Method for preparing protein fiber

Abstract

A method for preparing protein fibers is disclosed, which includes the steps of bringing a spinning solution containing protein and an organic solvent into contact with a coagulation solution and coagulating the protein. The protein content in the spinning solution is greater than 10% by weight based on the total amount of the spinning solution, and the coagulation solution contains water or an aqueous solution with a pH of greater than 0.25 and less than 10.00.

Description

Technical Field

[0001] The invention relates to a method for preparing protein fiber. Background Art

[0002] Conventionally, as methods for producing protein fibers containing structural proteins as their main component, there are known wet spinning methods and dry-wet spinning methods in which a spinning solution ejected from a nozzle is coagulated in a coagulation bath to form fibers.

[0003] In the wet spinning method and dry-wet spinning method of protein fibers, it is reported that a protein solution in which protein is dissolved in a solvent is used as a spinning solution (spinning solution), the spinning solution is extruded from a nozzle into a coagulation solution to remove the solvent from the spinning solution, and fibers are formed as undrawn yarns, thereby obtaining protein fibers (for example, refer to Patent Document 1, Patent Document 2, Non-Patent Document 1 and Non-Patent Document 2).

[0004] As solvents for the coagulation solution used in preparing protein fibers, lower alcohols such as methanol, ethanol, and 2-propanol, as well as ketones such as acetone, can generally be used. Non-Patent Document 1 reports, for example, a method for forming regenerated silk fibroin fibers by dissolving regenerated silk protein (regenerated silk fibroin) in formic acid and introducing it into a coagulation solution of a lower alcohol or acetone. Non-Patent Document 2, for example, reports a method for forming spider silk fibroin fibers by dissolving spider silk fibroin having an amino acid sequence derived from Nephila clavipes Masp1 in hexafluoroisopropanol (HFIP) and introducing it into a coagulation solution of methanol.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent No. 5540154

[0008] Patent Document 2: Japanese Patent No. 5584932

[0009] Non-patent literature

[0010] Non-patent document 1: Int. J. Biol. Macromol., Vol. 34, 2004, pp. 89-105

[0011] Non-patent document 2: PNAS, August 10, 2010, vol. 107, No. 32, pp. 14059-14063 Summary of the Invention

[0012] Problems to be solved by the invention

[0013] When producing protein fibers using wet spinning and wet-dry spinning methods using high-molecular-weight structural proteins such as silk fibroin, spider silk fibroin, and keratin, which are considered high-value materials for future applications, the coagulation solutions available are very limited. Among these, lower alcohols such as methanol, ethanol, and 2-propanol, as well as ketones such as acetone, are widely used as coagulation solutions for structural proteins because they are relatively inexpensive and have fiber-forming properties.

[0014] However, these coagulation solvents are designated as Class 4 hazardous substances under the Fire Services Act, posing risks such as explosion and fire. As the coagulation solvents consumed in the largest quantities during the production process, they are not suitable from a safety perspective. Further reductions in production costs and environmental impact are also desirable.

[0015] In view of the above problems in the prior art, an object of the present invention is to provide a method for preparing protein fibers, which uses a coagulation liquid containing water or an aqueous solution for preparation.

[0016] Solutions to Problems

[0017] The present inventors have conducted intensive research to solve the above-mentioned problems of the prior art and have found that protein fibers can be produced by combining a spinning solution containing protein and an organic solvent with a coagulation solution containing water or an aqueous solution with a pH of 0.25 or higher and 10.00 or lower.

[0018] That is, the present invention relates to, for example, the following inventions. [1]

[0020] A method for preparing protein fibers, comprising the steps of contacting a spinning solution containing protein and an organic solvent with a coagulation solution and coagulating the protein.

[0021] Based on the total amount of spinning solution, the protein content in the spinning solution is greater than 10% by mass.

[0022] The coagulation liquid contains water or an aqueous solution having a pH of 0.25 or higher and a pH of 10.00 or lower. [2]

[0024] The method according to [1], wherein the content of water or aqueous solution in the coagulation liquid is 60% by mass or more based on the total amount of the coagulation liquid. [3]

[0026] The method according to [1] or [2], wherein the aqueous solution is a saline solution, an acid solution or a mixed solution thereof. [4]

[0028] The method according to [3], wherein the acid aqueous solution is a carboxylic acid aqueous solution. [5]

[0030] The method according to [3] or [4], wherein the salt content in the coagulation liquid is 0.1% by mass or more based on the total amount of the coagulation liquid. [6]

[0032] The method according to [5], wherein the salt includes at least one selected from the group consisting of carboxylates and inorganic salts. [7]

[0034] The method according to [6], wherein the inorganic salt includes at least one selected from the group consisting of sulfate, chloride, nitrate, iodide, carbonate, hydrogen sulfate, hydrogen phosphate, hydrogen carbonate and thiocyanate. [8]

[0036] The method according to [6] or [7], wherein the inorganic salt includes at least one selected from the group consisting of sulfate, chloride, hydrogen phosphate and hydrogen carbonate. [9]

[0038] The method according to [7] or [8], wherein the chloride includes at least one selected from the group consisting of sodium chloride, calcium chloride, ammonium chloride, potassium chloride, lithium chloride, magnesium chloride and guanidine chloride.

[10]

[0040] The method according to any one of [7] to [9], wherein the sulfate includes at least one selected from the group consisting of ammonium sulfate, potassium sulfate, sodium sulfate, lithium sulfate, magnesium sulfate, and calcium sulfate.

[11]

[0042] The method according to any one of [3] to

[10] , wherein the brine solution comprises at least one selected from the group consisting of a sodium sulfate aqueous solution, a sodium chloride aqueous solution, brackish water, and seawater.

[12]

[0044] The method according to any one of [3] to

[11] , wherein the content of the organic solvent dissolved in the coagulation liquid from the spinning solution in contact with the coagulation liquid is 40% by mass or less, based on the total content of the saline solution in the coagulation liquid and the organic solvent dissolved in the coagulation liquid being 100% by mass.

[13]

[0046] The method according to any one of [1] to

[12] , wherein the average hydrophobicity index of the protein is greater than -1.3.

[14]

[0048] The method according to any one of [1] to

[13] , wherein the protein comprises at least one selected from the group consisting of spider silk protein, silk protein, collagen, elastic protein, elastin, and keratin.

[15]

[0050] The method according to any one of [1] to

[14] , wherein the protein is keratin or spider silk protein.

[16]

[0052] The method according to any one of [1] to

[15] , wherein the protein is spider silk protein.

[17]

[0054] The method according to any one of [1] to

[16] , wherein the average hydrophobicity index of the protein is greater than -0.8.

[18]

[0056] The method according to any one of [1] to

[17] , further comprising the step of stretching the coagulated protein.

[19]

[0058] The method according to any one of [1] to

[18] , wherein the organic solvent contains at least one selected from the group consisting of formic acid, dimethyl sulfoxide, and hexafluoroisopropanol.

[20]

[0060] The method according to any one of [1] to

[19] , wherein the organic solvent contains at least one selected from the group consisting of formic acid and dimethyl sulfoxide. [twenty one]

[0062] The method according to any one of [1] to

[20] , wherein the spinning solution further contains a dissolution accelerator. [twenty two]

[0064] A method for preparing protein fibers, comprising the steps of contacting a spinning solution containing protein and a solvent with a coagulation solution and coagulating the protein.

[0065] Based on the total amount of spinning solution, the protein content in the spinning solution is greater than 10% by mass.

[0066] The coagulation liquid contains water or an aqueous solution having a pH of 0.25 or higher and a pH of 2.50 or lower, or a pH of 7.50 or higher and a pH of 10.00 or lower.

[0067] Effects of the Invention

[0068] According to the present invention, a method for producing protein fibers using a coagulation solution containing water or an aqueous solution can be provided. By using a coagulation solution containing water or an aqueous solution, the risk of explosion, fire, etc., the production cost, and the environmental impact can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is a schematic diagram showing an example of the domain sequence of spider silk fibroin.

[0070] Figure 2 This is a schematic diagram showing an example of the domain sequence of spider silk fibroin.

[0071] Figure 3 This is a schematic diagram showing an example of the domain sequence of spider silk fibroin.

[0072] Figure 4 This is an explanatory diagram schematically showing an example of a spinning apparatus for producing protein fibers.

[0073] Figure 5 This is a graph showing an example of the results of the moisture absorption heat generation performance test. DETAILED DESCRIPTION

[0074] Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0075] (Method for preparing protein fiber)

[0076] The protein fiber production method of this embodiment includes contacting a spinning solution containing protein and an organic solvent with a coagulation solution to coagulate the protein. The protein content of the spinning solution is greater than 10% by mass, based on the total amount of the spinning solution. Furthermore, the coagulation solution contains water or an aqueous solution having a pH of 0.25 or higher and 10.00 or lower. The protein fiber production method of this embodiment can be performed using known spinning methods such as wet spinning and dry-wet spinning.

[0077] <Spinning solution>

[0078] The spinning solution according to this embodiment contains protein and an organic solvent.

[0079] (protein)

[0080] The protein fibers produced by the production method of this embodiment contain protein as a primary component. The protein contained in the spinning solution of this embodiment is artificially produced (synthetic protein), not natural protein or its purified form. The method for artificially producing protein is not particularly limited and can be produced using genetic recombination techniques, such as microorganisms, or synthetically.

[0081] The above-mentioned protein may be, for example, a structural protein or an artificial structural protein derived from the structural protein. The so-called structural protein refers to a protein that forms or maintains a structure, form, etc. in a living body.

[0082] Examples of the structural protein include spider silk proteins (such as spider silk fibroin), silk protein, collagen, elastic protein, elastin, elastin, and keratin.

[0083] The spider silk proteins of this embodiment include naturally derived spider silk proteins and modified spider silk proteins (hereinafter also referred to as "modified fibroin"). In this specification, the term "naturally derived spider silk protein" refers to a spider silk protein having the same amino acid sequence as a naturally derived spider silk protein (such as spider silk fibroin), and the term "modified spider silk protein" or "modified fibroin" refers to a spider silk protein having an amino acid sequence different from that of a naturally derived spider silk protein.

[0084] Naturally derived spider silk proteins include, for example, spidroin, transverse silk, and ampullate silk, produced by spider silk species. Dragline silk from the spinotube has repetitive regions composed of crystalline and amorphous regions (also known as amorphous regions), resulting in both high stress resistance and elasticity. Transverse silk, a spider silk protein, is characterized by its lack of crystalline regions but repetitive regions composed of amorphous regions. Transverse silk, compared to dragline silk from the spinotube, exhibits lower stress resistance but higher elasticity.

[0085] Produced by the spider's ampullate gland, large spindle silk proteins are characterized by excellent strength and toughness. Examples of large spindle silk proteins include MaSp1 and MaSp2, the large ampullate gland silk proteins from the goldspinning spider (Nephila clavipes), and ADF3 and ADF4, from the cross spider (Araneus diadematus). ADF3 is one of the two main dragline silk proteins of the cross spider. Spider silk proteins can also be derived from these dragline silk proteins. Spider silk proteins derived from ADF3 are relatively easy to synthesize and have excellent properties in terms of strength, elongation, and toughness.

[0086] Transverse silk proteins are produced by the flagelliform gland of spiders. Examples of transverse silk proteins include flagelliform silk proteins from the golden-spinning spider (Nephila clavipes).

[0087] As another example of spider silk fibroin produced by spider silk, for example, spiders belonging to the genus Araneus such as the ghost spider, cross spider, fat spider, five-striped spider and big-bellied orb spider, spiders belonging to the genus Neoscona such as the green spider, water-loving spider, bush spider and green spider-like spider, spiders belonging to the genus Pronus such as the brown spider, spiders belonging to the genus Cyrtarachne such as the toad spider and symmetrical spider, spiders belonging to the genus Cyrtarachne such as the Cuscuta spiny-bellied spider and the mastoid-spiny-bellied spider, etc. Spiders of the genus Gasteracantha, spiders belonging to the genus Ordgarius, such as Ho's knotweed spider and six-spined knotweed spider, spiders belonging to the genus Argiope, such as the pleasing-eyed spider, the small pleasing-eyed spider and the horizontal-striped spider, spiders belonging to the genus Arachnura, such as the double-peaked orbicularis argenteus, spiders belonging to the genus Acusilas, such as the brown hanging leaf spider, spiders belonging to the genus Cytophora, such as the Moluccan clouded spider, the square clouded spider and the full-color clouded spider, and the ugly cone spider. Spider silk proteins produced by spiders belonging to the genus Poltys, such as the head spider, spiders belonging to the genus Cyclosa, such as the eight-tuberculate dust spider, spider with four protrusions, spider with round abdomen, and spider with black tail, and spiders belonging to the genus Chorizops, such as the Japanese strong-headed spider, as well as spiders belonging to the genus Tetragnatha, such as the anterior tooth octopus, the cone-bellied octopus, the straight octopus, and the scale-marked octopus, spiders belonging to the genus Leucauge, such as the vertical-striped silver scale spider, the shoulder-spotted silver scale spider, and the small-shouldered silver scale spider, as well as spiders belonging to the genus Nephila and the spotted Spider silk proteins produced by spiders belonging to the genus Nephila, such as the Nephila nephila; spiders belonging to the genus Menosira, such as the beautiful wheat spider; spiders belonging to the genus Dyschiriognatha, such as the delicate thick-chelicerae; spiders belonging to the genus Latrodectus, such as the black widow spider, the redback spider, the geometric spider, and the interspotted spider; and spiders belonging to the family Tetragnathidae, such as the genus Euprosthenops.

[0088] More specific examples of spider silk proteins produced by spider silk include fibroin-3 (adf-3) [from Araneus diadematus] (GenBank Accession No. AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence), U37520 (base sequence)), major ampullate spidroin 1 [from Latrodectus hesperus] (GenBank Accession No. ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [from Nephila clavata] (GenBank Accession Nos. AAL32472 (amino acid sequence), AF441245 (base sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank Accession Nos. CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank Accession Nos. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephila clavipes] cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), etc.

[0089] The spider silk protein of this embodiment may be, for example, a protein comprising the formula 1: [(A) n motif-REP] m, or formula 2: [(A) n motif-REP] m -(A) n The spider silk protein of this embodiment may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited thereto, and are generally regions that do not have the characteristic amino acid motif repeats in fibroin and are composed of about 100 amino acid residues.

[0090] In this specification, the so-called "domain sequence" is a region that generates crystals unique to silk fibroin (typically, corresponding to the amino acid sequence (A) n motif) and an amino acid sequence of an amorphous region (typically, an amino acid sequence corresponding to REP), and is referred to as Formula 1: [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m -(A) n The amino acid sequence shown in the motif. Here, (A) n The motif indicates an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27. (A) n The number of amino acid residues in the motif may be 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. In addition, (A) n The ratio of the number of alanine residues in the motif to the total number of amino acid residues is sufficient as long as it is 40% or more, and can be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). n At least 7 of the motifs may be composed only of alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues, or an amino acid sequence composed of 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 120, 10 to 140, 10 to 160 or 10 to 180 amino acid residues. m represents an integer from 2 to 300, and may be an integer from 8 to 300, 10 to 300, 10 to 300, 20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to 160, 20 to 140 or 20 to 120. The presence of multiple (A) nThe motifs may be identical amino acid sequences or different amino acid sequences. The multiple REPs may be identical amino acid sequences or different amino acid sequences.

[0091] Modified spider silk proteins (modified fibroin) may be, for example, spider silk proteins obtained by modifying the amino acid sequence of naturally derived spider silk fibroin (e.g., spider silk proteins obtained by modifying the amino acid sequence of cloned naturally derived spider silk fibroin gene sequences). Alternatively, they may be artificially designed and synthesized spider silk proteins independent of naturally derived spider silk fibroin (e.g., spider silk proteins having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). Furthermore, in this embodiment, modified spider silk fibroin is preferably used as the modified fibroin because it also exhibits excellent thermal insulation properties, hygroscopic heat generation properties, and / or flame retardancy.

[0092] For example, the gene sequence of the cloned natural spider silk fibroin can be modified by, for example, replacing, deleting, inserting and / or adding one or more amino acid residues to obtain a modified fibroin. The replacement, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as local specific mutagenesis. Specifically, it can be carried out according to the methods described in documents such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0093] Specific examples of modified silk fibroin include modified silk fibroin of the large silk tube dragline protein produced by the large ampullate gland of spiders (first modified silk fibroin), modified silk fibroin with reduced glycine residue content (second modified silk fibroin), and modified silk fibroin with reduced (A) n The content of the modified silk fibroin (the third modified silk fibroin) with the motif reduced, the content of the glycine residues reduced and (A) n motif (the 4th modified silk fibroin), a modified silk fibroin having a domain sequence locally containing a region with a large hydrophobicity index (the 5th modified silk fibroin), and a modified silk fibroin having a domain sequence with reduced glutamine residue content (the 6th modified silk fibroin).

[0094] Examples of modified fibroin derived from the large spindle dragline silk protein produced by the large ampullate gland of spiders (first modified fibroin) include those comprising the formula 1: [(A) n motif-REP] mA protein having the domain sequence shown. In the first modified fibroin, n in Formula 1 is preferably an integer from 3 to 20, more preferably an integer from 4 to 20, further preferably an integer from 8 to 20, further preferably an integer from 10 to 20, further preferably an integer from 4 to 16, particularly preferably an integer from 8 to 16, and most preferably an integer from 10 to 16. In the first modified fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, further preferably 20 to 100 residues, and further preferably 20 to 75 residues. In the first modified fibroin, Formula 1: [(A) n motif-REP] m The total number of glycine residues, serine residues, and alanine residues contained in the amino acid sequence is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more relative to the total number of amino acid residues.

[0095] The first modified silk fibroin may be a protein comprising formula 1: [(A) n motif-REP] m The amino acid sequence is a unit of SEQ ID NOs: 1 to 3, and the C-terminal sequence is an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3, or an amino acid sequence having 90% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.

[0096] The amino acid sequence shown in sequence number 1 is identical to the amino acid sequence consisting of the 50 amino acid residues at the C-terminus of the amino acid sequence of ADF3 (GI: 1263287, NCBI), the amino acid sequence shown in sequence number 2 is identical to the amino acid sequence after removing 20 residues from the C-terminus of the amino acid sequence shown in sequence number 1, and the amino acid sequence shown in sequence number 3 is identical to the amino acid sequence after removing 29 residues from the C-terminus of the amino acid sequence shown in sequence number 1.

[0097] More specific examples of the first modified fibroin include modified fibroin comprising an amino acid sequence having 90% or greater sequence identity with (1-i) the amino acid sequence set forth in SEQ ID NO: 4 or (1-ii) the amino acid sequence set forth in SEQ ID NO: 4. The sequence identity is preferably 95% or greater.

[0098] The amino acid sequence set forth in SEQ ID NO: 4 is derived from the amino acid sequence of ADF3, to which an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added at the N-terminus. The amino acid sequence is mutated to increase the repeat region at positions 1 to 13 by approximately twofold and terminate translation at amino acid residue 1154. The amino acid sequence at the C-terminus of the amino acid sequence set forth in SEQ ID NO: 4 is identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0099] The modified silk fibroin of (1-i) can be composed of the amino acid sequence shown in SEQ ID NO:4.

