLPDDVSGLEKLESSITVEKLIEWTSSNLTEERNVK VYLPRLKIEEKYNLTSVLAALGITDLFSSSANLSGISTAE SLKLSRAVHESFVEIQEAGHEVEGPKEAGIEVTSALDEFR VDRPFLFVTKHNPTNSILFLGRCLSP PREDICTED: SEQIDNO:269 MGSISAASGEFCLDIFKELKVQHVNENIFYSPMVIVSALS Ovalbumin-like LVYLGARENTRAQIDKVIPFDKITGSSEAVESQCGTPVGA [Apalodermavittatum] HISLKDVFAQIAKRSDNYSLSFVNRLYAEETYPILPEYLQ CVKELYKGGLETISFQTAADQAREIINSWVESQTDGKIKN ILQPSSVDPQTKMVLVSAIYFKGLWEKSFKDEDTQAVPFR VTEQESKPVQMMYQIGSFKVAAIAAEKIKILELPYASEQL SMLVLLPDDVSGLEQLEKKISYEKLTEWTSSSVMEEKKIK VYLPRMKIEEKYNLTSILMSLGITDLFSSSANLSGISSTK SLKMSEAVHEASVEIYEAGSEASGITGDGMEATSVFGEFK VDHPFLFMIKHKPTNSILFFGRCISP Ovalbumin-like SEQIDNO:270 MGSIGPVSTEVCCDIFRELRSQSVQENVCYSPLLIISTLS [Corvuscornixcornix] MVYIGAKDNTKAQIEKAIHFDKIPGFGESTESQCGTSVSI HTSLKDIFTQITKPSDNYSISIARRLYAEEKYPILPEYIQ CVKELYKGGLESISFQTAAEKSRELINSWVESQTNGTIKN ILQPSSVSSQTDMVLVSAIYFKGLWEKAFKEEDTQTIPFR ITEQESKPVQMMSQIGTFKVAEIPSEKCRILELPYASGRL SLWVLLPDDISGLEQLETAITFENLKEWTSSSKMEERKIR VYLPRMKIEEKYNLTSVLKSLGITDLFSSSANLSGISSAE SLKVSAAFHEASVEIYEAGSKGVGSSEAGVDGTSVSEEIR ADHPFLFLIKHNPSDSILFFGRCFSP PREDICTED: SEQIDNO:271 MGSIGAASTEFCFDVFKELKVQHVNENIIISPLSIISALS Ovalbumin-like MVYLGAREDTRAQIDKVVHFDKITGFGEAIESQCPTSESV [Calypteanna] HASLKETFSQLTKPSDNYSLAFASRLYAEETYPILPEYLQ CVKELYKGGLETINFQTAAEQARQVINSWVESQTDGMIKS LLQPSSVDPQTEMILVNAIYFRGLWERAFKDEDTQELPFR ITEQESKPVQMMSQIGSFKVAVVASEKVKILELPYASGQL SMLVLLPDDVSGLEQLESSITVEKLIEWISSNTKEERNIK VYLPRMKIEEKYNLTSVLVALGITDLFSSSANLSGISSAE SLKISEAVHEAFVEIQEAGSEVVGSPGPEVEVTSVSEEWK ADRPFLFLIKHNPTNSILFFGRYISP PREDICTED: SEQIDNO:272 MGSIGPVSTEVCCDIFRELRSQSVQENVCYSPLLIISTLS Ovalbumin[Corvus MVYIGAKDNTKAQIEKAIHFDKIPGFGESTESQCGTSVSI brachyrhynchos] HTSLKDIFTQITKPSDNYSISIARRLYAEEKYPILQEYIQ CVKELYKGGLESISFQTAAEKSRELINSWVESQTNGTIKN ILQPSSVSSQTDMVLVSAIYFKGLWEKAFKEEDTQTIPFR ITEQESKPVQMMSQIGTFKVAEIPSEKCRILELPYASGRL SLWVLLPDDISGLEQLETSITFENLKEWTSSSKMEERKIR VYLPRMKIEEKYNLTSVLKSLGITDLFSSSANLSGISSAE SLKVSAVFHEASVEIYEAGSKGVGSSEAGVDGTSVSEEIR ADHPFLFLIKHNPSDSILFFGRCFSP Hypotheticalprotein SEQIDNO:273 MLNLMHPKQFCCTMGSIGPVSTEVCCDIFRELRSQSVQEN DUI87_08270 VCYSPLLIISTLSMVYIGAKDNTKAQIEKAIHFDKIPGFG [Hirundorustica