[0100] The modified silk fibroin with reduced glycine residue content (second modified silk fibroin) has an amino acid sequence in which the glycine residue content is reduced compared to naturally derived spider silk fibroin. The second modified silk fibroin can have an amino acid sequence equivalent to that of naturally derived spider silk fibroin, in which at least one or more glycine residues in REP are substituted with other amino acid residues.

[0101] The domain sequence of the second modified fibroin, compared to the naturally derived spider silk fibroin, may have an amino acid sequence corresponding to at least one or more glycine residues in the motif sequence selected from GGX and GPGXX (wherein G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP, in which one or more of the motif sequences is replaced with another amino acid residue.

[0102] In the second modified fibroin, the ratio of the motif sequence in which the glycine residue is substituted with another amino acid residue may be 10% or more relative to the entire motif sequence.

[0103] The second modified silk fibroin comprises formula 1: [(A) n motif-REP] m The domain sequence shown has the following amino acid sequence: When the amino acid residue (A) located at the most C-terminal side is removed from the above domain sequence, n The total number of amino acid residues of the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in all REPs in the sequence obtained by converting the motif to the C-terminus of the above-mentioned domain sequence is set to z, and (A) located most toward the C-terminus is removed from the above-mentioned domain sequence. n When the total number of amino acid residues in the sequence from the motif to the C-terminus of the above-mentioned domain sequence is w, z / w may be 30% or more, 40% or more, 50% or more, or 50.9% or more. (A) nThe number of alanine residues in the motif may be 83% or more, preferably 86% or more, more preferably 90% or more, further preferably 95% or more, and even more preferably 100% (meaning composed only of alanine residues) relative to the total number of amino acid residues.

[0104] The second modified fibroin preferably increases the proportion of amino acid sequences consisting of XGX by replacing one glycine residue in the GGX motif with another amino acid residue. In the second modified fibroin, the proportion of amino acid sequences consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, further preferably 6% or less, further preferably 4% or less, and particularly preferably 2% or less. The proportion of amino acid sequences consisting of GGX in the domain sequence can be calculated by the same method as the calculation method for the proportion of amino acid sequences consisting of XGX (z / w) described below.

[0105] The calculation method of z / w is further described in detail. First, in the formula 1: [(A) n motif-REP] m In the spider silk fibroin (modified fibroin or naturally derived spider silk fibroin) having the domain sequence shown, the C-terminal end (A) is removed from the domain sequence. n From all the REPs contained in the sequence obtained after the motif to the C-terminus of the domain sequence, an amino acid sequence consisting of XGX is extracted. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no repetition), z is 50×3=150. In addition, for example, as in the case of an amino acid sequence consisting of XGXGX, when X (the middle X) contained in 2 XGX exists, the calculation is performed by deducting the repeated part (in the case of XGXGX, it is 5 amino acid residues). w is the residue (A) located closest to the C-terminus is removed from the domain sequence. n The total number of amino acid residues contained in the sequence obtained after the sequence from the motif to the C-terminus of the domain sequence. Figure 1 In the case of the domain sequence shown, w is 4+50+4+100+4+10+4+20+4+30=230 (excluding the (A) located closest to the C-terminus). n Then, by dividing z by w, z / w (%) can be calculated.

[0106] In the second modified fibroin, z / w is preferably 50.9% or higher, more preferably 56.1% or higher, further preferably 58.7% or higher, further preferably 70% or higher, and further preferably 80% or higher. The upper limit of z / w is not particularly limited and may be, for example, 95% or lower.

[0107] For example, a second modified silk fibroin can be obtained by modifying the gene sequence of a cloned naturally-occurring spider silk fibroin by replacing at least a portion of the base sequence encoding a glycine residue with another amino acid residue. In this case, the modified glycine residue can be selected from either the GGX motif or the GPGXX motif, or the substitution can be performed to achieve a z / w ratio of 50.9% or greater. Alternatively, the modified silk fibroin can be obtained by designing an amino acid sequence that satisfies the aforementioned conditions based on the amino acid sequence of a naturally-occurring spider silk fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to modifications corresponding to substitution of a glycine residue in REP with another amino acid residue in the amino acid sequence of a naturally-occurring spider silk fibroin, further modifications corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues can be performed.

[0108] The other amino acid residues are not particularly limited as long as they are amino acid residues other than glycine residues. Preferred are hydrophobic amino acid residues such as valine (V) residues, leucine (L) residues, isoleucine (I) residues, methionine (M) residues, proline (P) residues, phenylalanine (F) residues, and tryptophan (W) residues; and hydrophilic amino acid residues such as glutamine (Q) residues, asparagine (N) residues, serine (S) residues, lysine (K) residues, and glutamic acid (E) residues. More preferred are valine (V) residues, leucine (L) residues, isoleucine (I) residues, and glutamine (Q) residues, and even more preferred is glutamine (Q) residues.

[0109] As a more specific example of the second modified silk fibroin, there can be listed a modified silk fibroin comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (2-i) sequence number 6, sequence number 7, sequence number 8 or sequence number 9, or (2-ii) the amino acid sequence shown in sequence number 6, sequence number 7, sequence number 8 or sequence number 9.

[0110] The modified silk fibroin of (2-i) is described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by replacing all GGX in REP corresponding to the amino acid sequence shown in SEQ ID NO: 10 of the naturally derived spider silk fibroin with GQX. The amino acid sequence shown in SEQ ID NO: 7 is obtained by replacing (A) from the N-terminal side to the C-terminal side in the amino acid sequence shown in SEQ ID NO: 6. n The motif is deleted every 2 times, and a [(A) n The amino acid sequence shown in SEQ ID NO: 8 is obtained in each of the amino acid sequences shown in SEQ ID NO: 7 (A) n Two alanine residues were inserted into the C-terminal side of the motif, and some glutamine (Q) residues were substituted with serine (S) residues, and some amino acids on the N-terminal side were deleted so that the molecular weight was almost the same as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a sequence in which a region of the 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 11 (wherein several amino acid residues on the C-terminal side of the region are substituted) is repeated four times, and a His tag is added to the C-terminus of the sequence.

[0111] The z / w value for the amino acid sequence set forth in SEQ ID NO: 10 (equivalent to naturally derived spider silk fibroin) is 46.8%. The z / w values ​​for the amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. Furthermore, the x / y values ​​for the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, with a sawtooth ratio (described below) of 1:1.8 to 11.3, are 15.0%, 15.0%, 93.4%, 92.7%, and 89.3%, respectively.

[0112] The modified silk fibroin of (2-i) can be composed of the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0113] The modified silk fibroin of (2-ii) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified silk fibroin of (2-ii) also comprises formula 1: [(A) n motif-REP] m The sequence identity is preferably 95% or greater.

[0114] The modified silk fibroin (2-ii) has a sequence identity of more than 90% with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and when the total number of amino acid residues of the amino acid sequence composed of XGX (wherein X represents an amino acid residue other than glycine) contained in REP is set to z and the total number of amino acid residues of REP in the above-mentioned domain sequence is set to w, z / w is preferably more than 50.9%.

[0115] The second modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus, thereby enabling separation, immobilization, detection, and visualization of the modified fibroin.

[0116] As a tag sequence, for example, an affinity tag that utilizes specific affinity (binding property, affinity) with other molecules can be listed. As a specific example of an affinity tag, a histidine tag (His tag) can be listed. The His tag is a short peptide composed of about 4 to 10 histidine residues arranged in an array, and has the property of specifically binding to metal ions such as nickel, and can therefore be used for the separation of modified silk fibroin by chelating metal chromatography. As a specific example of a tag sequence, for example, the amino acid sequence shown in sequence number 12 (an amino acid sequence comprising a His tag sequence and a hinge sequence) can be listed.

[0117] Alternatively, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can be used.

[0118] Furthermore, an "epitope tag" that utilizes an antigen-antibody reaction can also be used. By adding a peptide (epitope) that displays antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tags, myc tags, and FLAG tags. By utilizing epitope tags, modified silk fibroin can be easily purified with high specificity.

[0119] Furthermore, a tag obtained by removing the tag sequence using a specific protease can also be used. Alternatively, the protein adsorbed via the tag sequence can be treated with a protease to recover the modified silk fibroin after the tag sequence has been removed.

[0120] As a more specific example of the second modified silk fibroin containing a tag sequence, there can be listed a modified silk fibroin comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (2-iii) sequence number 13, sequence number 11, sequence number 14 or sequence number 15, or the amino acid sequence shown in (2-iv) sequence number 13, sequence number 11, sequence number 14 or sequence number 15.

[0121] The amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15 are obtained by adding the amino acid sequence shown in SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) to the N-termini of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0122] The modified silk fibroin of (2-iii) can be composed of the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.

[0123] The modified silk fibroin of (2-iv) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. The modified silk fibroin of (2-iv) also comprises formula 1: [(A) n motif-REP] m The sequence identity is preferably 95% or greater.

[0124] The modified silk fibroin (2-iv) has a sequence identity of more than 90% with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, and when the total number of amino acid residues of the amino acid sequence composed of XGX (wherein X represents an amino acid residue other than glycine) contained in REP is set to z and the total number of amino acid residues of REP in the above-mentioned domain sequence is set to w, z / w is preferably more than 50.9%.

[0125] The second modified silk fibroin may contain a secretion signal for releasing the protein produced by the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of host.

[0126] Reduced (A) n The modified silk fibroin (the third modified silk fibroin) having a motif content has a domain sequence compared with the natural spider silk fibroin (A) n The amino acid sequence of the modified silk fibroin has a reduced content of at least one or more (A) motifs compared to the natural spider silk fibroin. nThe amino acid sequence of the motif.

[0127] The third modified silk fibroin may have (A) equivalent to a 10-40% deletion from a naturally derived spider silk fibroin. n The amino acid sequence of the motif.

[0128] The domain sequence of the third modified silk fibroin may have at least one to three (A) residues per N-terminal to C-terminal end compared to the naturally derived spider silk fibroin. n One missing (A) in the motif n The amino acid sequence of the motif.

[0129] The domain sequence of the third modified silk fibroin may have the equivalent of two consecutive (A) repeated in sequence from at least the N-terminal side to the C-terminal side, compared to the naturally derived spider silk fibroin. n The deletion of the motif and a (A) n The amino acid sequence of the deletion motif.

[0130] The domain sequence of the third modified fibroin may have a deletion (A) equivalent to one every two at least from the N-terminal side to the C-terminal side. n The amino acid sequence of the motif.

[0131] The third modified silk fibroin comprises formula 1: [(A) n motif-REP] m The domain sequence shown has the following amino acid sequence: the adjacent two [(A) n The number of amino acid residues of REP in the motif-REP] unit is set to 1, and the number of amino acid residues of the REP with the smaller number of amino acid residues is set to 1, and the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 for the adjacent two [(A) n When the maximum value of the sum of the number of amino acid residues in the motif-REP] unit is x and the total number of amino acid residues in the domain sequence is y, x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) n The number of alanine residues in the motif may be 83% or more, preferably 86% or more, more preferably 90% or more, further preferably 95% or more, and even more preferably 100% (meaning composed only of alanine residues) relative to the total number of amino acid residues.

[0132] Reference Figure 1 The calculation method of x / y will be described in further detail. Figure 1 The domain sequence after removing the N-terminal sequence and the C-terminal sequence from the spider silk fibroin is shown. The domain sequence has (A) from the N-terminal side (left side)n Motif-1st REP (50 amino acid residues)-(A) n Motif-2 REP (100 amino acid residues)-(A) n Motif-3rd REP (10 amino acid residues)-(A) n Motif-4th REP (20 amino acid residues)-(A) n Motif-5th REP (30 amino acid residues)-(A) n The sequence of the motif.

[0133] Select two adjacent [(A) n Motif-REP] unit. In this case, there may also be unselected [(A) n motif-REP] unit. Figure 1 Mode 1 (comparison of the 1st REP and the 2nd REP, and comparison of the 3rd REP and the 4th REP), Mode 2 (comparison of the 1st REP and the 2nd REP, and comparison of the 4th REP and the 5th REP), Mode 3 (comparison of the 2nd REP and the 3rd REP, and comparison of the 4th REP and the 5th REP), and Mode 4 (comparison of the 1st REP and the 2nd REP) are shown. In addition, there are other selection methods.

[0134] Next, for each pattern, compare the selected two adjacent [(A) n The number of amino acid residues of each REP in the [motif-REP] unit. Comparison is performed by finding the ratio of the number of amino acid residues of the other REP when the number of amino acid residues of the REP with the relatively smaller number is set to 1. For example, when comparing the 1st REP (50 amino acid residues) and the 2nd REP (100 amino acid residues), when the 1st REP with the relatively smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the 2nd REP is 100 / 50 = 2. Similarly, when comparing the 4th REP (20 amino acid residues) and the 5th REP (30 amino acid residues), when the 4th REP with the relatively smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the 5th REP is 30 / 20 = 1.5.

[0135] Figure 1 In the figure, the solid line indicates that when the one with the relatively smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is 1.8 to 11.3 [(A) n motif-REP] unit. Such a ratio is hereinafter referred to as a sawtooth ratio. The dotted line indicates that when the side with the relatively smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other side is less than 1.8 or greater than 11.3 [(A) n motif-REP] units.

[0136] In each mode, two adjacent ones [(A) n The sum of all amino acid residues in the motif-REP unit (not just REP, but also (A) n The number of amino acid residues in the motif. ). Then, the summed values ​​are compared, and the sum of the pattern with the largest sum (the maximum sum) is set to x. Figure 1 In the example shown, the total value of mode 1 is the largest.

[0137] Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

[0138] In the third modified silk fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% ​​or more, further preferably 70% or more, further preferably 75% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited, for example, it can be 100% or less. When the sawtooth ratio is 1:1.9 to 11.3, x / y is preferably 89.6% or more, when the sawtooth ratio is 1:1.8 to 3.4, x / y is preferably 77.1% or more, when the sawtooth ratio is 1:1.9 to 8.4, x / y is preferably 75.9% or more, and when the sawtooth ratio is 1:1.9 to 4.1, x / y is preferably 64.2% or more.

[0139] When the third modified silk fibroin has multiple (A) in the domain sequence n For modified silk fibroin in which at least seven of the motifs consist exclusively of alanine residues, the ratio x / y is preferably 46.4% or greater, more preferably 50% or greater, even more preferably 55% or greater, even more preferably 60% or greater, even more preferably 70% or greater, and particularly preferably 80% or greater. The upper limit of x / y is not particularly limited, but may be 100% or less.

[0140] For example, the gene sequence encoding (A) can be modified so that x / y is 64.2% or more from the gene sequence of naturally-occurring spider silk fibroin cloned from the spider silk fibroin. n Alternatively, for example, one or more of the amino acid sequences of the naturally derived spider silk fibroin may be designed so that x / y is set to 64.2% or more. n The amino acid sequence in which the motif is deleted is obtained by chemically synthesizing the nucleic acid encoding the designed amino acid sequence. In any case, except for the deletion of (A) in the amino acid sequence of the spider silk fibroin from natural sources nIn addition to modifications corresponding to the motif, further modifications of the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed.

[0141] As a more specific example of the third modified silk fibroin, there can be listed a modified silk fibroin comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (3-i) sequence number 18, sequence number 7, sequence number 8 or sequence number 9, or the amino acid sequence shown in (3-ii) sequence number 18, sequence number 7, sequence number 8 or sequence number 9.

[0142] The modified silk fibroin of (3-i) is described. The amino acid sequence represented by SEQ ID NO: 18 is obtained by modifying (A) from the N-terminal side to the C-terminal side in the amino acid sequence represented by SEQ ID NO: 10 corresponding to the naturally derived spider silk fibroin. n The motif is deleted every 2 times, and a [(A) n The amino acid sequence shown in SEQ ID NO: 7 is obtained by replacing all GGX in REP of the amino acid sequence shown in SEQ ID NO: 18 with GQX. The amino acid sequence shown in SEQ ID NO: 8 is obtained by replacing all (A) in the amino acid sequence shown in SEQ ID NO: 7 with GQX. n Two alanine residues were inserted into the C-terminal side of the motif, and some glutamine (Q) residues were substituted with serine (S) residues, and some amino acids on the N-terminal side were deleted so that the molecular weight was almost the same as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a sequence in which a region of the 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 11 (wherein several amino acid residues on the C-terminal side of the region are substituted) is repeated four times, and a His tag is added to the C-terminus of the sequence.

[0143] The amino acid sequence represented by sequence number 10 (equivalent to naturally derived spider silk fibroin) has a sawtooth ratio of 1:1.8 to 11.3, and the x / y value is 15.0%. The x / y values ​​in the amino acid sequence represented by sequence number 18 and the amino acid sequence represented by sequence number 7 are both 93.4%. The x / y value in the amino acid sequence represented by sequence number 8 is 92.7%. The x / y value in the amino acid sequence represented by sequence number 9 is 89.3%. The z / w values ​​in the amino acid sequences represented by sequence numbers 10, 18, 7, 8, and 9 are 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively.

[0144] The modified silk fibroin of (3-i) can be composed of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0145] The modified silk fibroin of (3-ii) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified silk fibroin of (3-ii) also comprises formula 1: [(A) n motif-REP] m The sequence identity is preferably 95% or greater.

[0146] The modified silk fibroin of (3-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and the adjacent two [(A) n The number of amino acid residues of REP in the motif-REP] unit is set to 1, and the ratio of the number of amino acid residues of the REP with the smaller number of amino acid residues is 1.8 to 11.3 (the sawtooth ratio is 1:1.8 to 11.3). n When the maximum value of the total value obtained by adding the number of amino acid residues in the [motif-REP] unit is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

[0147] The third modified fibroin may contain the tag sequence at either or both of the N-terminus and the C-terminus.

[0148] As a more specific example of the third modified silk fibroin containing a tag sequence, there can be listed a modified silk fibroin comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (3-iii) sequence number 17, sequence number 11, sequence number 14 or sequence number 15, or the amino acid sequence shown in (3-iv) sequence number 17, sequence number 11, sequence number 14 or sequence number 15.

[0149] The amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15 are obtained by adding the amino acid sequence shown in SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) to the N-termini of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0150] The modified silk fibroin of (3-iii) can be composed of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.

[0151] The modified silk fibroin of (3-iv) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. The modified silk fibroin of (3-iv) also comprises formula 1: [(A) n motif-REP] m The sequence identity is preferably 95% or greater.

[0152] The modified silk fibroin of (3-iv) has a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, and two adjacent [(A) n The number of amino acid residues of REP in the motif-REP] unit is set to 1, and the number of amino acid residues of the REP with the smaller number of amino acid residues is set to 1, and the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 for the adjacent two [(A) n When the maximum value of the total value obtained by adding the number of amino acid residues in the [motif-REP] unit is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

[0153] The third modified silk fibroin may contain a secretion signal for releasing the protein produced by the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of host.

[0154] Will reduce the content of glycine residues and (A) n The domain sequence of the modified silk fibroin (the fourth modified silk fibroin) with the motif content is compared with the natural spider silk fibroin, which has the following characteristics: n The fourth modified silk fibroin domain sequence has an amino acid sequence with a reduced content of glycine residues in addition to the motif. Compared with the natural spider silk fibroin, the modified silk fibroin domain sequence has at least one or more (A) n In addition to the motif deletion, it may also have an amino acid sequence corresponding to at least one or more glycine residues in REP being replaced by other amino acid residues. That is, the fourth modified fibroin is a modified fibroin having both the above-mentioned reduced glycine residue content (the second modified fibroin) and reduced (A) n Modified silk fibroin having a motif content (third modified silk fibroin) with characteristics. Specific embodiments are as described for the second modified silk fibroin and the third modified silk fibroin.

[0155] More specific examples of the fourth modified fibroin include modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence set forth in (4-i) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 57, or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 57. Specific embodiments of the modified fibroin comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 57 are as described above.

[0156] The domain sequence of the modified silk fibroin (the 5th modified silk fibroin) having a domain sequence locally containing a region with a large hydrophobicity index can have an amino acid sequence equivalent to one or more amino acid residues in REP being replaced with amino acid residues with a large hydrophobicity index and / or one or more amino acid residues with a large hydrophobicity index being inserted into REP, and locally containing a region with a large hydrophobicity index, compared to the spider silk fibroin of natural origin.

[0157] The region with a large local hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

[0158] The amino acid residue having a large hydrophobicity index is more preferably an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A).

[0159] The fifth modified silk fibroin, compared with the naturally derived spider silk fibroin, is equivalent to replacing one or more amino acid residues in REP with amino acid residues with a large hydrophobicity index and / or inserting one or more amino acid residues with a large hydrophobicity index into REP. In addition, compared with the naturally derived spider silk fibroin, it can also be modified in the amino acid sequence by replacing, deleting, inserting and / or adding one or more amino acid residues.

[0160] For example, the fifth modified silk fibroin can be obtained by replacing one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index) from the gene sequence of a cloned naturally derived spider silk fibroin, and / or inserting one or more hydrophobic amino acid residues into REP. Alternatively, an amino acid sequence equivalent to replacing one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues and / or inserting one or more hydrophobic amino acid residues into REP from the amino acid sequence of a naturally derived spider silk fibroin can be designed and chemically synthesized into a nucleic acid encoding the designed amino acid sequence. In any case, in addition to modifications equivalent to replacing one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues and / or inserting one or more hydrophobic amino acid residues into REP from the amino acid sequence of a naturally derived spider silk fibroin, further modifications corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues can be performed.

[0161] The fifth modified silk fibroin comprises formula 1: [(A) n motif-REP] m The domain sequence shown in FIG, and having the following amino acid sequence: (A) located at the most C-terminal side is removed from the above domain sequence n Among all REPs contained in the sequence obtained from the sequence from the motif to the C-terminus of the above-mentioned domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the residues (A) located closest to the C-terminus are removed from the above-mentioned domain sequence. n When the total number of amino acid residues contained in the sequence from the motif to the C-terminus of the domain sequence is q, p / q is 6.2% or more.

[0162] The hydrophobicity index of amino acid residues was determined using a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132). Specifically, the hydrophobicity index (hydrophilic index, hereinafter also referred to as "HI") for each amino acid is shown in Table 1 below.

[0163] [Table 1]

[0164] amino acids HI amino acids HI Isoleucine (Ile) 4.5 Tryptophan (Trp) -0.9 Valine (Val) 4.2 Tyrosine (Tyr) -1.3 Leucine (Leu) 3.8 Proline (Pro) -1.6 Phenylalanine (Phe) 2.8 Histidine (His) -3.2 Cysteine ​​(Cys) 2.5 Asparagine (Asn) -3.5 Methionine (Met) 1.9 Aspartic acid (Asp) -3.5 Alanine (Ala) 1.8 Glutamine (Gln) -3.5 Glycine (Gly) -0.4 Glutamate (Glu) -3.5 Threonine (Thr) -0.7 Lysine (Lys) -3.9 Serine (Ser) -0.8 Arginine (Arg) -4.5

[0165] The calculation method of p / q is further explained in detail. In the calculation, the formula 1 is used: [(A) n motif-REP] m The domain sequence shown is excluding the domain located closest to the C-terminus (A). n The sequence obtained by following the sequence from the motif to the C-terminus of the domain sequence (hereinafter referred to as "sequence A"). First, in all REPs contained in sequence A, the average value of the hydrophobicity index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the sum of the HI of each amino acid residue contained in the 4 consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all 4 consecutive amino acid residues (each amino acid residue is used for the calculation of 1 to 4 average values). Next, the region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is determined. Even if a certain amino acid residue belongs to multiple "4 consecutive amino acid residues with an average value of the hydrophobicity index of 2.6 or more", it will be included in the region as one amino acid residue. Moreover, the total number of amino acid residues contained in the region is p. In addition, the total number of amino acid residues contained in sequence A is q.

[0166] For example, when 20 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" are extracted (without duplication), the region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more contains 20 consecutive 4 amino acid residues (without duplication), and p is 20×4=80. In addition, for example, when only one amino acid residue is repeated in 2 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", the region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more contains 7 amino acid residues (p=2×4-1=7, "-1" is the deduction of the repeated part). For example, in Figure 2 In the case of the domain sequence shown in FIG, there are 7 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" without repetition, so p is 7×4=28. Figure 2 In the case of the domain sequence shown, q is 4+50+4+40+4+10+4+20+4+30=170 (excluding the last (A) at the C-terminal side). n Then, by dividing p by q, p / q (%) can be calculated. Figure 2 In this case, 28 / 170=16.47%.

[0167] In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, further preferably 20% or more, and further preferably 30% or more. The upper limit of p / q is not particularly limited and may be, for example, 45% or less.

[0168] For example, the amino acid sequence of a cloned naturally-occurring spider silk fibroin is modified by replacing one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP so that the p / q condition is satisfied, and then modifying the amino acid sequence to include a region with a high hydrophobicity index. Alternatively, for example, an amino acid sequence satisfying the p / q condition can be designed based on the amino acid sequence of a naturally-occurring spider silk fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence to obtain the fifth modified fibroin. In any case, in addition to modifications corresponding to substitution of one or more amino acid residues in REP with amino acid residues with a high hydrophobicity index and / or insertion of one or more amino acid residues with a high hydrophobicity index into REP, modifications corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues can also be performed compared to the naturally-occurring spider silk fibroin.

[0169] There are no particular restrictions on the amino acid residues with a large hydrophobicity index, but preferred are isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A), and more preferred are valine (V), leucine (L) and isoleucine (I).

[0170] As specific examples of the fifth modified silk fibroin, there can be listed modified silk fibroin comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (5-i) sequence number 19, sequence number 20 or sequence number 21, or the amino acid sequence shown in (5-ii) sequence number 19, sequence number 20 or sequence number 21.

[0171] The modified silk fibroin of (5-i) is described. The amino acid sequence shown in SEQ ID NO: 22 is obtained by modifying (A) of the naturally derived spider silk fibroin. nThe amino acid sequence of consecutive alanine residues in the motif is deleted, so that the number of consecutive alanine residues becomes 5. The amino acid sequence shown in SEQ ID NO: 19 is relative to the amino acid sequence shown in SEQ ID NO: 22, in which an amino acid sequence (VLI) consisting of three amino acid residues is inserted at every other REP, and part of the amino acids on the C-terminal side are deleted so that the molecular weight is approximately the same as that of the amino acid sequence shown in SEQ ID NO: 22. The amino acid sequence shown in SEQ ID NO: 23 is relative to the amino acid sequence shown in SEQ ID NO: 22, in which each (A) n Two alanine residues are inserted into the C-terminal side of the motif, and further, some glutamine (Q) residues are replaced with serine (S) residues, and some amino acids on the C-terminal side are deleted so that the molecular weight is substantially the same as that of the amino acid sequence set forth in SEQ ID NO: 22. The amino acid sequence set forth in SEQ ID NO: 20 has an amino acid sequence (VLI) consisting of three amino acid residues inserted into every other REP relative to the amino acid sequence set forth in SEQ ID NO: 23. The amino acid sequence set forth in SEQ ID NO: 21 has an amino acid sequence (VLI) consisting of three amino acid residues inserted into every other REP relative to the amino acid sequence set forth in SEQ ID NO: 23.

[0172] The modified silk fibroin of (5-i) can be composed of the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0173] The modified silk fibroin of (5-ii) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified silk fibroin of (5-ii) also comprises formula 1: [(A) n motif-REP] m The sequence identity is preferably 95% or greater.

[0174] The modified silk fibroin of (5-ii) has a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and preferably, the amino acid sequence of (A) located closest to the C-terminus is removed from the domain sequence. n Of all REPs contained in the sequence obtained from the sequence from the motif to the C-terminus of the domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the residues located closest to the C-terminus (A) are removed from the domain sequence. n When the total number of amino acid residues contained in the sequence from the motif to the C-terminus of the domain sequence is q, p / q is 6.2% or more.

[0175] The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.

[0176] As a more specific example of the 5th modified silk fibroin containing a tag sequence, there can be listed a modified silk fibroin including an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (5-iii) sequence number 24, sequence number 25 or sequence number 26, or the amino acid sequence shown in (5-iv) sequence number 24, sequence number 25 or sequence number 26.

[0177] The amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26 is obtained by adding the amino acid sequence represented by SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, respectively.

[0178] The modified silk fibroin of (5-iii) can be composed of the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0179] The modified silk fibroin of (5-iv) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. The modified silk fibroin of (5-iv) also comprises formula 1: [(A) n motif-REP] m The sequence identity is preferably 95% or greater.

[0180] The modified silk fibroin (5-iv) has a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and preferably, the amino acid residue (A) located closest to the C-terminus is removed from the domain sequence. n Of all REPs contained in the sequence obtained from the sequence from the motif to the C-terminus of the domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the residues located closest to the C-terminus (A) are removed from the domain sequence. n When the total number of amino acid residues contained in the sequence from the motif to the C-terminus of the domain sequence is q, p / q is 6.2% or more.

[0181] The fifth modified silk fibroin may contain a secretion signal for releasing the protein produced by the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of host.

[0182] The modified silk fibroin having a domain sequence for reducing the content of glutamine residues (the sixth modified silk fibroin) has an amino acid sequence for reducing the content of glutamine residues compared to naturally-derived spider silk fibroin.

[0183] The sixth modified fibroin preferably contains at least one motif selected from the group consisting of a GGX motif and a GPGXX motif in the amino acid sequence of REP.

[0184] When the sixth modified fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited and may be 50% or less or 30% or less.

[0185] In this specification, the "GPGXX motif content rate" is a value calculated by the following method.

[0186] In the formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n In the spider silk fibroin having the domain sequence shown in the motif, the domain sequence is excluding the C-terminal end (A) n Among all REPs contained in the sequence obtained from the sequence from the GPGXX motif to the C-terminus of the domain sequence, the number obtained by multiplying the total number of GPGXX motifs contained in the region by 3 (i.e., the total number of G and P in the GPGXX motif) is set as s, and the (A) located closest to the C-terminus is removed from the domain sequence. n The sequence from the motif to the C-terminus of the domain sequence is removed (A) n When the total number of amino acid residues of all REPs obtained after the motif is set as t, the GPGXX motif content rate is calculated as s / t.

[0187] In the calculation of the GPGXX motif content, the motif (A) located closest to the C-terminus was removed from the domain sequence. n The purpose of "sequences from the motif to the C-terminus of the domain sequence" is to exclude the following influence: "(A) located at the most C-terminal side" n The sequence from the motif to the C-terminus of the domain sequence (equivalent to the sequence of REP) may contain sequences with low relevance to the characteristic sequences of spider silk fibroin. When m is small (that is, when the domain sequence is short), this may affect the calculation results of the GPGXX motif content. In addition, when the "GPGXX motif" is located at the C-terminus of REP, it is treated as a "GPGXX motif" even if "XX" is "AA", for example.

[0188] Figure 3 This is a schematic diagram showing the domain sequence of spider silk fibroin. Figure 3 Let's explain in detail how to calculate the GPGXX motif content. Figure 3 The domain sequence of spider silk fibroin shown ("[(A) n motif-REP] m -(A) n motif" type), all REPs are included in the "removal from the domain sequence from the (A) located at the most C-terminal side" n "(The sequence obtained by the sequence from the motif to the C-terminus of the domain sequence)" Figure 3 In the sequence shown in "region A" in the figure, the number of GPGXX motifs used to calculate s is 7, and s is 7×3=21. Similarly, since all REPs are included in the "region A" located closest to the C-terminal side of the domain sequence, n "(The sequence obtained by the sequence from the motif to the C-terminus of the domain sequence)" Figure 3 Therefore, (A) is further removed from the sequence. n The total number of amino acid residues of all REPs obtained after the motif is 50+40+10+20+30=150. Next, s / t (%) can be calculated by dividing s by t. Figure 3 In the case of fibroin, it is 21 / 150=14.0%.

[0189] In the sixth modified fibroin, the glutamine residue content is preferably 9% or less, more preferably 7% or less, further preferably 4% or less, and particularly preferably 0%.

[0190] In this specification, the "glutamine residue content" is a value calculated by the following method.

[0191] In the formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n In the spider silk fibroin having the domain sequence shown in the motif, the domain sequence is excluding the C-terminal end (A) n The sequence obtained by the sequence from the motif to the C-terminus of the domain sequence (equivalent to Figure 3 Among all REPs contained in the "region A" of the domain sequence, when the total number of glutamine residues contained in the region is set to u, and the residues located closest to the C-terminus (A) are removed from the domain sequence. n The sequence from the motif to the C-terminus of the domain sequence is removed (A) nWhen the total number of amino acid residues of all REPs obtained after the motif is set as t, the glutamine residue content is calculated as u / t. In the calculation of the glutamine residue content, the ratio is calculated as "removing the residues located closest to the C-terminus (A) from the domain sequence". n The reason for targeting the "sequence obtained from the sequence from the motif to the C-terminus of the domain sequence" is the same as the above reason.

[0192] The domain sequence of the sixth modified fibroin may have an amino acid sequence corresponding to a deletion or substitution of one or more glutamine residues in REP with other amino acid residues, compared to naturally-derived spider silk fibroin.

[0193] The "other amino acid residues" may be any amino acid residue other than a glutamine residue, and are preferably amino acid residues having a hydrophobicity index greater than that of the glutamine residue. The hydrophobicity indexes of the amino acid residues are shown in Table 1.

[0194] As shown in Table 1, examples of amino acid residues having a hydrophobicity index greater than that of glutamine residues include amino acid residues selected from the group consisting of isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). Among these, more preferred are amino acid residues selected from the group consisting of isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), and alanine (A), and even more preferred are amino acid residues selected from the group consisting of isoleucine (I), valine (V), leucine (L), and phenylalanine (F).

[0195] The hydrophobicity of REP in the sixth modified fibroin is preferably -0.8 or higher, more preferably -0.7 or higher, further preferably 0 or higher, even more preferably 0.3 or higher, and particularly preferably 0.4 or higher. The upper limit of REP is not particularly limited and may be 1.0 or lower or 0.7 or lower.

[0196] In this specification, the "hydrophobicity degree of REP" is a value calculated by the following method.

[0197] In the formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n In the spider silk fibroin having the domain sequence shown in the motif, the domain sequence is excluding the C-terminal end (A) nThe sequence obtained by the sequence from the motif to the C-terminus of the domain sequence (equivalent to Figure 3 Among all REPs contained in the "region A" of the domain sequence, when the sum of the hydrophobicity indexes of the amino acid residues in the region is set to v, and the residues located closest to the C-terminus (A) are removed from the domain sequence, n The sequence from the motif to the C-terminus of the domain sequence is removed (A) n When the total number of amino acid residues of all REPs obtained after the motif is set as t, the hydrophobicity of REP is calculated as v / t. In the calculation of the hydrophobicity of REP, the hydrophobicity of REP is calculated by "removing the residues (A) located at the most C-terminal side from the domain sequence". n The reason for targeting the "sequence obtained from the sequence from the motif to the C-terminus of the domain sequence" is the same as the above reason.

[0198] Compared with the naturally derived spider silk fibroin, the domain sequence of the 6th modified fibroin can be further modified by replacing, deleting, inserting and / or adding one or more amino acid residues in the amino acid sequence, in addition to the modification equivalent to the deletion of one or more glutamine residues in REP and / or the replacement of one or more glutamine residues in REP with other amino acid residues.

[0199] For example, the sixth modified silk fibroin can be obtained by deleting one or more glutamine residues in REP and / or replacing one or more glutamine residues in REP with other amino acid residues in the gene sequence of a cloned naturally-occurring spider silk fibroin. Furthermore, for example, the sixth modified silk fibroin can be obtained by designing an amino acid sequence equivalent to the amino acid sequence of a naturally-occurring spider silk fibroin in which one or more glutamine residues in REP are deleted and / or replaced with other amino acid residues, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0200] As more specific examples of the 6th modified silk fibroin, there can be listed modified silk fibroin comprising the amino acid sequence shown in (6-i) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43, or modified silk fibroin comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (6-ii) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43.

[0201] The modified silk fibroin (6-i) will be described.

[0202] The amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) was based on the base sequence and amino acid sequence of natural silk fibroin from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415). n The amino acid sequence consisting of consecutive alanine residues in the motif was modified with amino acids to improve productivity, such as increasing the number of consecutive alanine residues to 5. On the other hand, Met-PRT410 was not modified with glutamine residues (Q), and therefore the glutamine residue content was the same as that of naturally derived silk fibroin.

[0203] The amino acid sequence shown in SEQ ID NO: 27 (M_PRT888) is an amino acid sequence obtained by replacing all QQ residues in Met-PRT410 (SEQ ID NO: 7) with VL residues.

[0204] The amino acid sequence shown in SEQ ID NO: 28 (M_PRT965) is an amino acid sequence obtained by replacing all QQ in Met-PRT410 (SEQ ID NO: 7) with TS and replacing the remaining Q with A.

[0205] The amino acid sequence shown in SEQ ID NO: 29 (M_PRT889) is an amino acid sequence obtained by replacing all QQ in Met-PRT410 (SEQ ID NO: 7) with VL, and replacing the remaining Q with I.

[0206] The amino acid sequence shown in SEQ ID NO: 30 (M_PRT916) is an amino acid sequence obtained by replacing all QQ in Met-PRT410 (SEQ ID NO: 7) with VI, and replacing the remaining Q with L.

[0207] The amino acid sequence shown in SEQ ID NO: 31 (M_PRT918) is an amino acid sequence obtained by replacing all QQ in Met-PRT410 (SEQ ID NO: 7) with VF, and replacing the remaining Q with I.

[0208] The amino acid sequence shown in SEQ ID NO: 34 (M_PRT525) is an amino acid sequence obtained by inserting two alanine residues into the region (A5) composed of consecutive alanine residues relative to Met-PRT410 (SEQ ID NO: 7), deleting two domain sequences on the C-terminal side so that the molecular weight is almost the same as that of Met-PRT410, and replacing the glutamine residue (Q) at position 13 with a serine residue (S) or a proline residue (P).

[0209] The amino acid sequence shown in SEQ ID NO: 32 (M_PRT699) is an amino acid sequence obtained by replacing all QQ in M_PRT525 (SEQ ID NO: 34) with VL.

[0210] The amino acid sequence shown in SEQ ID NO: 33 (M_PRT698) is an amino acid sequence obtained by replacing all QQ in M_PRT525 (SEQ ID NO: 34) with VL, and replacing the remaining Q with I.

[0211] The amino acid sequence shown in sequence number 43 (Met-PRT966) is an amino acid sequence obtained by replacing all QQ in the amino acid sequence shown in sequence number 9 (the amino acid sequence before the amino acid sequence shown in sequence number 42 is added to the C-terminus) with VF, and replacing the remaining Q with I.