ESTESQCGTSVSIHTSLKDIFTQITKPSDNYSISIASRLY rustica] AEEKYPILPEYIQCVKELYKGGLESISFQTAAEKSRELIN SWVESQTNGTIKNILQPSSVSSQTDMVLVSAIYFKGLWEK AFKEEDTQTVPFRITEQESKPVQMMSQIGTFKVAEIPSEK CRILELPYASGRLSLWVLLPDDISGLEQLETAITSENLKE WTSSSKMEERKIKVYLPRMKIEEKYNLTSVLKSLGITDLF SSSANLSGISSAESLKVSGAFHEAFVEIYEAGSKAVGSSG AGVEDTSVSEEIRADHPFLFFIKHNPSDSILFFGRCFSP OstrichOVA SEQIDNO:274 EAEAGSIGTASAEFCFDVFKELKVHHVNENIFYSPLSIIS sequenceassecreted ALSMVYLGARENTKTQMEKVIHFDKITGLGESMESQCGTG frompichia VSIHTALKDMLSEITKPSDNYSLSLASRLYAEQTYAILPE YLQCIKELYKESLETVSFQTAADQARELINSWIESQTNGV IKNFLQPGSVDSQTELVLVNAIYFKGMWEKAFKDEDTQEV PFRITEQESRPVQMMYQAGSFKVATVAAEKIKILELPYAS GELSMLVLLPDDISGLEQLETTISFEKLTEWTSSNMMEDR NMKVYLPRMKIEEKYNLTSVLIALGMTDLFSPAANLSGIS AAESLKMSEAIHAAYVEIYEADSEIVSSAGVQVEVTSDSE EFRVDHPFLFLIKHNPTNSVLFFGRCISP Ostrichconstruct SEQIDNO:275 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG (secretionsignal YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV matureprotein) SLEKREAEAGSIGTASAEFCFDVFKELKVHHVNENIFYSP LSIISALSMVYLGARENTKTQMEKVIHFDKITGLGESMES QCGTGVSIHTALKDMLSEITKPSDNYSLSLASRLYAEQTY AILPEYLQCIKELYKESLETVSFQTAADQARELINSWIES QTNGVIKNFLQPGSVDSQTELVLVNAIYFKGMWEKAFKDE DTQEVPFRITEQESRPVQMMYQAGSFKVATVAAEKIKILE LPYASGELSMLVLLPDDISGLEQLETTISFEKLTEWTSSN MMEDRNMKVYLPRMKIEEKYNLTSVLIALGMTDLFSPAAN LSGISAAESLKMSEAIHAAYVEIYEADSEIVSSAGVQVEV TSDSEEFRVDHPFLFLIKHNPTNSVLFFGRCISP DuckOVAsequence SEQIDNO:276 EAEAGSIGAASTEFCFDVFRELRVQHVNENIFYSPFSIIS assecretedfrompichia ALAMVYLGARDNTRTQIDKVVHFDKLPGFGESMEAQCGTS VSVHSSLRDILTQITKPSDNFSLSFASRLYAEETYAILPE YLQCVKELYKGGLESISFQTAADQARELINSWVESQINGI IKNILQPSSVDSQTTMVLVNAIYFKGMWEKAFKDEDTQAM PFRMTEQESKPVQMMYQVGSFKVAMVTSEKMKILELPFAS GMMSMFVLLPDEVSGLEQLESTISFEKLTEWTSSTMMEER RMKVYLPRMKMEEKYNLTSVFMALGMTDLFSSSANMSGIS STVSLKMSEAVHAACVEIFEAGRDVVGSAEAGMDVTSVSE EFRADHPFLFFIKHNPTNSILFFGRWMSP Duckconstruct SEQIDNO:277 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG (secretionsignal YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV matureprotein) SLEKREAEAGSIGAASTEFCFDVFRELRVQHVNENIFYSP FSIISALAMVYLGARDNTRTQIDKVVHFDKLPGFGESMEA QCGTSVSVHSSLRDILTQITKPSDNFSLSFASRLYAEETY AILPEYLQCVKELYKGGLESISFQTAADQARELINSWVES QTNGIIKNILQPSSVDSQTTMVLVNAIYFKGMWEKAFKDE DTQAMPFRMTEQESKPVQMMYQVGSFKVAMVTSEKMKILE LPFASGMMSMFVLLPDEVSGLEQLESTISFEKLTEWTSST MMEERRMKVYLPRMKMEEKYNLTSVFMALGMTDLFSSSAN MSGISSTVSLKMSEAVHAACVEIFEAGRDVVGSAEAGMDV TSVSEEFRADHPFLFFIKHNPTNSILFFGRWMSP OvoglobulinG2 SEQIDNO:278 TRAPDCGGILTPLGLSYLAEVSKPHAEVVLRQDLMAQRAS DLFLGSMEPSRNRITSVKVADLWLSVIPEAGLRLGIEVEL RIAPLHAVPMPVRISIRADLHVDMGPDGNLQLLTSACRPT VQAQSTREAESKSSRSILDKVVDVDKLCLDVSKLLLFPNE QLMSLTALFPVTPNCQLQYLPLAAPVFSKQGIALSLQTTF QVAGAVVPVPVSPVPFSMPELASTSTSHLILALSEHFYTS LYFTLERAGAFNMTIPSMLTTATLAQKITQVGSLYHEDLP ITLSAALRSSPRVVLEEGRAALKLFLTVHIGAGSPDFQSF LSVSADVTAGLQLSVSDTRMMISTAVIEDAELSLAASNVG LVRAALLEELFLAPVCQQVPAWMDDVLREGVHLPHLSHFT YTDVNVVVHKDYVLVPCKLKLRSTMA* OvoglobulinG3 SEQIDNO:279 MDSISVTNAKFCFDVFNEMKVHHVNENILYCPLSILTALA MVYLGARGNTESQMKKVLHFDSITGAGSTTDSQCGSSEYV HNLFKELLSEITRPNATYSLEIADKLYVDKTFSVLPEYLS CARKFYTGGVEEVNFKTAAEEARQLINSWVEKETNGQIKD LLVSSSIDFGTTMVFINTIYFKGIWKIAFNTEDTREMPFS MTKEESKPVQMMCMNNSFNVATLPAEKMKILELPYASGDL SMLVLLPDEVSGLERIEKTINFDKLREWTSTNAMAKKSMK VYLPRMKIEEKYNLTSILMALGMTDLFSRSANLTGISSVD NLMISDAVHGVFMEVNEEGTEATGSTGAIGNIKHSLELEE FRADHPFLFFIRYNPTNAILFFGRYWSP* -ovomucin SEQIDNO:280 CSTWGGGHFSTFDKYQYDFTGTCNYIFATVCDESSPDFNI QFRRGLDKKIARIIIELGPSVIIVEKDSISVRSVGVIKLP YASNGIQIAPYGRSVRLVAKLMEMELVVMWNNEDYLMVLT EKKYMGKTCGMCGNYDGYELNDFVSEGKLLDTYKFAALQK MDDPSEICLSEEISIPAIPHKKYAVICSQLLNLVSPTCSV PKDGFVTRCQLDMQDCSEPGQKNCTCSTLSEYSRQCAMSH QVVFNWRTENFCSVGKCSANQIYEECGSPCIKTCSNPEYS CSSHCTYGCFCPEGTVLDDISKNRTCVHLEQCPCTLNGET YAPGDTMKAACRTCKCTMGQWNCKELPCPGRCSLEGGSFV TTFDSRSYRFHGVCTYILMKSSSLPHNGTLMAIYEKSGYS HSETSLSAIIYLSTKDKIVISQNELLTDDDELKRLPYKSG DITIFKQSSMFIQMHTEFGLELVVQTSPVFQAYVKVSAQF QGRTLGLCGNYNGDTTDDFMTSMDITEGTASLFVDSWRAG NCLPAMERETDPCALSQLNKISAETHCSILTKKGTVFETC HAVVNPTPFYKRCVYQACNYEETFPYICSALGSYARTCSS MGLILENWRNSMDNCTITCTGNQTFSYNTQACERTCLSLS NPTLECHPTDIPIEGCNCPKGMYLNHKNECVRKSHCPCYL EDRKYILPDQSTMTGGITCYCVNGRLSCTGKLQNPAESCK APKKYISCSDSLENKYGATCAPTCQMLATGIECIPTKCES GCVCADGLYENLDGRCVPPEECPCEYGGLSYGKGEQIQTE CEICTCRKGKWKCVQKSRCSSTCNLYGEGHITTFDGQRFV FDGNCEYILAMDGCNVNRPLSSFKIVTENVICGKSGVTCS