[0212] The glutamine residue content of the amino acid sequences represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 43 was 9% or less (Table 2).

[0213] [Table 2]

[0214]

[0215] The modified silk fibroin (6-i) can be composed of the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43.

[0216] The modified silk fibroin of (6-ii) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43. The modified silk fibroin of (6-ii) also comprises Formula 1: [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m -(A) n The protein has a domain sequence represented by the motif. The sequence identity is preferably 95% or more.

[0217] The glutamine residue content of the modified silk fibroin of (6-ii) is preferably 9% or less. Furthermore, the GPGXX motif content of the modified silk fibroin of (6-ii) is preferably 10% or more.

[0218] The sixth modified silk fibroin may comprise a tag sequence at either or both of the N-terminus and the C-terminus, thereby enabling separation, immobilization, detection, and visualization of the modified silk fibroin.

[0219] As more specific examples of the 6th modified silk fibroin containing a tag sequence, there can be listed modified silk fibroin comprising the amino acid sequence shown in (6-iii) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 55 or SEQ ID NO: 56, or modified silk fibroin comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (6-iv) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 55 or SEQ ID NO: 56.

[0220] The amino acid sequences set forth in SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, and 44 have the amino acid sequence set forth in SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, and 43, respectively, with the amino acid sequence set forth in SEQ ID NOs: 12 (including a His-tag sequence and a hinge sequence) added to the N-terminus. Since only the tag sequence was added to the N-terminus, the glutamine residue content did not change, and the glutamine residue content of the amino acid sequences set forth in SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, and 44 was 9% or less (Table 3).

[0221] In the amino acid sequence (PRT1107) shown in sequence number 55, the serine residue (S) of the amino acid sequence (Met-PRT918) shown in sequence number 31 is replaced with an alanine residue (A), a valine residue (V), a leucine residue (L) or an isoleucine residue (I), and a tag sequence is further added to the N-terminus.

[0222] In the amino acid sequence (PRT1083) shown in sequence number 56, the proline residue (P) of the amino acid sequence (Met-PRT918) shown in sequence number 31 is replaced with a threonine residue (T) or a leucine residue (L), and a tag sequence is further added to the N-terminus.

[0223] [Table 3]

[0224]

[0225] The modified silk fibroin (6-iii) can be composed of the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 55 or SEQ ID NO: 56.

[0226] The modified silk fibroin of (6-iv) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 55, or SEQ ID NO: 56. The modified silk fibroin of (6-iv) also comprises Formula 1: [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m -(A) n The protein has a domain sequence represented by the motif. The sequence identity is preferably 95% or more.

[0227] The glutamine residue content of the modified silk fibroin (6-iv) is preferably 9% or less. Furthermore, the GPGXX motif content of the modified silk fibroin (6-iv) is preferably 10% or more.

[0228] The sixth modified silk fibroin may contain a secretion signal for releasing the protein produced by the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of host.

[0229] The modified silk fibroin may be a modified silk fibroin having at least two or more characteristics among the characteristics of the first modified silk fibroin, the second modified silk fibroin, the third modified silk fibroin, the fourth modified silk fibroin, the fifth modified silk fibroin, and the sixth modified silk fibroin.

[0230] Spider silk protein can be a hydrophilic spider silk protein or a hydrophobic spider silk protein. The so-called hydrophobic spider silk protein refers to a spider silk protein having a value (average HI) greater than -0.8 after the sum of the hydrophobicity index (HI) of all amino acid residues constituting the spider silk protein is divided by the total number of amino acid residues, more preferably a protein having an average HI of -0.6 or more, more preferably a protein having an average HI of -0.4 or more, further preferably a protein having an average HI of -0.2 or more, and particularly preferably a spider silk protein having an average HI of 0 or more. The hydrophobicity index is shown in Table 1. In addition, a hydrophilic spider silk protein refers to a spider silk protein having an average HI of -0.8 or less. The average hydrophobicity index (HI) of the protein involved in this embodiment is preferably -1.3 or greater, or preferably -1.0 or greater, or preferably -0.8 or greater, or preferably greater than -0.8, or preferably -0.7 or greater, or preferably -0.6 or greater, more preferably -0.5 or greater, or preferably -0.4 or greater, or preferably -0.3 or greater, or preferably -0.2 or greater, or preferably -0.1 or greater, more preferably 0 or greater, or more preferably 0.1 or greater, or more preferably 0.2 or greater, further preferably 0.3 or greater, and particularly preferably 0.4 or greater.

[0231] The HIs of the amino acid sequences represented by SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41 are shown in Table 4. When calculating the HI of each amino acid sequence, the calculation was performed after excluding sequences unrelated to the modified fibroin (i.e., sequences corresponding to the amino acid sequence represented by SEQ ID NO: 12).

[0232] [Table 4]

[0233] Amino acid sequence Degree of hydrophobicity Amino acid sequence shown in SEQ ID NO: 11 -0.8 Amino acid sequence shown in SEQ ID NO: 15 -0.8 Amino acid sequence shown in SEQ ID NO: 35 0.07 Amino acid sequence shown in SEQ ID NO: 36 -0.16 Amino acid sequence shown in SEQ ID NO: 37 0.55 Amino acid sequence shown in SEQ ID NO: 38 0.54 Amino acid sequence shown in SEQ ID NO: 39 0.49 Amino acid sequence shown in SEQ ID NO: 40 0.21 Amino acid sequence shown in SEQ ID NO: 41 0.48 Amino acid sequence shown in SEQ ID NO: 45 -0.74 Amino acid sequence shown in SEQ ID NO: 47 -1.2 Amino acid sequence shown in SEQ ID NO: 48 0.47 Amino acid sequence shown in SEQ ID NO: 49 -0.531

[0234] Examples of hydrophobic spider silk proteins include the sequence of the first modified silk protein, the sequence of the second modified silk protein, the sequence of the third modified silk protein, the sequence of the fifth modified silk protein, and the sequence of the sixth modified silk protein. More specific examples of hydrophobic spider silk proteins include spider silk proteins comprising the amino acid sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43, and the amino acid sequence set forth in SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 44.

[0235] Examples of hydrophilic spider silk proteins include the sequence of the fourth modified fibroin described above. More specific examples of hydrophilic spider silk proteins include spider silk proteins comprising the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 47.

[0236] The above-mentioned spider silk proteins can be used alone or in combination of two or more.

[0237] For example, spider silk protein can be produced by expressing the nucleic acid in a host transformed with an expression vector having a nucleic acid sequence encoding the spider silk protein and one or more regulatory sequences operably linked to the nucleic acid sequence.

[0238] The preparation method of the nucleic acid encoding spider silk protein is not particularly limited. For example, the nucleic acid can be manufactured by utilizing a gene encoding natural spider silk protein and amplifying and cloning by polymerase chain reaction (PCR), or by a method for chemical synthesis. The chemical synthesis method of nucleic acid is also not particularly limited. For example, the gene can be chemically synthesized by connecting the amino acid sequence information of spider silk protein obtained from the network database of NCBI, etc. and oligonucleotides automatically synthesized by AKTA oligopilot plus10 / 100 (GE Healthcare Japan Co., Ltd.) using PCR, etc. At this time, in order to easily purify and / or confirm spider silk protein, the nucleic acid encoding the spider silk protein consisting of the amino acid sequence consisting of the amino acid sequence consisting of the start codon and the His10 tag can be synthesized at the N-terminus.

[0239] Regulatory sequences are sequences that control the expression of recombinant proteins in the host (e.g., promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.), and can be appropriately selected according to the type of host. As promoters, inducible promoters that function in host cells and can express the target spider silk protein can also be used. Inducible promoters are promoters that can control transcription by physical factors such as the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or an increase or decrease in temperature, osmotic pressure, or pH.

[0240] The type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, etc. As the expression vector, a vector that can autonomously replicate in the host cell or can be integrated into the host chromosome and contains a promoter at a position capable of transcribing the nucleic acid encoding the spider silk protein is preferably used.

[0241] As the host, any of prokaryotes and eukaryotic organisms such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be appropriately used.

[0242] When using prokaryotes such as bacteria as hosts, the expression vector is preferably one that can autonomously replicate in prokaryotes and contains a promoter, a ribosome binding sequence, a nucleic acid encoding a spider silk protein, and a transcription termination sequence. It may also contain a gene that controls the promoter.

[0243] Examples of prokaryotes include microorganisms belonging to the genera Escherichia, Bacillus pumilus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Bacillus pumilus include Bacillus pumilus in soil. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphilum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium formicum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

[0244] When using prokaryotes as hosts, examples of vectors for introducing nucleic acids encoding spider silk proteins include pBTrp2 (manufactured by Boehringer Ingelheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (Japanese Patent Application Publication No. 2002-238569).

[0245] Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.). Examples of yeast include yeast belonging to the genera Saccharomyces, Pichia, and Schizosaccharomyces. Examples of filamentous fungi include filamentous fungi belonging to the genera Aspergillus, Penicillium, and Trichoderma.

[0246] When using eukaryotic cells as hosts, examples of vectors for introducing nucleic acids encoding spider silk proteins include YEp13 (ATCC 37115) and YEp24 (ATCC 37051). Any method for introducing an expression vector into such host cells can be used, as long as it is a method for introducing DNA into such host cells. Examples include methods using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, spheroplasting, protoplasting, lithium acetate, and competent cell methods.

[0247] As methods for expressing nucleic acids using a host transformed with an expression vector, in addition to direct expression, secretory production and fusion protein expression can be performed according to the methods described in Molecular Cloning, 2nd edition.

[0248] For example, the transformed host can be cultured in a culture medium, and the spider silk protein can be produced and accumulated in the culture medium, and then collected from the culture medium, thereby preparing the spider silk protein. The method of culturing the transformed host in the culture medium can be carried out according to the method commonly used for culturing the host.

[0249] When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, any natural medium or synthetic medium can be used as a culture medium as long as it contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host and can efficiently culture the host.

[0250] The carbon source may be any substance assimilable by the host, for example, carbohydrates such as glucose, fructose, sucrose, and molasses containing these sugars, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.

[0251] As nitrogen sources, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermentation bacteria and their digestion products can be used.

[0252] As the inorganic salts, for example, potassium hydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

[0253] Cultivation of prokaryotes such as Escherichia coli or eukaryotic organisms such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40°C. The culture time is generally 16 hours to 7 days. The pH of the culture medium during cultivation is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.

[0254] Furthermore, during culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed. When culturing microorganisms transformed with an expression vector using an inducible promoter, an inducer may be added to the culture medium as needed. For example, when culturing microorganisms transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside may be added to the culture medium; when culturing microorganisms transformed with an expression vector using the trp promoter, indoleacrylic acid may be added to the culture medium.

[0255] The spider silk protein produced by the transformed host can be isolated and purified using methods commonly used for protein isolation and purification. For example, when the spider silk protein is expressed in a dissolved state within the cells, after completion of the culture, the host cells are recovered by centrifugation, suspended in an aqueous buffer, and then disrupted using an ultrasonic disruptor, French press, Manton-Gaulin high-pressure homogenizer, or Dyno mill to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified product can be obtained by using methods commonly used for the separation and purification of proteins, namely, solvent extraction, salting-out using ammonium sulfate or the like, desalting, precipitation using an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-agarose and DIAION HPA-75 (Mitsubishi Chemical Corporation), cation exchange chromatography using resins such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using resins such as butyl agarose and phenyl agarose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric point electrophoresis, alone or in combination.

[0256] As the above-mentioned chromatography, column chromatography using Phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), or Sephadex G-150 (Pharmacia Biotech) is preferably used.

[0257] Alternatively, if the spider silk protein forms an insoluble form within the cell during expression, the host cells can be similarly recovered and then disrupted and centrifuged to recover the insoluble form of the spider silk protein in the form of a precipitate fraction. The recovered insoluble form of the spider silk protein can be solubilized using a protein denaturant. Following this operation, a purified standard product of the spider silk protein can be obtained using the same separation and purification methods as described above.

[0258] When spider silk proteins are secreted outside the cells, they can be recovered from the culture supernatant. Specifically, the culture is treated by centrifugation or the like to obtain a culture supernatant, and a purified standard can be obtained from the culture supernatant using the same separation and purification method as described above.

[0259] As a structural protein (collagen) derived from collagen, for example, there can be mentioned a protein comprising the formula 3: [REP3] p A structural protein having the domain sequence shown in Formula 3 (wherein, p represents an integer from 5 to 300; REP3 represents an amino acid sequence consisting of Gly-XY; X and Y represent any amino acid residue other than Gly; and a plurality of REP3 residues may be identical or different amino acid sequences). Specifically, a structural protein comprising the amino acid sequence shown in SEQ ID NO: 45 can be exemplified. The amino acid sequence shown in SEQ ID NO: 45 has the amino acid sequence shown in SEQ ID NO: 46 (tag sequence and hinge sequence) added to the N-terminus of the amino acid sequence from residue 301 to residue 540 corresponding to the repeating portion and motif of the partial sequence of human type IV collagen obtained from the NCBI database (NCBI GenBank Accession No.: CAA56335.1, GI: 3702452). Furthermore, as a structural protein derived from collagen, a structural protein comprising the amino acid sequence shown in SEQ ID NO: 59 can be exemplified.

[0260] Examples of structural proteins derived from resilin (resilin) ​​include those comprising the formula 4: [REP4] qThe structural protein of the domain sequence shown in Formula 4 (wherein, in Formula 4, q represents an integer of 4 to 300. REP4 represents an amino acid sequence consisting of Ser-JJ-Tyr-Gly-U-Pro. J represents an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr. U represents an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser. The multiple REP4s that exist may have the same amino acid sequence or different amino acid sequences. Specifically, the amino acid sequence shown in SEQ ID NO: 47 can be listed. The amino acid sequence shown in sequence number 47 adds the amino acid sequence (tag sequence and hinge sequence) shown in sequence number 46 to the N-terminus of the amino acid sequence of the sequence obtained after the Thr of the 87th residue is replaced with Ser and the Asn of the 95th residue is replaced with Asp. In addition, as the structural protein from elastic protein, the structural protein comprising the amino acid sequence shown in sequence number 60 can be listed.

[0261] As structural proteins derived from elastin (elastin), for example, structural proteins having amino acid sequences such as NCBI GenBank Accession Nos. AAC98395 (human), I47076 (sheep), and NP786966 (cattle) can be cited. Specifically, a structural protein comprising the amino acid sequence set forth in SEQ ID NO: 48 can be cited. The amino acid sequence set forth in SEQ ID NO: 48 has the amino acid sequence set forth in SEQ ID NO: 46 (tag sequence and hinge sequence) added to the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of NCBI GenBank Accession No. AAC98395.

[0262] As structural proteins (keratins) derived from keratin, for example, type I keratin from goats (Caprahircus) can be cited. Specifically, a structural protein comprising the amino acid sequence shown in SEQ ID NO: 49 (amino acid sequence of NCBI GenBank Accession No. ACY30466) can be cited. The amino acid sequence shown in SEQ ID NO: 49 has the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 46 added to the N-terminus of the amino acid sequence of NCBI GenBank Accession No. ACY30466. In addition, for example, as structural proteins derived from keratin, a structural protein comprising the amino acid sequence shown in SEQ ID NO: 58 can be cited. The amino acid sequence shown in SEQ ID NO: 58 has an amino acid sequence consisting of amino acid residues 1 to 292 from the N-terminus of SEQ ID NO: 49, an amino acid sequence in which leucine or valine is substituted with alanine or glycine, an amino acid sequence in which three amino acid residues from the 1st to 246th amino acid residues from the N-terminus are substituted, and an amino acid sequence obtained by inserting an amino acid sequence consisting of GAAAAAG (SEQ ID NO: 62) is repeated.

[0263] Collagen, elastic protein, elastin and keratin can be either hydrophilic or hydrophobic proteins. A hydrophobic protein is a protein having a value (average HI) greater than -0.8 after the sum of the hydrophobicity index (HI) of all amino acid residues constituting the protein is divided by the total number of amino acid residues. More preferably, the average HI is greater than -0.6, more preferably the average HI is greater than -0.4, further preferably the average HI is greater than -0.2, and particularly preferably the average HI is greater than 0. The hydrophobicity index is shown in Table 1. Furthermore, a hydrophilic protein is a protein having an average HI of less than -0.8.

[0264] Examples of the hydrophobic collagen, hydrophobic elastic protein, hydrophobic elastin, and hydrophobic keratin include proteins comprising the amino acid sequence shown in SEQ ID NO: 45, SEQ ID NO: 48, or SEQ ID NO: 49.

[0265] Examples of the hydrophilic collagen, hydrophilic elastic protein, hydrophilic elastin, and hydrophilic keratin include proteins containing the amino acid sequence represented by SEQ ID NO: 47.

[0266] The HIs of the amino acid sequences represented by SEQ ID NOs: 45, 47, 48, and 49 are shown in Table 4. When calculating the HI of each amino acid sequence, sequences unrelated to collagen, resilin, elastin, and keratin (i.e., sequences corresponding to the amino acid sequence represented by SEQ ID NO: 12) were excluded.

[0267] Furthermore, structural proteins include hydrophobic proteins and polypeptides that tend to self-aggregate in polar solvents, and the structural proteins are preferably hydrophobic proteins. Structural proteins or structural proteins derived therefrom can be used alone or in combination of two or more. By combining two or more structural proteins, the overall hydrophobicity can be adjusted to a desired value. The hydrophobicity can be calculated using the above-described method.

[0268] (Organic Solvent)

[0269] The organic solvent of the spinning solution involved in the present embodiment can be used as long as it is an organic solvent that can dissolve artificial protein. As an organic solvent, for example, hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl-2-imidazolidinone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine N-oxide (NMO) and formic acid can be listed. These solvents can be used alone or in combination of two or more. For example, the organic solvent can include at least one selected from the group consisting of formic acid, DMSO and HFIP, or at least one selected from the group consisting of formic acid, DMSO and HFIP, or formic acid. These organic solvents can also be water-containing.

[0270] When the total amount of the spinning solution is set to 100% by mass, the protein content in the spinning solution involved in this embodiment is preferably 10-40% by mass, more preferably 10-35% by mass, more preferably 12-35% by mass, further preferably 15-35% by mass, more preferably 15-30% by mass, further preferably 20-35% by mass, and particularly preferably 20-30% by mass. When the content of structural protein is 10% by mass or more, the influence of the accompanying flow generated in the coagulation bath on the fibers formed can be further reduced, and productivity can be improved. When the content of structural protein is 40% by mass or less, the spinning solution can be ejected more stably from the spinning spinneret, and productivity can be improved.

[0271] (Dissolution Accelerator) The spinning solution according to this embodiment may further contain a dissolution accelerator. By containing a dissolution accelerator, the preparation of the spinning solution becomes easy.

[0272] The dissolution promoter may be an inorganic salt composed of a Lewis acid and a Lewis base as shown below. Examples of the Lewis base include halide ions. Examples of the Lewis acid include metal ions such as alkali metal ions, halides, and alkaline earth metal ions. Examples of the inorganic salt include alkali metal halides and alkaline earth metal halides. Specific examples of alkali metal halides include lithium chloride and lithium bromide. Specific examples of alkaline earth halides include magnesium chloride and calcium chloride. The dissolution promoter may be used alone or in combination of two or more.

[0273] These inorganic salts can be used as dissolution promoters for structural proteins in formic acid or DMSO, with lithium chloride and calcium chloride being particularly preferred. By incorporating these dissolution promoters (these inorganic salts) into the spinning solution, structural proteins can be dissolved at high concentrations in the spinning solution. This further improves the production efficiency of protein fibers and is expected to enhance the quality and physical properties of the protein fibers, such as stress resistance.

[0274] Relative to the total amount of the spinning solution, the content of the dissolution promoter can be more than 0.1 mass%, more than 1 mass%, more than 2 mass%, more than 3 mass%, more than 4 mass%, more than 7 mass%, more than 10 mass% or more than 15 mass%, and can also be less than 20 mass%, less than 16 mass%, less than 12 mass% or less than 9 mass%.

[0275] (Various Additives) The spinning solution may further contain various additives as needed. Examples of additives include plasticizers, leveling agents, crosslinking agents, crystallization nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers, and synthetic resins. The additive content may be 50 parts by mass or less relative to 100 parts by mass of the total protein content in the spinning solution.

[0276] The viscosity of the spinning solution according to the present embodiment can be appropriately set according to the intended use of the fiber to be produced, the spinning method, etc. For example, at 20° C., it can be 60,000 to 130,000 mPa·sec or 65,000 to 125,000 mPa·sec. In addition, for example, at 35°C, it can be 500-35,000 mPa·sec, 1,000-35,000 mPa·sec, 3,000-30,000 mPa·sec, 500-20,000 mPa·sec, 500-15,000 mPa·sec, 1,000-15,000 mPa·sec, 1,000-12,000 mPa·sec, 1,500-12,000 mPa·sec, 1,500-10,000 mPa·sec, 1,500-8,000 mPa·sec, etc. For example, at 40°C, the viscosity may be 500 to 35,000 mPa·sec, 1,000 to 35,000 mPa·sec, 5,000 to 35,000 mPa·sec, 10,000 to 30,000 mPa·sec, 12,000 to 30,000 mPa·sec, or 12,000 to 28,000 mPa·sec. Furthermore, for example, at 70°C, the viscosity may be 1,000 to 6,000 mPa·sec, or 1,500 to 5,000 mPa·sec. The viscosity of the spinning solution can be measured using, for example, an "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

[0277] To promote dissolution, the spinning solution may be stirred or shaken for a period of time. In this case, the spinning solution may be heated to a temperature at which the protein can be dissolved, depending on the structural protein and solvent used. The spinning solution may be heated, for example, to 30°C or higher, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, or 90°C or higher. To further prevent decomposition of the artificial protein, the temperature is preferably 40°C. The upper limit of the heating temperature is, for example, below the boiling point of the solvent.

[0278] <Coagulation liquid>

[0279] The coagulation liquid involved in this embodiment contains water or an aqueous solution with a pH of 0.25 or higher and a pH of 10.00 or lower. Thus, a method for producing protein fibers can be provided that reduces the risk of explosion / fire, manufacturing costs, and environmental impact. The aqueous solution can be a saline solution, an acid solution, or a mixed solution of a saline solution and an acid solution. It can also be a saline solution or a mixed solution of a saline solution and an acid solution, or a saline solution. Here, the mixed solution of a saline solution and an acid solution is not limited to a solution in which a saline solution and an acid solution are mixed, but also includes a solution in which an acid is mixed in a saline solution, a solution in which a salt is mixed in an acid solution, and a solution in which a salt and an acid are dissolved in water.

[0280] (aqueous acid solution)

[0281] Examples of the acidic aqueous solution include aqueous solutions of carboxylic acids, and specific examples of carboxylic acids include formic acid, acetic acid, propionic acid, citric acid, and oxalic acid. These solvents may be used alone or in admixture of two or more. For example, the acidic aqueous solution may be an aqueous solution of citric acid or an aqueous solution of formic acid.

[0282] (Saline solution)

[0283] Examples of the saline solution include saline solutions of organic salts or inorganic salts, and mixed aqueous solutions of organic salts and inorganic salts.

[0284] Examples of the organic salt include carboxylates, and specific examples of the carboxylates include formates, acetates, propionates, citrates, and oxalates. For example, the organic salt may also include formates, acetates, and citrates.

[0285] Specific examples of the formates include ammonium formate, potassium formate, sodium formate, lithium formate, magnesium formate, and calcium formate.

[0286] Specific examples of acetates include ammonium acetate, potassium acetate, sodium acetate, lithium acetate, magnesium acetate, and calcium acetate.

[0287] Specific examples of propionate salts include ammonium propionate, potassium propionate, sodium propionate, lithium propionate, magnesium propionate, and calcium propionate.

[0288] Specific examples of citrate salts include ammonium citrate, potassium citrate, sodium citrate, lithium citrate, magnesium citrate, and calcium citrate. For example, the citrate salt may include at least one selected from the group consisting of ammonium citrate, potassium citrate, sodium citrate, magnesium citrate, and calcium citrate, may further include at least one selected from the group consisting of ammonium citrate, potassium citrate, and sodium citrate, may further include at least one selected from the group consisting of potassium citrate and sodium citrate, and may also be sodium citrate.

[0289] Specific examples of oxalates include ammonium oxalate, potassium oxalate, sodium oxalate, lithium oxalate, magnesium oxalate, and calcium oxalate. Carboxylates are more preferably sodium carboxylates, and specific examples of sodium carboxylates include sodium formate, sodium acetate, sodium propionate, and sodium oxalate.

[0290] Specific examples of the inorganic salt include normal salts, acidic salts, and basic salts.

[0291] Specific examples of normal salts include sulfates, chlorides, nitrates, iodides, thiocyanates, and carbonates.

[0292] Specific examples of sulfates include ammonium sulfate, potassium sulfate, sodium sulfate, lithium sulfate, magnesium sulfate, and calcium sulfate. For example, the sulfate may include at least one selected from the group consisting of ammonium sulfate, sodium sulfate, magnesium sulfate, and calcium sulfate, and may also include at least one selected from the group consisting of ammonium sulfate and sodium sulfate, or may be sodium sulfate.

[0293] Specific examples of chlorides include ammonium chloride, potassium chloride, sodium chloride, lithium chloride, magnesium chloride, calcium chloride, and guanidine chloride. For example, the chloride may include at least one selected from the group consisting of ammonium chloride, potassium chloride, sodium chloride, lithium chloride, calcium chloride, magnesium chloride, and guanidine chloride. The chloride may also include at least one selected from the group consisting of ammonium chloride, potassium chloride, sodium chloride, lithium chloride, calcium chloride, and magnesium chloride. The chloride may also include at least one selected from the group consisting of potassium chloride, sodium chloride, and calcium chloride. The chloride may also include at least one selected from the group consisting of sodium chloride and calcium chloride, or may be sodium chloride.

[0294] Specific examples of nitrates include ammonium nitrate, potassium nitrate, sodium nitrate, lithium nitrate, magnesium nitrate, and calcium nitrate.

[0295] Specific examples of the iodide salt include ammonium iodide, potassium iodide, sodium iodide, lithium iodide, magnesium iodide, and calcium iodide.

[0296] Specific examples of the thiocyanate include ammonium thiocyanate, potassium thiocyanate, sodium thiocyanate, lithium thiocyanate, magnesium thiocyanate, calcium thiocyanate, and guanidine thiocyanate.

[0297] Specific examples of carbonates include ammonium carbonate, potassium carbonate, sodium carbonate, lithium carbonate, magnesium carbonate, and calcium carbonate.

[0298] Specific examples of the acidic salt include hydrogen sulfate, hydrogen phosphate, and hydrogen carbonate.

[0299] Specific examples of hydrogen sulfate include ammonium hydrogen sulfate, potassium hydrogen sulfate, sodium hydrogen sulfate, lithium hydrogen sulfate, magnesium hydrogen sulfate, and calcium hydrogen sulfate.

[0300] Specific examples of hydrogen phosphates include sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, diammonium hydrogen phosphate, magnesium dihydrogen phosphate, dimagnesium hydrogen phosphate, calcium dihydrogen phosphate, and dicalcium hydrogen phosphate.

[0301] Specific examples of the bicarbonate include ammonium bicarbonate, potassium bicarbonate, sodium bicarbonate, lithium bicarbonate, magnesium bicarbonate, and calcium bicarbonate.

[0302] Specific examples of the basic salt include calcium hydroxide chloride, magnesium hydroxide chloride, and the like.

[0303] The above-mentioned acids, acid aqueous solutions, salts, and saline aqueous solutions may be used alone or in combination of two or more.

[0304] Examples of salt mixed aqueous solutions obtained by mixing two or more salts or salt solutions include mixed aqueous solutions of the above-mentioned organic salts, mixed aqueous solutions of the above-mentioned inorganic salts, and mixed aqueous solutions of the above-mentioned organic and inorganic salts. From the perspective of reducing production costs, brackish water and seawater are particularly preferred. Brackish water and seawater are known to primarily contain potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, and calcium sulfate.

[0305] The coagulating liquid preferably contains a saline solution, more preferably a saline solution. The inclusion of a salt further increases the desolvation rate. The salt more preferably includes at least one member selected from the group consisting of carboxylates, sulfates, chlorides, hydrogen phosphates, and hydrogen carbonates, further preferably at least one member selected from the group consisting of carboxylates, sulfates, and chlorides, and particularly preferably at least one member selected from the group consisting of sulfates and chlorides. The inclusion of these salts further enhances fiber-forming ability and the resulting fiber's elongation.

[0306] As carboxylates, sodium carboxylates are more preferred; as sulfates, ammonium sulfate, sodium sulfate, magnesium sulfate, and calcium sulfate are more preferred; as chlorides, potassium chloride, sodium chloride, magnesium chloride, and calcium chloride are more preferred; as bicarbonates, sodium bicarbonate is more preferred; and as mixed aqueous solutions, brackish water and seawater are particularly preferred. The use of these salts and mixed aqueous solutions not only improves fiber-forming properties but also further reduces production costs.

[0307] The salt content is 0.1% by mass or more, 0.3% by mass or more, 0.5% by mass or more, 0.7% by mass or more, 1% by mass or more, 1.3% by mass or more, 1.5% by mass or more, 1.7% by mass or more, 2% by mass or more, 2.3% by mass or more, 2.5% by mass or more, 2.7% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, or 20% by mass or more relative to the total amount of the coagulation liquid. As an upper limit, it can be 30% by mass or less, 25% by mass or less, or a content below the solubility. For example, the salt content relative to the total amount of the coagulation liquid may be 0.1% by mass or more and 30% by mass or less, 0.3% by mass or more and 25% by mass or less, 1% by mass or more and 25% by mass or less, 3% by mass or more and 25% by mass or less, 5% by mass or more and 25% by mass or less, 8% by mass or more and 25% by mass or less, 10% by mass or more and 25% by mass or less, 1% by mass or more and 20% by mass or less, 3% by mass or more and 20% by mass or less, 5% by mass or more and 20% by mass or less, 8% by mass or more and 20% by mass or less, 10% by mass or more and 20% by mass or less, 15% by mass or more and 20% by mass or less, or 16% by mass or more and 20% by mass or less. For example, the salt content relative to the total amount of the coagulation liquid is preferably 0.05 mol / L or more, can be 0.05 mol / L or more and 5.5 mol / L or less, can be 0.1 mol / L or more and 5.0 mol / L or less, can be 0.1 mol / L or more and 4.5 mol / L or less, and can be 0.1 mol / L or more and 4.0 mol / L or less.

[0308] For example, when sodium chloride is used, the salt content relative to the total amount of the coagulation liquid may be 0.1 mol / L to 5.0 mol / L, 0.1 mol / L to 4.5 mol / L, or 0.1 mol / L to 4.0 mol / L.

[0309] For example, when potassium chloride is used, the salt content can be 0.1 mol / L or more and 3.9 mol / L or less relative to the total amount of the coagulation liquid.

[0310] For example, when calcium chloride is used, the salt content relative to the total amount of the coagulation liquid may be 0.1 mol / L or more and 14.3 mol / L or less, 0.1 mol / L or more and 13.0 mol / L or less, 0.1 mol / L or more and 12.0 mol / L or less, 0.1 mol / L or more and 11.0 mol / L or less, 0.1 mol / L or more and 10.0 mol / L or less, or 0.1 mol / L or more and 9.0 mol / L or less. It can also be 0.1 mol / L or more and 8.0 mol / L or less, 0.1 mol / L or more and 7.0 mol / L or less, 0.1 mol / L or more and 6.0 mol / L or less, 0.1 mol / L or more and 5.0 mol / L or less, 0.1 mol / L or more and 4.0 mol / L or less, 0.1 mol / L or more and 3.0 mol / L or less, or 0.1 mol / L or more and 2.0 mol / L or less.

[0311] For example, when sodium sulfate is used, the salt content relative to the total amount of the coagulation liquid can be 0.1 mol / L to 3.4 mol / L, 0.1 mol / L to 3.0 mol / L, 0.1 mol / L to 2.5 mol / L, or 0.1 mol / L to 2.0 mol / L. Furthermore, for example, the salt content relative to the total amount of the coagulation liquid can be 3% to 28% by mass, 3% to 25% by mass, 3% to 20% by mass, 5% to 20% by mass, or 8% to 20% by mass. Furthermore, the sodium sulfate content relative to the total amount of the coagulation liquid is preferably 10% to 20% by mass, further preferably 11% to 19% by mass, more preferably 11% to 18% by mass, further preferably 12% to 18% by mass, and particularly preferably 12% to 17% by mass. When the content of sodium sulfate relative to the total amount of the coagulating solution is 10% by mass or more, sufficient coagulation speed can be obtained, and the increase in cost due to equipment investment can be avoided. When the content of sodium sulfate relative to the total amount of the coagulating solution is 20% by mass or less, the occurrence of broken threads at the interface between the spinning solution and the coagulated filaments (threads) caused by the rapid coagulation of the spinning solution can be avoided. In addition, from the perspective of improving the recovery efficiency of the solvent, in the above case, the water content equivalent to the total amount of the coagulating solution is preferably 60% by mass or more and 80% by mass or less, more preferably 60% by mass or more and 70% by mass or less. In addition, the concentration of the sodium sulfate aqueous solution when using sodium sulfate is preferably 10% by mass or more and 22% by mass or less, further preferably 10% by mass or more and 20% by mass or less, more preferably 12% by mass or more and 20% by mass or less, further preferably 14% by mass or more and 20% by mass or less, and particularly preferably 16% by mass or more and 20% by mass or less. When the concentration of the sodium sulfate aqueous solution is 10% by mass or more, sufficient coagulation speed can be obtained, and the increase in cost due to equipment investment can be avoided. When the content of the sodium sulfate aqueous solution is 22% by mass or less, it is possible to avoid yarn breakage at the interface between the spinning solution and the coagulated yarn (thread) due to rapid coagulation of the spinning solution.

[0312] For example, when sodium citrate is used, the salt content may be 0.1 mol / L to 1.6 mol / L, or 0.1 mol / L to 1.3 mol / L, relative to the total amount of the coagulation liquid.

[0313] The aqueous solution contained in the coagulation liquid of this embodiment can be selected from the group consisting of, for example, carboxylic acid aqueous solution, bicarbonate aqueous solution, formate aqueous solution, acetate aqueous solution, chloride aqueous solution, sulfate aqueous solution, hydrogen phosphate aqueous solution, citrate aqueous solution, brackish water, seawater, and mixtures thereof. Furthermore, the aqueous solution contained in the coagulation liquid of this embodiment can be selected from the group consisting of, for example, citric acid aqueous solution, formic acid aqueous solution, sodium bicarbonate aqueous solution, sodium formate aqueous solution, sodium acetate aqueous solution, sodium chloride aqueous solution, sodium sulfate aqueous solution, ammonium sulfate aqueous solution, potassium hydrogen phosphate aqueous solution, calcium chloride aqueous solution, sodium citrate aqueous solution, brackish water, seawater, and mixtures thereof.

[0314] The coagulation liquid before contacting with the spinning solution may contain an organic solvent or may not contain an organic solvent. In addition, even if the coagulation liquid before contacting with the spinning solution does not contain an organic solvent, in the process of contacting the spinning solution with the coagulation liquid, there may be a situation where the organic solvent is dissolved from the contacted spinning solution into the coagulation liquid. Taking the total mass of the coagulation liquid (when the organic solvent is dissolved from the spinning solution into the coagulation liquid, the total content of the coagulation liquid before contacting with the spinning solution and the organic solvent dissolved from the spinning solution into the coagulation liquid) as 100 mass%, the content of the organic solvent contained in the coagulation liquid (including the case where the spinning solution in contact with the coagulation liquid is dissolved into the coagulation liquid) is preferably 0 mass% or more and 40 mass% or less, 0 mass% or more and 30 mass% or less, 5 mass% or more and 30 mass% or less, 5 mass% or more and 25 mass% or less. % or less, 0% or more and 20% or less, 5% or more and 20% or less, 5% or more and 15% or less, 10% or more and 40% or less, 15% or more and 40% or less, 20% or more and 40% or less, 10% or more and 30% or less, 10% or more and 20% or less, 0% or more and 10% or less, 0% or more and 5% or less, 0% or more and 2% or less. When the content of the organic solvent is within the above range, the effect of the present invention is more significant. Based on the total mass of the coagulation liquid as 100% by mass, the content of the organic solvent contained in the coagulation liquid can be 10% or more and 40% or less, 15% or more and 40% or less, or 20% or more and 40% or less. When the content of the organic solvent is within the above range, the fiber-forming ability of the structural protein is further improved. The organic solvent is preferably formic acid, DMSO or HFIP, more preferably formic acid.

[0315] The pH of the aqueous solution contained in the coagulation liquid may be 0.25 to 10.00, or 0.25 to 9.50.

[0316] The pH of the acid aqueous solution in the coagulation liquid may be, for example, 0.25 to less than 7.00, 0.50 to less than 7.00, 1.00 to less than 7.00, 1.50 to less than 7.00, 2.00 to less than 7.00, or 3.00 to less than 7.00.

[0317] The pH of the saline solution in the coagulation liquid may be, for example, 0.50 to 10.00, 1.00 to 10.00, 2.00 to 10.00, 3.00 to 10.00, 3.50 to 10.00, 4.00 to 10.00, 4.50 to 10.00, 5.00 to 10.00, 5.50 to 10.00, 6.00 to 10.00, 6.50 to 10.00, or 6.50 to 9.50.

[0318] The content of the water or aqueous solution in the coagulation liquid is preferably 60% by mass or greater, more preferably 70% by mass or greater, even more preferably 80% by mass or greater, even more preferably 90% by mass or greater, and particularly preferably 95% by mass or greater, relative to the total amount of the coagulation liquid. When the content of the water or aqueous solution is within the above range, the fiber-forming ability of the structural protein is further enhanced. The content of the water or aqueous solution in the coagulation liquid can be, for example, 60% by mass or greater and 100% by mass or less, 70% by mass or greater and 100% by mass or less, 80% by mass or greater and 100% by mass or less, or 95% by mass or greater and 100% by mass or less, relative to the total amount of the coagulation liquid.

[0319] The coagulation liquid may contain formic acid. From the viewpoint of improving solvent recovery efficiency, the formic acid content is preferably 15 to 25% by mass, more preferably 16 to 25% by mass, further preferably 16 to 24% by mass, and particularly preferably 18 to 24% by mass relative to the total amount of the coagulation liquid.