RSISIYLGNLTIILRDETYSISGKNLQVKYNVKKNALHLM FDIIIPGKYNMTLIWNKHMNFFIKISRETQETICGLCGNY NGNMKDDFETRSKYVASNELEFVNSWKENPLCGDVYFVVD PCSKNPYRKAWAEKTCSIINSQVFSACHNKVNRMPYYEAC VRDSCGCDIGGDCECMCDAIAVYAMACLDKGICIDWRTPE FCPVYCEYYNSHRKTGSGGAYSYGSSVNCTWHYRPCNCPN QYYKYVNIEGCYNCSHDEYFDYEKEKCMPCAMQPTSVTLP TATQPTSPSTSSASTVLTETTNPPV* Lysozyme SEQIDNO:281 KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNENT QATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPC SALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDV QAWIRGCRL* Lysozyme SEQIDNO:282 KVFGRCELAAAMKRHGLDNYRGYSLGNWVCVAKFESNFNT QATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPC SALLSSDITASVNCAKKIVSDGNGMSAWVAWRNRCKGTDV QAWIRGCRL* LysozymeC(Human) SEQIDNO:283 KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNT RATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLS CSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRD VRQYVQGCGV* LysozymeC(Bos SEQIDNO:284 KVFERCELARTLKKLGLDGYKGVSLANWLCLTKWESSYNT taurus) KATNYNPSSESTDYGIFQINSKWWCNDGKTPNAVDGCHVS CRELMENDIAKAVACAKHIVSEQGITAWVAWKSHCRDHDV SSYVEGCTL* Ovoinhibitor SEQIDNO:285 IEVNCSLYASGIGKDGTSWVACPRNLKPVCGTDGSTYSNE CGICLYNREHGANVEKEYDGECRPKHVMIDCSPYLQVVRD GNTMVACPRILKPVCGSDSFTYDNECGICAYNAEHHTNIS KLHDGECKLEIGSVDCSKYPSTVSKDGRTLVACPRILSPV CGTDGFTYDNECGICAHNAEQRTHVSKKHDGKCRQEIPEI DCDQYPTRKTTGGKLLVRCPRILLPVCGTDGFTYDNECGI CAHNAQHGTEVKKSHDGRCKERSTPLDCTQYLSNTQNGEA ITACPFILQEVCGTDGVTYSNDCSLCAHNIELGTSVAKKH DGRCREEVPELDCSKYKTSTLKDGRQVVACTMIYDPVCAT NGVTYASECTLCAHNLEQRTNLGKRKNGRCEEDITKEHCR EFQKVSPICTMEYVPHCGSDGVTYSNRCFFCNAYVQSNRT LNLVSMAAC* Cystatin SEQIDNO:286 MAGARGCVVLLAAALMLVGAVLGSEDRSRLLGAPVPVDEN DEGLQRALQFAMAEYNRASNDKYSSRVVRVISAKRQLVSG IKYILQVEIGRTTCPKSSGDLQSCEFHDEPEMAKYTTCTF VVYSIPWLNQIKLLESKCQ* PorcineLipase SEQIDNO:287 SEVCFPRLGCFSDDAPWAGIVQRPLKILPWSPKDVDTRFL LYTNQNQNNYQELVADPSTITNSNFRMDRKTRFIIHGFID KGEEDWLSNICKNLFKVESVNCICVDWKGGSRTGYTQASQ NIRIVGAEVAYFVEVLKSSLGYSPSNVHVIGHSLGSHAAG EAGRRTNGTIERITGLDPAEPCFQGTPELVRLDPSDAKFV DVIHTDAAPIIPNLGFGMSQTVGHLDFFPNGGKQMPGCQK NILSQIVDIDGIWEGTRDFVACNHLRSYKYYADSILNPDG FAGFPCDSYNVFTANKCFPCPSEGCPQMGHYADRFPGKTN