[0320] The temperature of the coagulation liquid may be room temperature, 0°C to 90°C, 0°C to 80°C, 5°C to 80°C, 10°C to 80°C, 15°C to 80°C, 20°C to 80°C, 25°C to 80°C, 30°C to 80°C, 40°C to 80°C, 50°C to 80°C, 60°C to 80°C, 70°C to 80°C, 20°C to 70°C, 30°C to 70°C, 40°C to 70°C, 50°C to 70°C, 20°C to 60°C, 30°C to 60°C, 40°C to 60°C, or 50°C to 60°C. The lower limit of the coagulation liquid temperature can be above the melting point of the organic solvent contained in the spinning solution, and the upper limit of the temperature can be below the boiling point of the organic solvent contained in the spinning solution. By increasing the temperature of the coagulation liquid, the desolvation rate of the spinning solution can be accelerated. In addition, the temperature of the coagulation liquid is preferably 55°C to 65°C, more preferably 45°C to 55°C, and even more preferably 35°C to 45°C. When the coagulation liquid temperature is above 35°C, an appropriate desolvation rate can be achieved, and productivity can be further improved. When the coagulation liquid temperature is below 65°C, rapid softening of the spinning solution caused by heating the spinning solution in the coagulation liquid can be prevented.

[0321] The coagulation solution may further contain the above-mentioned dissolution accelerator which can be added to the spinning solution.

[0322] (Spinning process)

[0323] The method for producing protein fibers according to this embodiment can be produced by a known wet spinning method or a dry-wet spinning method. That is, in the spinning process, the spinning solution is brought into contact with the coagulation solution to coagulate the protein. Including the spinning process, the method for producing protein fibers according to this embodiment can be produced, for example, by using Figure 4 The spinning device shown is used for implementation.

[0324] Figure 4 This is an explanatory diagram schematically showing an example of a spinning apparatus for producing protein fibers. Figure 4 The spinning device 10 shown is an example of a spinning device for wet spinning, and includes an extruder 1, a coagulation bath 20, a washing bath (stretching bath) 21, and a drying device 4 in this order from the upstream side.

[0325] The extrusion device 1 has a storage tank 7 in which a spinning solution (spinning liquid) 6 is stored. A coagulation liquid 11 is stored in a coagulation bath 20. The spinning solution 6 is extruded from a nozzle 9 provided in the coagulation liquid 11 by a gear pump 8 installed at the lower end of the storage tank 7. The extruded spinning solution 6 is supplied (introduced) into the coagulation liquid 11 in the coagulation bath 20. The solvent is removed from the spinning solution in the coagulation liquid 11, and the spider silk protein coagulates. The coagulated spider silk protein is introduced into the washing bath 21, washed with the washing liquid 12 in the washing bath 21, and then conveyed to the drying device 4 by the first roller 13 and the second roller 14 provided in the washing bath 21. At this time, for example, if the rotation speed of the second roller 14 is set faster than the rotation speed of the first roller 13, a protein fiber 36 stretched at a ratio corresponding to the rotation speed ratio can be obtained. After being stretched in the washing liquid 12, the protein fibers are removed from the washing bath 21 and dried while passing through the drying device 4. They are then taken up by the winding machine. This process ultimately produces the protein fibers as a winding material 5 wound on the winding machine through the spinning device 10. Reference numerals 18a to 18g denote yarn guides.

[0326] The temperature of the coagulation liquid 11 is not particularly limited and may be 80°C or less, 70°C or less, 60°C or less, 50°C or less, 40°C or less, 30°C or less, 25°C or less, 20°C or less, 10°C or less, or 5°C or less. From the perspectives of operability and cooling costs, it is preferably 0°C or less. Furthermore, the temperature of the coagulation liquid 11 can be adjusted, for example, by using a spinning apparatus 10 comprising a coagulation bath 20 equipped with a heat exchanger and a cooling circulation device. For example, by passing a medium cooled to a predetermined temperature by the cooling circulation device through a heat exchanger disposed within the coagulation bath, the temperature of the coagulation liquid 11 can be adjusted to within the aforementioned range through heat exchange between the coagulation liquid 11 and the heat exchanger. In this case, by circulating a solvent serving as the medium in the coagulation liquid 11, more efficient cooling can be achieved.

[0327] A plurality of coagulation baths for storing the coagulation liquid may be provided.

[0328] The coagulated artificial structural protein can be directly wound up by a winding machine after leaving the coagulation bath or the washing bath, or can be dried by a drying device and then wound up by a winding machine.

[0329] The distance that the coagulated artificial structural protein passes through the coagulation solution is sufficient as long as the desolvation can be carried out efficiently, and can be determined based on, for example, the extrusion speed (discharge speed) of the spinning solution from the nozzle. The residence time of the coagulated artificial structural protein (or spinning solution) in the coagulation solution can be determined based on, for example, the distance that the coagulated artificial structural protein passes through the coagulation solution, the extrusion speed of the spinning solution from the nozzle, and the like.

[0330] (Stretching process)

[0331] The method for producing the artificial structural protein fiber of this embodiment may further include a step of stretching the coagulated artificial structural protein (stretching step). Examples of stretching methods include wet heat stretching and dry heat stretching. The stretching step may be performed, for example, in the coagulation bath 20 or in the washing bath 21. The stretching step may also be performed in air.

[0332] Stretching in the washing bath 21 can also be performed in warm water or in a solution containing an organic solvent or the like, so-called wet heat stretching. The wet heat stretching temperature is preferably 50-90°C. A temperature of 50°C or higher allows for a stable reduction in the pore diameter of the yarn. A temperature of 90°C or lower facilitates temperature control and improves spinning stability. A more preferred temperature is 75-85°C.

[0333] Wet heat stretching can be carried out in warm water, or in a solution of an organic solvent or the like added to warm water, or in steam heating. The temperature can be, for example, 40 to 200°C, 50 to 180°C, 50 to 150°C, or 75 to 90°C. The stretching ratio during wet heat stretching can be, for example, 1 to 30 times, 2 to 25 times, 2 to 20 times, 2 to 15 times, 2 to 10 times, 2 to 8 times, 2 to 6 times, or 2 to 4 times relative to the unstretched yarn (or pre-stretched yarn). However, the stretching ratio is not limited as long as it is within the range of the desired fiber thickness, mechanical properties, and other characteristics.

[0334] Dry heat stretching can be performed using a device such as a contact hot plate or a non-contact oven, but is not particularly limited as long as the device can heat the fiber to a predetermined temperature and stretch it at a predetermined ratio. The temperature can be, for example, 100°C to 270°C, 140°C to 230°C, 140°C to 200°C, 160°C to 200°C, or 160°C to 180°C.

[0335] The draw ratio in the dry heat drawing step can be, for example, 1 to 30 times, 2 to 30 times, 2 to 20 times, or 3 to 15 times relative to the undrawn yarn (or pre-drawn yarn), preferably 3 to 10 times, more preferably 3 to 8 times, and even more preferably 4 to 8 times. However, the draw ratio is not limited as long as it is within a range that can achieve the desired fiber characteristics such as thickness and mechanical properties.

[0336] In the stretching step, wet heat stretching and dry heat stretching may be performed separately, or they may be performed in multiple stages, or they may be performed in combination. That is, as the stretching step, the first stage stretching may be performed by wet heat stretching, and the second stage stretching may be performed by dry heat stretching, or the first stage stretching may be performed by wet heat stretching, the second stage stretching may be performed by wet heat stretching, and the third stage stretching may be performed by dry heat stretching. The wet heat stretching and dry heat stretching may be appropriately combined.

[0337] Relative to the unstretched yarn (or pre-stretched yarn), the lower limit of the final stretching ratio of the artificial structural protein fiber after the stretching process is preferably 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or 9 times. The upper limit of the final stretching ratio of the modified silk core protein fiber after the stretching process is preferably 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times or 10 times. In addition, for example, the final stretching ratio can be 3 to 40 times, 3 to 30 times, 5 to 30 times, 5 to 20 times, 5 to 15 times, or 5 to 13 times. However, the stretching ratio is not limited as long as it is within the range of the desired fiber thickness, mechanical properties and other characteristics.

[0338] In the spinning step, the spinneret shape, hole shape, number of holes, etc. of the spinning spinneret are not particularly limited and can be appropriately selected according to the desired fiber diameter and number of filaments.

[0339] Before or after drying, an oil may be applied to the undrawn yarn (or pre-drawn yarn) or the drawn yarn, as needed, for the purpose of imparting charge suppression, astringency, lubricity, etc. The type and amount of the oil applied are not particularly limited and can be appropriately adjusted based on the intended use of the fiber and the handleability of the fiber.

[0340] When the spinneret's holes are circular, the spinneret's pore diameter can be, for example, 0.01 mm or greater and 0.6 mm or less. A pore diameter of 0.01 mm or greater reduces pressure loss and equipment costs. A pore diameter of 0.6 mm or less reduces the need for stretching operations to reduce fiber diameter and reduces the likelihood of tensile fractures during the process from ejection to winding.

[0341] The temperature of the spinning solution as it passes through the spinneret and the temperature of the spinning nozzle are not particularly limited and can be adjusted appropriately depending on the concentration and viscosity of the spinning solution, the type of organic solvent used, and other factors. To prevent degradation of structural proteins, the temperature is preferably between 30°C and 100°C. Furthermore, to reduce the possibility of pressure increases due to solvent volatilization and clogging of the piping due to solidification of the spinning solution, the upper limit of the temperature is preferably below the boiling point of the solvent used. This improves process stability.

[0342] The production method according to the present embodiment may further include a step of filtering the spinning solution before ejecting the spinning solution (filtration step) and / or a step of degassing the spinning solution before ejecting the spinning solution (degassing step).

[0343] (Evaluation of fiber physical properties)

[0344] The physical properties of artificial structural protein fibers can be measured and evaluated in the following manner.

[0345] The fiber fineness and elongation of randomly sampled fibers were measured using a single-filament strength and elongation measuring device "FAVIMAT" manufactured by Textechno under an environment of 20°C and 65% relative humidity, and the average value was calculated. The preferred conditions for measuring strength and elongation are: a load cell capacity of 210 cN, a needle length of 20 mm, and a tensile speed of 10 mm / min. The area enclosed by the stress-strain curve and the horizontal axis, where the strength at break is defined as the breaking strength [g / d], the elongation at break is defined as the breaking elongation [%], the horizontal axis is the strain [%], and the vertical axis is the stress [g / d], is multiplied by the density [kg / m 3 ] and the obtained value is defined as toughness [MJ / m 3 ]. Preferably, the measured value is calculated as an average value of, for example, a sampling number n=10.

[0346] (Evaluation of fiber shrinkage)

[0347] Artificial structural protein fibers have the property of shrinking when in contact with (wetting) water below the boiling point. In artificial structural protein fibers, the less such shrinkage is, the more preferable it is.

[0348] The shrinkage property can be evaluated using, for example, a shrinkage ratio determined by the following method as an indicator.

[0349] A plurality of artificial structural protein fibers of about 30 cm in length are bundled together to form a fiber bundle with a fineness of 150 deniers. A plumb bob weighing 0.8 g is installed on this fiber bundle, and in this state, the fiber bundle is immersed in 40°C water for 90 seconds to shrink it. Then, each fiber bundle is taken out of the water, dried with a plumb bob weighing 0.8 g installed, and the length of each fiber bundle after drying is measured. The shrinkage rate is calculated according to the following formula. In addition, L0 represents the fiber length before contact with water (after spinning) (in this case, 30 cm), L D It indicates the length of the fiber after shrinkage (dry fiber after immersion in water).

[0350] Formula: Shrinkage [%] = {1-(L D / L0)}×100

[0351] (product)

[0352] The protein fibers involved in this embodiment can be used as fibers (long fibers, short fibers, monofilaments, or multifilaments) or yarns (woven yarns, twisted yarns, false twisted yarns, processed yarns, mixed yarns, or blended yarns) in fabrics such as woven, knitted, braided, or non-woven fabrics, paper, and cotton. Furthermore, they can be used in high-strength applications such as ropes, surgical sutures, hemostats, flexible fasteners for electrical components, and physiologically active materials for transplantation (e.g., artificial ligaments and aortic bands). These can be prepared by known methods.

[0353] Example

[0354] Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.

[0355] (Preparation of artificial structural proteins)

[0356] (1) Preparation of expression vector

[0357] Based on the base sequence and amino acid sequence of the silk fibroin from the golden-spinning spider (Nephila clavipes) (GenBank accession number: P46804.1, GI: 1174415), an artificial structural protein (modified silk fibroin) having sequence number 44 (hereinafter also referred to as "PRT966"), a modified silk fibroin having sequence number 15 (hereinafter also referred to as "PRT799"), a modified silk fibroin having sequence number 37 (hereinafter also referred to as "PRT918"), a modified silk fibroin having sequence number 50 (hereinafter also referred to as "PRT705"), a modified silk fibroin having sequence number 51 (hereinafter also referred to as "PRT52"), and a modified silk fibroin having sequence number 52 (hereinafter also referred to as "PRT53") were sequenced. The following are also referred to as "PRT826" ), a modified silk fibroin with sequence number 52 (hereinafter referred to as "PRT853"), a modified silk fibroin with sequence number 53 (hereinafter referred to as "PRT1103"), a modified silk fibroin with sequence number 54 (hereinafter referred to as "PRT1104"), a modified silk fibroin with sequence number 55 (hereinafter referred to as "PRT1107"), a modified silk fibroin with sequence number 56 (hereinafter referred to as "PRT1083"), and a modified silk fibroin with sequence number 57 (hereinafter referred to as "PRT1125") for design. Furthermore, artificial structural proteins include keratin with sequence number 58 (hereinafter referred to as "PRT855"), collagen with sequence number 59 (hereinafter referred to as "PRT537"), elastic protein with sequence number 60 (hereinafter referred to as "PRT366"), and interferon gamma with sequence number 61 (hereinafter referred to as "PRT662").

[0358] In addition, the amino acid sequence shown in sequence number 44 has an amino acid sequence in which all QQ in the amino acid sequence shown in sequence number 9 (the amino acid sequence before the amino acid sequence shown in sequence number 42 is added to the C-terminus) is replaced with VF, and the remaining Q is replaced with I, and the amino acid sequence shown in sequence number 12 is added to the N-terminus for the purpose of improving hydrophobicity.

[0359] In addition, the amino acid sequence shown in sequence number 15 has an amino acid sequence in which amino acid residues are replaced, inserted, and deleted in the amino acid sequence of the silk core protein from the golden spinning spider in order to improve productivity, and the amino acid sequence shown in sequence number 12 (tag sequence and hinge sequence) is added to the N-terminus.

[0360] The amino acid sequence (PRT1103) shown in sequence number 53 is a sequence in which the tyrosine residue (Y) of the amino acid sequence shown in sequence number 7 (Met-PRT410) is replaced by a phenylalanine residue (F), and most of the serine residues (S) are replaced by alanine residues (A) or glycine residues (G), and a tag sequence is further added to the N-terminus.

[0361] In the amino acid sequence shown in sequence number 54 (PRT1104), most of the serine residues (S) in the amino acid sequence shown in sequence number 7 are replaced with alanine residues (A) or glycine residues (G), and a tag sequence is further added to the N-terminus.

[0362] In the amino acid sequence (PRT1107) shown in sequence number 55, the serine residue (S) of the amino acid sequence (Met-PRT918) shown in sequence number 31 is replaced with an alanine residue (A), a valine residue (V), a leucine residue (L) or an isoleucine residue (I), and a tag sequence is further added to the N-terminus.

[0363] In the amino acid sequence (PRT1083) shown in sequence number 56, the proline residue (P) of the amino acid sequence (Met-PRT918) shown in sequence number 31 is replaced with a threonine residue (T) or a leucine residue (L), and a tag sequence is further added to the N-terminus.

[0364] The amino acid sequence (PRT855) shown in sequence number 58 has an amino acid sequence consisting of amino acid residues 1 to 292 from the N-terminus of PRT798 (sequence number 49), and an amino acid sequence in which leucine or valine is replaced by alanine or glycine, and has an amino acid sequence in which three amino acid residues among the amino acid residues 1 to 246 from the N-terminus are replaced, and an amino acid sequence obtained by inserting an amino acid sequence consisting of GAAAAAG (sequence number 62) is repeated.

[0365] Next, nucleic acids encoding the designed artificial structural proteins PRT966, PRT799, PRT918, PRT826, PRT853, PRT1104, PRT705, PRT1125, PRT1103, PRT1107 and PRT1083 (modified fibroin), PRT855 (keratin), PRT537 (collagen), PRT366 (elastin), and interferon gamma (PRT662) were synthesized. In this nucleic acid, an NdeI site was added to the 5' end, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Then, after the nucleic acid was treated with NdeI and EcoRI restriction enzymes and excised, it was recombined into the protein expression vector pET-22b (+) to obtain an expression vector.

[0366] (2) Expression of artificial structural proteins

[0367] The expression vector obtained in (1) was used to transform E. coli BLR (DE3). The transformed E. coli was cultured in 2 mL LB medium containing ampicillin for 15 hours. The culture solution was measured at OD 600 The flask culture was continued until the OD was 0.005 and the culture temperature was maintained at 30°C. 600 Change to 5 (about 15 hours) to obtain seed culture solution.

[0368] [Table 5]

[0369]

[0370] The seed culture solution was measured at OD 600 The 500 mL of production medium (Table 6) was added to a small fermenter at a concentration of 0.05. The culture temperature was maintained at 37°C and the pH was controlled at 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the saturated dissolved oxygen concentration.

[0371] [Table 6]

[0372]

[0373] After the glucose in the production medium is completely consumed, the feed solution (glucose 455g / 1L, Yeat Extract 120g / 1L) is immediately added at a rate of 1mL / min. The culture temperature is maintained at 37°C and the pH is constantly controlled at 6.9 for cultivation. In addition, the dissolved oxygen concentration in the culture solution is maintained at 20% of the dissolved oxygen saturation concentration and cultured for 20 hours. Then, 1M isopropyl-β-thiogalactopyranoside (IPTG) is added to the culture solution to make its final concentration 1mM, and the induced modified silk core protein is expressed. After 20 hours after the addition of IPTG, the culture solution is centrifuged and the bacteria are recovered. SDS-PAGE is performed using bacteria prepared from the culture solution before and after IPTG addition, and the expression of the target artificial structural protein is confirmed by the appearance of bands depending on the size of the target modified silk core protein added by IPTG.

[0374] (3) Purification of artificial structural proteins

[0375] Two hours after adding IPTG, the cells were washed with 20mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20mM Tris-HCl buffer (pH 7.4) containing approximately 1mM PMSF and disrupted using a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20mM Tris-HCl buffer (pH 7.4) until it reached high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10mM sodium dihydrogen phosphate, 20mM NaCl, 1mM Tris-HCl, pH 7.0) to a concentration of 100mg / mL and stirred at 60°C for 30 minutes to dissolve it. After dissolution, the cells were dialyzed against water using dialysis tubing (Sanko Junyaku Co., Ltd., cellulose tubing 36 / 32). The white aggregated protein obtained after dialysis was recovered by centrifugation, the moisture was removed using a freeze dryer, and the freeze-dried powder was recovered to obtain modified silk fibroin (PRT966, PRT799, PRT918PRT826, PRT853, PRT1104, PRT705, PRT1125, PRT1103, PRT1107 and PRT1083), PRT855 (keratin), PRT537 (collagen), PRT366 (elastin) and PRT662 (interferon γ).

[0376] (Evaluation of Fiber Forming Ability (Coagulation Solution))

[0377] (1-1) Preparation of spinning solution

[0378] 26% by mass of the modified fibroin (PRT966) obtained in the artificial structural protein preparation step described above was mixed with 74% by mass of formic acid (Asahi Chemical Co., Ltd., purity 98%) as a dissolving solvent. The mixture was heated at 70°C for 1 hour with stirring using an aluminum block heater to dissolve the mixture. The mixture was then filtered and degassed using a metal filter with a 1 μm mesh to obtain a spinning solution.

[0379] (1-2) Spinning solution ejection test

[0380] The spinning solution obtained in (1-1) was filled into a 10 ml syringe and ejected from a nozzle with a nozzle diameter of 0.2 μm into the coagulation solution to coagulate the modified silk fibroin at room temperature. The coagulated fibrils were wound at a linear speed of 2.39 m / min. The obtained fibrils were observed and the fiber forming ability was visually judged. The extrusion rate of the spinning solution was 0.075 ml / min. The types of coagulation solutions used are shown in Table 7. In addition, the brackish water was brackish water collected from the estuary of Sakata City, Yamagata Prefecture, and the seawater was seawater collected from the ocean in Kamo City, Yamagata Prefecture. The concentrations [wt%] of brackish water and seawater represent approximate values ​​of the concentrations of all solutes. The mixed solutions of Test Examples 26 to 28 are solutions in which the formic acid in the spinning solution in contact is dissolved in the sodium chloride aqueous solution, and the ratio of the total mass of the coagulation solution (mixed solution) is set to 60% to 80% by mass of the sodium chloride aqueous solution and 20% to 40% by mass of formic acid. The formic acid aqueous solution of Test Example 29 was prepared by dissolving formic acid in the spinning solution in contact in water, with the total mass of the coagulation solution (mixed solution) being 80% by mass of water and 20% by mass of formic acid.

[0381] The evaluation results of the fiber forming ability are shown in Table 7. The evaluation criteria for the fiber forming ability are as follows.

[0382] ⊙: Fiber formation. The resulting fiber is flexible and homogeneous.

[0383] ○: Fibers were formed. The obtained fibers were flexible.

[0384] △: Fibers are formed.

[0385] ×: No fibers were formed.

[0386] [Table 7]

[0387]

[0388] As shown in Table 7, when any one of water, acid aqueous solution, saline solution and mixed solution is used, flexible fibers can be formed (Test Examples 1 to 26). When the coagulation liquid is a saline solution, flexible and homogeneous fibers can be formed, and extremely good fiber forming ability is shown (Test Examples 4 to 26). In particular, by using abundant and inexpensive water, sodium chloride aqueous solution, brackish water and seawater in the coagulation liquid, the manufacturing cost can be greatly reduced. In addition, it is shown that even when the organic solvent in the spinning solution in contact is dissolved in the coagulation liquid, flexible fibers can be formed (Test Examples 27 to 30). In particular, when the coagulation liquid in which formic acid is dissolved is a sodium chloride aqueous solution, flexible and homogeneous fibers can be formed (Test Examples 28 to 30).

[0389] (Evaluation of Fibril Forming Ability (Protein))

[0390] (2-1) Preparation of spinning solution

[0391] Modified silk fibroin (PRT966, PRT826, PRT853, PRT1104, PRT705, PRT1125, PRT1103, PRT1107, and PRT1083), keratin (PRT855), collagen (PRT537), and elastin (PRT366), or 26% interferon gamma (PRT662), or commercially available structural protein MediGelatin (manufactured by Nippi Co., Ltd.), egg white albumin, or 26% casein, obtained in the above-described artificial structural protein preparation process, were mixed with 74% formic acid or 74% dimethyl sulfoxide solution (lithium chloride concentration: 4.0% by mass) containing lithium chloride as a dissolving solvent. The mixture was heated at 70°C for 1 hour with stirring using an aluminum block heater to dissolve the mixture. Filtration and degassing were performed using a metal filter with a mesh size of 1 μm to obtain a spinning solution. Table 8 shows the prepared spinning solution.

[0392] (2-2) Spinning liquid ejection test

[0393] The spinning solution obtained in (2-1) is filled into a 10 ml syringe and ejected from a nozzle with a nozzle diameter of 0.2 μm into a coagulation solution (a 30% sodium sulfate aqueous solution or water) to coagulate the modified silk core protein at room temperature. The obtained protofibrils are observed and the fiber forming ability is visually judged. The extrusion rate of the spinning solution is 0.075 ml / min. The evaluation criteria for fiber forming ability are as follows. The evaluation results of fiber forming ability are shown in Table 8. In addition, the average HI in Table 8 is a value calculated by calculating the sum of the hydrophobicity index (HI) of all amino acid residues constituting the structural protein and then dividing this sum by the total number of amino acid residues. It is a value calculated by calculating the hydrophobicity index of the amino acid sequence (a sequence containing a tag sequence or a hinge sequence) recorded in the sequence table.

[0394] ○: Fibers were formed and the coagulation properties were good.

[0395] △: Fibers were formed, but the coagulation property was low.

[0396] ×: Spinning solution is gelled or the solute is insoluble

[0397] [Table 8]

[0398]

[0399] As shown in Table 8, when the coagulation liquid was an aqueous sodium sulfate solution, fiber formation was confirmed for all structural proteins with an average HI value of -1.229 to 0.95, and fiber-forming ability was confirmed (Test Examples 83 to 109). Furthermore, when the coagulation liquid was water, fiber formation was confirmed for modified fibroin with an average HI value of 0.466 to 0.95, and fiber-forming ability was confirmed (Test Examples 107 to 109). When an aqueous sodium sulfate solution was used as the coagulation liquid, fiber formation was possible regardless of the type of structural protein, and excellent fiber-forming ability was demonstrated.

[0400] (Preparation and evaluation of artificial structural protein fibers)

[0401] (1) Preparation of spinning solution

[0402] A spinning dope was prepared by the same procedure as that used in the evaluation of the fiber-forming ability, except that the concentration of the modified fibroin (PRT966) was 26% by mass and the concentration of formic acid was 74% by mass.

[0403] (2) Wet spinning

[0404] The prepared spinning solution is filled into a storage tank, and a gear pump is used to eject the spinning solution from a single-hole nozzle with a diameter of 0.2 mm in a desktop spinning device in a coagulation bath, and a precursor is formed. Next, the coagulated precursor is stretched in a water bath. After washing and stretching in a water bath, a drying plate is used to dry the modified silk core protein fiber (artificial protein fiber) obtained by curling the modified silk core protein fiber using a desktop spinning device. The conditions for wet spinning are as follows, and the coagulation solution used is as shown in Table 9.

[0405] Extrusion nozzle diameter: 0.2mm

[0406] Temperature of coagulation liquid: room temperature

[0407] Stretching ratio in water bath: 3.5 to 5.5 times

[0408] Water bath temperature: 40°C

[0409] Drying temperature: 60℃

[0410] (3) Evaluation of physical properties of artificial structural protein fibers

[0411] The physical properties of the fiber obtained in (2) above were measured by the following method. In an environment of temperature of 20°C and relative humidity of 65%, the single-filament strength and elongation measuring device "FAVIMAT" manufactured by Textechno was used to measure the fineness and strength and elongation of 10 randomly sampled fibers (number of samples = 10), and the average value was calculated. The conditions for measuring strength and elongation are: the capacity of the weighing sensor is 210 cN, the needle length is 20 mm, and the stretching speed is 10 mm / min. The strength at break is defined as the breaking strength [g / d], and the elongation at break is defined as the breaking elongation [%]. In addition, the value of the area enclosed by the stress-strain curve and the horizontal axis when the horizontal axis is set to strain [%] and the vertical axis is set to stress [g / d] is multiplied by the density [kg / m 3 ] and the obtained value is defined as toughness [MJ / m 3 The density of modified silk fibroin (PRT966) is 1.34[g / cm 3 ].

[0412] The results of the mechanical and physical property evaluations of the modified silk fibroin fibers are shown in Tables 9, 10, 11, and 12. The elongation values ​​in Tables 9 and 10 are relative values, with the elongation at break of the modified silk fibroin fibers in Comparative Example 2 (fibers prepared using 100% methanol in the coagulation bath) set to 100. Furthermore, the elongation at break for Test Example 33 (potassium chloride aqueous solution) and Test Example 36 (sodium sulfate aqueous solution) in Table 9, and in Table 10, are values ​​obtained when the coagulation bath temperature was set to 60°C.

[0413] The toughness values ​​in Tables 11 and 12 are relative values, with the toughness of the modified fibroin fiber in Comparative Example 3 (fiber prepared using 100% methanol in the coagulation bath) being set to 100. Furthermore, the relative toughness values ​​for Test Example 44 (potassium chloride aqueous solution) and Test Example 47 (sodium sulfate aqueous solution) in Table 11, and in Table 12, are those obtained when the coagulation bath temperature was set to 60°C.

[0414] [Table 9]

[0415]

[0416] As shown in Table 9, the modified silk fibroin fibers (Test Examples 31 to 40) prepared by using carboxylate aqueous solutions (sodium citrate and sodium formate aqueous solutions), chloride aqueous solutions (potassium chloride, sodium chloride and calcium chloride aqueous solutions), sulfate aqueous solutions (sodium sulfate and ammonium sulfate aqueous solutions), hydrogen phosphate aqueous solutions (buffered solution), and mixed solutions (brackish water and seawater) in the coagulation liquid, and the modified silk fibroin fibers (Test Example 41) prepared by using water in the coagulation liquid, compared with the modified silk fibroin fibers (Comparative Example 2) prepared by using methanol in the coagulation liquid, all have the effect of further improving the elongation and can obtain unexpectedly excellent results.

[0417] [Table 10]

[0418]

[0419] As shown in Table 10, the modified silk fibroin fibers prepared using a mixed aqueous solution of sodium chloride and formic acid as the coagulation solution (Test Examples 43-44) and the modified silk fibroin fibers prepared using a mixed aqueous solution of water and formic acid as the coagulation solution (Test Examples 45-46) showed a further improvement in elongation compared to the modified silk fibroin fibers prepared using methanol as the coagulation solution (Comparative Example 2). These results indicate that even when the formic acid in the spinning solution is dissolved in water (Test Example 41) and the coagulation solution is a sodium chloride aqueous solution (Test Example 42), an excellent elongation improvement effect can be observed.

[0420] [Table 11]

[0421]

[0422] As shown in Table 11, the modified silk fibroin fibers (Test Examples 47 to 56) prepared by using carboxylate aqueous solutions (sodium citrate aqueous solutions and sodium formate aqueous solutions), chloride aqueous solutions (potassium chloride aqueous solutions and sodium chloride aqueous solutions), sulfate aqueous solutions (sodium sulfate aqueous solutions and ammonium sulfate aqueous solutions), phosphate buffer solutions, and mixed solutions (brackish water and seawater) in the coagulation liquid, and the modified silk fibroin fibers (Test Example 56) prepared by using water in the coagulation liquid, when compared with the modified silk fibroin fibers (Comparative Example 3) prepared by using methanol in the coagulation liquid, all have the effect of further improving the toughness value and can obtain unexpectedly excellent results.

[0423] [Table 12]

[0424]

[0425] As shown in Table 12, the modified silk fibroin fibers prepared using a mixed aqueous solution of sodium chloride and formic acid as the coagulation solution (Test Examples 58-59) and the modified silk fibroin fibers prepared using a mixed aqueous solution of water and formic acid as the coagulation solution (Test Examples 60-61) showed a further improvement in toughness values ​​compared to the modified silk fibroin fibers prepared using methanol as the coagulation solution (Comparative Example 3). These results indicate that even when formic acid in the spinning solution is dissolved in water (Test Example 41) and the coagulation solution is a sodium chloride aqueous solution (Test Example 34), an excellent improvement in toughness values ​​can be observed.

[0426] (4) Evaluation of shrinkage of artificial structural protein fibers

[0427] The modified silk fibroin fibers obtained in (2) above are gathered into a length of about 30 cm and bundled to obtain a silk fibroin fiber bundle with a fineness of 150D. A plumb bob weighing 0.8 g is installed on each silk fibroin fiber bundle, and in this state, the fiber bundle is immersed in water at 40°C for 90 seconds to shrink it. Then, each fiber bundle is taken out of the water, dried with a plumb bob weighing 0.8 g installed, and the length of each fiber bundle after drying is measured. The shrinkage rate is calculated according to the following formula. In addition, L0 represents the length of the modified silk fibroin fiber before contact with water (after spinning) (in this case, 30 cm), L D It indicates the length of the fiber after shrinkage (dry fiber after immersion in water).

[0428] Formula: Shrinkage [%] = {1-(L D / L0)}×100

[0429] The shrinkage rates of the modified fibroin fibers are shown in Tables 11 and 12. The shrinkage rates in Tables 13 and 14 are shown as relative values, with the shrinkage rate of the modified fibroin fiber in Comparative Example 4 (fibers prepared using methanol in the coagulation bath) (17.0%) being set to 100. Furthermore, the shrinkage rates of Test Example 64 (potassium chloride aqueous solution) and Test Example 73 (sodium sulfate aqueous solution) in Table 13, and in Table 14, are shown when the coagulation bath temperature is set to 60°C.

[0430] [Table 13]

[0431]

[0432] As shown in Table 13, the modified silk fibroin fibers (Test Examples 62 to 76) prepared by using carboxylate aqueous solutions (sodium citrate aqueous solution and sodium formate aqueous solution), chloride aqueous solutions (potassium chloride aqueous solution and sodium chloride aqueous solution), sulfate aqueous solutions (sodium sulfate aqueous solution and ammonium sulfate aqueous solution), phosphate aqueous solutions, and mixed solutions (brackish water and seawater) in the coagulation liquid, and the modified silk fibroin fiber (Test Example 77) prepared by using water in the coagulation liquid, compared with the modified silk fibroin fiber (Comparative Example 4) prepared by using methanol in the coagulation liquid, have an excellent effect of reducing the shrinkage rate to water, and unexpectedly excellent results can be obtained.

[0433] In particular, by using abundant and inexpensive water, sodium chloride aqueous solution, sodium sulfate aqueous solution, brackish water and seawater in the coagulation liquid, in addition to significantly reducing the manufacturing cost, the elongation can be improved (see Table 9), the toughness can be improved (see Table 11), and the shrinkage to water can be reduced (see Table 13).

[0434] [Table 14]

[0435]

[0436] As shown in Table 14, the modified fibroin fibers prepared using a mixed aqueous solution of sodium chloride and formic acid as the coagulation solution (Test Examples 78-80) and the modified fibroin fibers prepared using a mixed aqueous solution of water and formic acid as the coagulation solution (Test Examples 81-82) exhibited an excellent effect of reducing shrinkage in water at all mixing ratios, compared to the modified fibroin fibers prepared using methanol as the coagulation solution (Comparative Example 4), yielding unexpectedly excellent results. This demonstrates that even when formic acid in the spinning solution is dissolved in water (Test Example 77) and the coagulation solution is a sodium chloride aqueous solution (Test Example 65), an excellent effect of reducing shrinkage in water can be confirmed.

[0437] Reference Example 1: Flammability Test of Modified Silk Fiber Protein

[0438] Freeze-dried powder of modified silk fibroin (PRT799) was added to a lithium chloride solution in dimethyl sulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass and mixed with a shaker for 3 hours to dissolve it. Insoluble matter and bubbles were then removed to obtain a modified silk fibroin solution (spinning solution).

[0439] The resulting spinning solution was heated to 90°C, filtered through a metal filter with a mesh size of 5 μm, placed in a 30 mL stainless steel syringe, allowed to stand and degas, and then ejected from a solid nozzle with a needle diameter of 0.2 mm into a 100% methanol coagulation bath. The ejection temperature was 90°C. After coagulation, the resulting raw yarn was wound and naturally dried to obtain modified silk fibroin fibers (raw material fibers).

[0440] Using twisted yarn obtained by twisting raw material fibers together, circular knitting was performed using a circular knitting machine to produce a knitted fabric (thickness: 180 denier, gauge: 18). 20 g of the obtained knitted fabric was cut out and used as a test piece.

[0441] The flammability test was conducted in accordance with the "Test Methods for Powdered or Particle-Formed or Low-Melting-Point Synthetic Resins" outlined in "Fire and Disaster Management Agency Hazardous Substances Regulation No. 50 (May 31, 1995)." The test was conducted at a temperature of 22°C, a relative humidity of 45%, and an atmospheric pressure of 1021 hPa. The measurement results (oxygen concentration (%), combustion rate (%), and combustion conversion rate (%)) are shown in Table 15.

[0442] [Table 15]

[0443] Oxygen concentration (%) Combustion rate (%) Combustion conversion rate (%) 20.0 39.1 40.1 27.0 48.1 49.3 27.0 51.9 53.2 30.0 53.6 54.9 50.0 61.2 62.7 70.0 91.1 93.3 100.0 97.6 100.0

[0444] The results of the flammability test showed that the critical oxygen index (LOI) value of the knitted fabric using modified silk fibroin (PRT799) fibers was 27.2. Generally, an LOI value of 26 or higher indicates flame retardancy. This indicates that modified silk fibroin has excellent flame retardancy.

[0445] Reference Example 2: Evaluation of Hygroscopic and Heat-generating Properties of Modified Silk Fiber Protein

[0446] The freeze-dried powder of modified fibroin was added to a lithium chloride dimethyl sulfoxide solution (concentration: 4.0% by mass) to a concentration of 24% by mass and mixed with a shaker for 3 hours to dissolve it. Insoluble matter and bubbles were then removed to obtain a modified fibroin solution (spinning solution).

[0447] The resulting spinning solution was heated to 60°C, filtered through a metal filter with a mesh size of 5 μm, placed in a 30 mL stainless steel syringe, allowed to stand and degas, and then ejected from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The ejection temperature was 60°C. After coagulation, the resulting raw yarn was wound and naturally dried to obtain modified silk fibroin fibers (raw material fibers).

[0448] For comparison, commercially available wool fiber, cotton fiber, Tencel fiber, rayon fiber, and polyester fiber were prepared as raw material fibers.

[0449] Knitted fabrics were produced using various raw material fibers by flat knitting on a flat knitting machine. The thickness and gauge of knitted fabrics using PRT918 or PRT799 fibers are shown in Table 16. The thickness and gauge of knitted fabrics using other raw material fibers were adjusted to achieve a cover factor approximately the same as that of knitted fabrics using modified fibroin fibers. Specifically, the following is described.

[0450] [Table 16]

[0451] Raw fiber Thickness [N] Stitch length [GG] PRT918 1 / 30 (wool yarn count monofilament) 18 PRT799 1 / 30 (wool yarn count monofilament) 16 wool 2 / 30 (double wire) 14 cotton 2 / 34 (double wire) 14 Tencel 2 / 30 (double wire) 15 Man-made fibers 1 / 38 (monofilament) 14 polyester 1 / 60 (monofilament) 14

[0452] Two pieces of knitted fabric cut to 10 cm x 10 cm were bonded together and sewn together at the four edges to create a test piece (specimen). The test piece was placed in a low-humidity environment (temperature 20±2°C, relative humidity 40±5%) for at least 4 hours, then moved to a high-humidity environment (temperature 20±2°C, relative humidity 90±5%). The temperature was measured at one-minute intervals for 30 minutes using a temperature sensor installed in the center of the test piece.