GVSQVFYLNTGDASNFARWRYKVSVTLSGKKVTGHILVSL FGNEGNSRQYEIYKGTLQPDNTHSDEFDSDVEVGDLQKVK FIWYNNNVINPTLPRVGASKITVERNDGKVYDFCSQETVR EEVLLTLNPC* KidLipase SEQIDNO:288 GLVAADRITGGKDFRDIESKFALRTPEDTAEDTCHLIPGV TESVANCHFNHSSKTFVVIHGWTVTGMYESWVPKLVAALY KREPDSNVIVVDWLSRAQQHYPVSAGYTKLVGQDVAKFMN WMADEFNYPLGNVHLLGYSLGAHAAGIAGSLTSKKVNRIT GLDPAGPNFEYAEAPSRLSPDDADFVDVLHTFTRGSPGRS IGIQKPVGHVDIYPNGGTFQPGCNIGEALRVIAERGLGDV DQLVKCSHERSVHLFIDSLLNEENPSKAYRCNSKEAFEKG LCLSCRKNRCNNMGYEINKVRAKRSSKMYLKTRSQMPYKV FHYQVKIHFSGTESNTYTNQAFEISLYGTVAESENIPFTL PEVSTNKTYSFLLYTEVDIGELLMLKLKWISDSYFSWSNW WSSPGFDIGKIRVKAGETQKKVIFCSREKMSYLQKGKSPV IFVKCHDKSLNRKSG* PorcineLactoferrin SEQIDNO:289 APKKGVRWCVISTAEYSKCRQWQSKIRRTNPMFCIRRASP TDCIRAIAAKRADAVTLDGGLVFEADQYKLRPVAAEIYGT EENPQTYYYAVAVVKKGFNFQLNQLQGRKSCHTGLGRSAG WNIPIGLLRRFLDWAGPPEPLQKAVAKFFSQSCVPCADGN AYPNLCQLCIGKGKDKCACSSQEPYFGYSGAFNCLHKGIG DVAFVKESTVFENLPQKADRDKYELLCPDNTRKPVEAFRE CHLARVPSHAVVARSVNGKENSIWELLYQSQKKFGKSNPQ EFQLFGSPGQQKDLLFRDATIGFLKIPSKIDSKLYLGLPY LTAIQGLRETAAEVEARQAKVVWCAVGPEELRKCRQWSSQ SSQNLNCS LASTTEDCIVQVLKGEADAMSLDGGFIYTAGKCGLVPVLA ENQKSRQSSSSDCVHRPTQGYFAVAVVRKANGGITWNSVR GTKSCHTAVDRTAGWNIPMGLLVNQTGSCKFDEFFSQSCA PGSQPGSNLCALCVGNDQGVDKCVPNSNERYYGYTGAFRC LAENAGDVAFVKDVTVLDNTNGQNTEEWARELRSDDFELL CLDGTRKPVTEAQNCHLAVAPSHAVVSRKEKAAQVEQVLL TEQAQFGRYGKDCPDKFCLFRSETKNLLFNDNTEVLAQLQ GKTTYEKYLGSEYVTAIANLKQCSVSPLLEACAFMMR* BovineLactoferrin SEQIDNO:290 APRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRA FALECIRAIAEKKADAVTLDGGMVFEAGRDPYKLRPVAAE IYGTKESPQTHYYAVAVVKKGSNFQLDQLQGRKSCHTGLG RSAGWIIPMGILRPYLSWTESLEPLQGAVAKFFSASCVPC IDRQAYPNLCQLCKGEGENQCACSSREPYFGYSGAFKCLQ DGAGDVAFVKETTVFENLPEKADRDQYELLCLNNSRAPVD AFKECHLAQVPSHAVVARSVDGKEDLIWKLLSKAQEKFGK NKSRSFQLFGSPPGQRDLLFKDSALGFLRIPSKVDSALYL GSRYLTTLKNLRETAEEVKARYTRVVWCAVGPEEQKKCQQ WSQQSGQNVTCATASTTDDCIVLVLKGEADALNLDGGYIY TAGKCGLVPVLAENRKSSKHSSLDCVLRPTEGYLAVAVVK KANEGLTWNSLKDKKSCHTAVDRTAGWNIPMGLIVNQTGS CAFDEFFSQSCAPGADPKSRLCALCAGDDQGLDKCVPNSK EKYYGYTGAFRCLAEDVGDVAFVKNDTVWENTNGESTADW AKNLNREDFRLLCLDGTRKPVTEAQSCHLAVAPNHAVVSR SDRAAHVKQVLLHQQALFGKNGKNCPDKFCLFKSETKNLL FNDNTECLAKLGGRPTYEEYLGTEYVTAIANLKKCSTSPL LEACAFLTR* Saccharomyces SEQIDNO:291 APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNST cerevisiae NNGLLFINTTIASIAAKEEGVSLDKR a-mating factorsignalpeptide andsecretionsignal Saccharomyces SEQIDNO:292 APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNST cerevisiae NNGLLFINTTIASIAAKEEGVSLDKREAEA a-mating factorsignalpeptide andsecretionsignal endingwithEAEA EndoH- SEQIDNO:293 MTIAHHCIFLVILAFLALINVASGAPAPVKQGPTSVAYVE Saccharomyces VNNNSMLNVGKYTLADGGGNAFDVAVIFAANINYDTGTKT cerevisiae AYLHFNENVQRVLDNAVTQIRPLQQQGIKVLLSVLGNHQG Flo5fusion AGFANFPSQQAASAFAKQLSDAVAKYGLDGVDFDDEYAEY (fullORF,including GNNGTAQPNDSSFVHLVTALRANMPDKIISLYNIGPAASR peptidesthatare LSYGGVDVSDKFDYAWNPYYGTWQVPGIALPKAQLSPAAV cleavedoffpost- EIGRTSRSTVADLARRTVDEGYGVYLTYNLDGGDRTADVS translationally) AFTRELYGSEAVRTPGSSGSSGSSGSSGSSGSSGSSGSSE AAAREAAAREAAAREAAARGGGGSGGGGSGGGGSATEACL PAGQRKSGMNINFYQYSLKDSSTYSNAAYMAYGYASKTKL GSVGGQTDISIDYNIPCVSSSGTFPCPQEDSYGNWGCKGM GACSNSQGIAYWSTDLFGFYTTPTNVTLEMTGYFLPPQTG SYTFSFATVDDSAILSVGGSIAFECCAQEQPPITSTNFTI NGIKPW DGSLPDNITGTVYMYAGYYYPLKVVYSNAVSWGTLPISVE LPDGTTVSDNFEGYVYSFDDDLSQSNCTIPDPSIHTTSTI TTTTEPWTGTFTSTSTEMTTITDTNGQLTDETVIVIRTPT TASTITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVIV IRTPTSEGLITTTTEPWTGTFTSTSTEMTTVTGTNGQPTD ETVIVIRTPTSEGLITTTTEPWTGTFTSTSTEVTTITGTN GQPTDETVIVIRTPTSEGLITTTTEPWTGTFTSTSTEMTT VTGTNGQPTDETVIVIRTPTSEGLISTTTEPWTGTFTSTS TEVTTITGTNGQPTDETVIVIRTPTSEGLITTTTEPWTGT FTSTSTEMTTVTGTNGQPTDETVIVIRTPTSEGLITRTTE PWTGTFTSTSTEVTTITGTNGQPTDETVIVIRTPTTAISS SLSSSSGQITSSITSSRPIITPFYPSNGTSVISSSVISSS VTSSLVTSSSFISSSVISSSTTTSTSIFSESSTSSVIPTS SSTSGSSESKTSSASSSSSSSSISSESPKSPTNSSSSLPP VTSATTGQETASSLPPATTTKTSEQTTLVTVTSCESHVCT ESISSAIVSTATVTVSGVTTEYTTWCPISTTETTKQTKGT TEQTKGTTEQTTETTKQTTVVTISSCESDICSKTASPAIV STSTATINGVTTEYTTWCPISTTESKQQTTLVTVTSCESG VCSETTSPAIVSTATATVNDVVTVYPTWRPQTTNEQSVSS KMNSATSETTTNTGAAETKTAVTSSLSRFNHAETQTASAT DVIGHSSSVVSVSETGNTMSLTSSGLSTMSQQPRSTPASS MVGSSTASLEISTYAGSANSLLAGSGLSVFIASLLLAII AflexibleGSlinker SEQIDNO:294 GSSGSSGSSGSSGSSGSSGSSGSS withhigherScontent AflexibleGSlinker SEQIDNO:295 GGGGSGGGGSGGGGS withmuchhigherG content