[0453] The maximum heat generation due to moisture absorption was determined from the measurement results using the following formula A.

[0454] Formula A: Maximum heat generation by hygroscopic absorption = {(the maximum temperature of the sample when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium, and then moved to a high humidity environment) - (the temperature of the sample when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium, and then moved to a high humidity environment)} (°C) / sample weight (g)

[0455] Figure 5 This is a graph showing an example of the results of the hygroscopic heat generation performance test. The horizontal axis of the graph represents the placement time (minutes) in a high humidity environment, where 0 is the time it takes for the sample to be transferred from a low humidity environment to a high humidity environment. The vertical axis of the graph represents the temperature measured by the temperature sensor (sample temperature). Figure 5 In the graph shown, the point indicated by M corresponds to the maximum value of the sample temperature.

[0456] The calculation results of the maximum heat generation by moisture absorption of each knitted fabric are shown in Table 16.

[0457] [Table 17]

[0458]

[0459] As shown in Table 17, it can be seen that the modified silk fibroin (PRT918 and PRT799) has a higher maximum heat generation by hygroscopic absorption than the existing materials and is excellent in heat generation by hygroscopic absorption.

[0460] Reference Example 3: Evaluation of thermal insulation properties of modified silk fibroin

[0461] The freeze-dried powder of modified fibroin was added to a lithium chloride dimethyl sulfoxide solution (concentration: 4.0% by mass) to a concentration of 24% by mass and mixed with a shaker for 3 hours to dissolve it. Insoluble matter and bubbles were then removed to obtain a modified fibroin solution (spinning solution).

[0462] The resulting spinning solution was heated to 60°C, filtered through a metal filter with a mesh size of 5 μm, placed in a 30 mL stainless steel syringe, allowed to stand and degas, and then ejected from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The ejection temperature was 60°C. After coagulation, the resulting raw yarn was wound and naturally dried to obtain modified silk fibroin fibers (raw material fibers).

[0463] For comparison, commercially available wool fibers, silk fibers, cotton fibers, rayon fibers, and polyester fibers were prepared as raw material fibers.

[0464] Knitted fabrics were produced using various raw material fibers by flat knitting on a flat knitting machine. Table 18 shows the count, twist count, gauge, and weight per unit area of ​​knitted fabrics using PRT966 or PRT799 fibers. Knitted fabrics using other raw material fibers were adjusted to have a cover factor approximately the same as that of knitted fabrics using modified fibroin fibers. Specifically, the following is described.

[0465] [Table 18]

[0466] Raw fiber Count [Nm] Number of twists Stitch length [GG] <![CDATA[Grammage [g / m 2 > PRT966 30 1 18 90.1 PRT799 30 1 16 111.0 wool 30 2 14 242.6 silk 60 2 14 225.2 cotton 34 2 14 194.1 Man-made fibers 38 1 14 181.8 polyester 60 1 14 184.7

[0467] Thermal insulation performance was evaluated using a KES-F7 Thermolab II tester manufactured by Kado Technology Co., Ltd. using the dry contact method (a method that assumes direct contact between skin and clothing in a dry state). A piece of knitted fabric cut into a 20cm x 20cm rectangle was used as a test piece (specimen). The test piece was placed on a hot plate set to a constant temperature (30°C) and the amount of heat dissipated through the test piece was determined under a wind tunnel wind speed of 30cm / second (a). Without the test piece, the amount of heat dissipated under the same conditions as above was determined (b), and the thermal insulation rate (%) was calculated using the following formula (B).

[0468] Formula B: Heat retention rate (%) = (1-a / b) × 100

[0469] The thermal insulation performance index was calculated based on the following formula C from the measurement results.

[0470] Formula C: Thermal insulation performance index = thermal insulation rate (%) / unit area weight of sample (g / m 2 )

[0471] The calculation results of the thermal insulation performance index are shown in Table 18. It can be assessed that the higher the thermal insulation performance index of a material, the better the thermal insulation performance.

[0472] [Table 19]

[0473] Raw fiber Thermal insulation performance index PRT966 0.33 PRT799 0.22 wool 0.16 silk 0.11 cotton 0.13 Man-made fibers 0.02 polyester 0.18

[0474] As shown in Table 19, it can be seen that the modified silk fibroin (PRT966 and PRT799) has a higher thermal insulation index than the existing materials and is excellent in thermal insulation performance.

[0475] As shown in Reference Examples 1 to 3, when the modified silk fibroin is a modified spider silk fibroin, thermal insulation, hygroscopic heat generation, and / or flame retardancy can be further enhanced. When modified spider silk fibroin is used as the protein and water or an aqueous solution with a pH of 0.25 or higher and 10.00 or lower (particularly aqueous sodium chloride solution, aqueous sodium sulfate solution, aqueous ammonium sulfate solution, aqueous potassium chloride solution, aqueous calcium chloride solution, aqueous sodium formate solution, aqueous sodium citrate solution, aqueous formic acid solution, mixed aqueous formic acid solutions, brackish water, and seawater) is used as the coagulation solution to produce fibers, the resulting fibers exhibit further enhanced thermal insulation, hygroscopic heat generation, and / or flame retardancy, as well as toughness and elongation, and can further reduce shrinkage due to water.

[0476] Explanation of symbols

[0477] 1 Extrusion device

[0478] 2 Undrawn yarn manufacturing device

[0479] 3. Wet heat stretching device

[0480] 4 Drying device

[0481] 6 Spinning solution

[0482] 10 Spinning device

[0483] 20 coagulation bath

[0484] 21 Washing bath

[0485] 36 Protein Fiber Sequence Listing <110> Sibabo Co., Ltd. <120> Preparation method of protein fiber <130> FP19-0989-00 <150> JP2018-185598 <151> 2018-09-28 <160> 62 <170> PatentIn version 3.5 <210> 1 <211> 50 <212> PRT <213> Araneus diadematus <400> 1 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala 1 5 10 15 Leu Val Ser Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Gly 20 25 30 Ala Ser Ala Gln Tyr Thr Gln Met Val Gly Gln Ser Val Ala Gln Ala 35 40 45 Leu Ala 50 <210> 2 <211> 30 <212> PRT <213> Araneus diadematus <400> 2 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala 1 5 10 15 Leu Val Ser Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn 20 25 30 <210> 3 <211> 21 <212> PRT <213> Araneus diadematus <400> 3 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala 1 5 10 15 Leu Val Ser Ile Leu 20 <210> 4 <211> 1154 <212> PRT <213> Artificial sequence <220> <223> Recombinant spider silk protein ADF3KaiLargeNRSH1 <400> 4 Met His His His His His His His His His His Ser Ser Gly Ser Ser 1 5 10 15 Leu Glu Val Leu Phe Gln Gly Pro Ala Arg Ala Gly Ser Gly Gln Gln 20 25 30 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 35 40 45 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 50 55 60 Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln 65 70 75 80 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 85 90 95 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro 100 105 110 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 115 120 125 Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln 130 135 140 Gly Pro Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 145 150 155 160 Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly 165 170 175 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 180 185 190 Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln 195 200 205 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 210 215 220 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 225 230 235 240 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 245 250 255 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 260 265 270 Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 275 280 285 Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly 290 295 300 Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 305 310 315 320 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 325 330 335 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 340 345 350 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 355 360 365 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 370 375 380 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 385 390 395 400 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 405 410 415 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 420 425 430 Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly 435 440 445 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 450 455 460 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 465 470 475 480 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly 485 490 495 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 500 505 510 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 515 520 525 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser 530 535 540 Val Ser Arg Ala Arg Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln 545 550 555 560 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 565 570 575 Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly 580 585 590 Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 595 600 605 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly 610 615 620 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 625 630 635 640 Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly 645 650 655 Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln 660 665 670 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 675 680 685 Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 690 695 700 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 705 710 715 720 Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 725 730 735 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 740 745 750 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 755 760 765 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 770 775 780 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly 785 790 795 800 Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 805 810 815 Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln 820 825 830 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro 835 840 845 Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser 850 855 860 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 865 870 875 880 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 885 890 895 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly 900 905 910 Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 915 920 925 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 930 935 940 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly 945 950 955 960 Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly 965 970 975 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 980 985 990 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 995 1000 1005 Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser 1010 1015 1020 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 1025 1030 1035 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 1040 1045 1050 Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser Val 1055 1060 1065 Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala 1070 1075 1080 Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser 1085 1090 1095 Ser Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala 1100 1105 1110 Ala Leu Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala 1115 1120 1125 Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu 1130 1135 1140 Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu 1145 1150 <210> 5 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> His tag and start codon <400> 5 Met His His His His His His His His His Ser Ser Gly Ser Ser 1 5 10 15 Leu Glu Val Leu Phe Gln Gly Pro 20 <210> 6 <211> 597 <212> PRT <213> Artificial sequence <220> <223> Met-PRT380 <400> 6 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 20 25 30 Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly 35 40 45 Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro 50 55 60 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala 65 70 75 80 Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala 85 90 95 Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 100 105 110 Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly 115 120 125 Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro 130 135 140 Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 145 150 155 160 Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 165 170 175 Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly 180 185 190 Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly 195 200 205 Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala 210 215 220 Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr 225 230 235 240 Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly 245 250 255 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro 260 265 270 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 275 280 285 Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly 290 295 300 Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro 305 310 315 320 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro 325 330 335 Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Ala Ala Ala 340 345 350 Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala 355 360 365 Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly 370 375 380 Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro 385 390 395 400 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 405 410 415 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 420 425 430 Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala 435 440 445 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr 450 455 460 Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly 465 470 475 480 Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 485 490 495 Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro 500 505 510 Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly 515 520 525 Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser 530 535 540 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala 545 550 555 560 Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly 565 570 575 Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln 580 585 590 Gly Pro Gly Ala Ser 595 <210> 7 <211> 590 <212> PRT <213> Artificial Sequence <220> <223> Met-PRT410 <400> 7 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 20 25 30 Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 35 40 45 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro 50 55 60 Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 65 70 75 80 Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln 85 90 95 Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 100 105 110 Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala 115 120 125 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr 130 135 140 Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly 145 150 155 160 Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro 165 170 175 Gly Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 180 185 190 Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly 195 200 205 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala 210 215 220 Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser 225 230 235 240 Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly 245 250 255 Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln 260 265 270 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 275 280 285 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 290 295 300 Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly 305 310 315 320 Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser 325 330 335 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 340 345 350 Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln 355 360 365 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly 370 375 380 Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 385 390 395 400 Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 405 410 415 Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr 420 425 430 Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro 435 440 445 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro 450 455 460 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 465 470 475 480 Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly 485 490 495 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser 500 505 510 Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly 515 520 525 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly 530 535 540 Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser 545 550 555 560 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala 565 570 575 Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 580 585 590 <210> 8 <211> 565 <212> PRT <213> Artificial Sequence <220> <223> Met-PRT468 <400> 8 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Gly Ser Asn Gly Pro Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 35 40 45 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 50 55 60 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala 65 70 75 80 Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly 85 90 95 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 100 105 110 Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly 115 120 125 Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro 130 135 140 Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly 145 150 155 160 Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 165 170 175 Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly 180 185 190 Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser 195 200 205 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly 210 215 220 Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser 245 250 255 Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Tyr Gly Pro Gly Gln 260 265 270 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser 275 280 285 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Pro Ser Ala Ala Ala 290 295 300 Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala 305 310 315 320 Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln 325 330 335 Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly 340 345 350 Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser 355 360 365 Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala 370 375 380 Ala Ala Ala Ala Ala Gly Ser Tyr Gln Gln Gly Pro Gly Gln Gln Gly 385 390 395 400 Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly 405 410 415 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr 420 425 430 Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala 435 440 445 Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro 450 455 460 Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly 465 470 475 480 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro 485 490 495 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala 500 505 510 Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln 515 520 525 Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly 530 535 540 Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln 545 550 555 560 Gly Pro Gly Ala Ser 565 <210> 9 <211> 2364 <212> PRT <213> Artificial sequence <220> <223> Met-PRT799 <400> 9<​​​​​​​​​​​​​​50 55 60 Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 65 70 75 80 Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln 85 90 95 Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 100 105 110 Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala 115 120 125 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr 130 135 140 Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly 145 150 155 160 Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro 165 170 175 Gly Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 180 185 190 Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly 195 200 205 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala 210 215 220 Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser 225 230 235 240 Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly 245 250 255 Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln 260 265 270 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 275 280 285 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 290 295 300 Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly 305 310 315 320 Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser 325 330 335 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 340 345 350 Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln 355 360 365 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly 370 375 380 Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 385 390 395 400 Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 405 410 415 Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr 420 425 430 Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro 435 440 445 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro 450 455 460 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 465 470 475 480 Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly 485 490 495 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser 500 505 510 Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly 515 520 525 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly 530 535 540 Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser 545 550 555 560 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala 565 570 575 Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln 580 585 590 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln 595 600 605 Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Gly Gln Tyr 610 615 620 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala 625 630 635 640 Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 645 650 655 Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly 660 665 670 Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 675 680 685 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser 690 695 700 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly 705 710 715 720 Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 725 730 735 Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala 740 745 750 Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro 755 760 765 Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly 770 775 780 Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly 785 790 795 800 Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro 805 810 815 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 820 825 830 Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 835 840 845 Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln 850 855 860 Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln 865 870 875 880 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 885 890 895 Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly 900 905 910 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala 915 920 925 Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln 930 935 940 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln 945 950 955 960 Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr 965 970 975 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly 980 985 990 Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln 995 1000 1005 Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln 1010 1015 1020 Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro 1025 1030 1035 Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln 1040 1045 1050 Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly 1055 1060 1065 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly 1070 1075 1080 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 1085 1090 1095 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln 1100 1105 1110 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 1115 1120 1125 Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 1130 1135 1140 Gln Ser Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr 1145 1150 1155 Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly 1160 1165 1170 Pro Gly Ala Ser Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser 1175 1180 1185 Ala Ala Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Gln 1190 1195 1200 Gly Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 1205 1210 1215 Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro 1220 1225 1230 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 1235 1240 1245 Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 1250 1255 1260 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 1265 1270 1275 Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro 1280 1285 1290 Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro 1295 1300 1305 Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 1310 1315 1320 Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser 1325 1330 1335 Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr 1340 1345 1350 Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser 1355 1360 1365 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser 1370 1375 1380 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala 1385 1390 1395 Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser 1400 1405 1410 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly Gln 1415 1420 1425 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly 1430 1435 1440 Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala 1445 1450 1455 Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 1460 1465 1470 Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln 1475 1480 1485 Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln 1490 1495 1500 Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln 1505 1510 1515 Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala 1520 1525 1530 Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly 1535 1540 1545 Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr 1550 1555 1560 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr 1565 1570 1575 Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala 1580 1585 1590 Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro 1595 1600 1605 Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr 1610 1615 1620 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro 1625 1630 1635 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala 1640 1645 1650 Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln 1655 1660 1665 Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 1670 1675 1680 Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro 1685 1690 1695 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 1700 1705 1710 Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly 1715 1720 1725 Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly 1730 1735 1740 Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln 1745 1750 1755 Gln Gly Pro Gly Ala Ser Gly Gln Gln Gly Pro Tyr Gly Pro Gly 1760 1765 1770 Ala Ser Ala Ala Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly 1775 1780 1785 Gln Gln Gly Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln 1790 1795 1800 Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 1805 1810 1815 Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser 1820 1825 1830 Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 1835 1840 1845 Ala Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 1850 1855 1860 Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser 1865 1870 1875 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala 1880 1885 1890 Gly Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly 1895 1900 1905 Ala Ser Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser 1910 1915 1920 Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly 1925 1930 1935 Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 1940 1945 1950 Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser 1955 1960 1965 Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser 1970 1975 1980 Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro 1985 1990 1995 Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro 2000 2005 2010 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly 2015 2020 2025 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 2030 2035 2040 Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly 2045 2050 2055 Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly 2060 2065 2070 Gln Gln Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly 2075 2080 2085 Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 2090 2095 2100 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln 2105 2110 2115 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln 2120 2125 2130 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly 2135 2140 2145 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 2150 2155 2160 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala 2165 2170 2175 Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr 2180 2185 2190 Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly 2195 2200 2205 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 2210 2215 2220 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala 2225 2230 2235 Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser 2240 2245 2250 Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln 2255 2260 2265 Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln 2270 2275 2280 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 2285 2290 2295 Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro 2300 2305 2310 Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly Pro Gly Gln 2315 2320 2325 Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser 2330 2335 2340 Gly Gln Gln Gly Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu 2345 2350 2355 His His His His His His 2360 <210> 10 <211> 597 <212> PRT <213> Artificial sequence <220> <223> Met-PRT313 <400> 10 Met Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15<0002​​​​​​​​​​​​​​​​​​​​​​​​​115 120 125 Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro 130 135 140 Gly Ser Gly Gly Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 145 150 155 160 Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro 165 170 175 Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly 180 185 190 Gly Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly 195 200 205 Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gly Ser Ala Ala 210 215 220 Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr 225 230 235 240 Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gly Gln Gly Pro Tyr Gly 245 250 255 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Tyr Gly Pro 260 265 270 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 275 280 285 Gly Gly Asn Gly Pro Gly Ser Gly Gly Tyr Gly Pro Gly Gln Gln Gly 290 295 300 Pro Gly Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Gly Gln Gly Pro 305 310 315 320 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 325 330 335 Gly Gly Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ser Ala Ala Ala 340 345 350 Ala Ala Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala 355 360 365 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly 370 375 380 Pro Gly Gly Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gln Gln Gly Pro 385 390 395 400 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 405 410 415 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 420 425 430 Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro Ser Ala 435 440 445 Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Ser Gly Pro Gly Gly Tyr 450 455 460 Gly Pro Tyr Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly 465 470 475 480 Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 485 490 495 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro 500 505 510 Gly Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gly Tyr Gly 515 520 525 Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser 530 535 540 Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser Ala Ala Ala 545 550 555 560 Ala Ala Gly Gly Tyr Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 565 570 575 Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln 580 585 590 Gly Pro Gly Ala Ser 595 <210> 11 <211> 601 <212> PRT <213> Artificial sequence <220> <223> PRT410 <400> 11 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gln 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln 20 25 30 Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Gly Gln Tyr 35 40 45 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala 50 55 60 Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 65 70 75 80 Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly 85 90 95 Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 100 105 110 [[ID=4,7]]Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser 115 120 125 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly 130 135 140 Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 145 150 155 160 Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala 165 170 175 Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro 180 185 190 Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly 195 200 205 Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly 210 215 220 Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro 225 230 235 240 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 245 250 255 Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 260 265 270 Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln 275 280 285 Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln 290 295 300 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 305 310 315 320 Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly 325 330 335 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala 340 345 350 Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln 355 360 365 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln 370 375 380 Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr 385 390 395 400 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly 405 410 415 Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln 420 425 430 Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser 435 440 445 Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala 450 455 460 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 465 470 475 480 Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly 485 490 495 Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln 500 505 510 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 515 520 525 Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 530 535 540 Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln 545 550 555 560 Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly Pro 565 570 575 Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 580 585 590 Ser Gly Gln Gln Gly Pro Gly Ala Ser 595 600 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <220> <223> His tag <400> 12 Met His His His His His His Ser Ser Gly Ser Ser 1 5 10 <210> 13 <211> 608 <212> PRT <213> Artificial sequence <220> <223> PRT380 <400> 13 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gln 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln 20 25 30 Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala 35 40 45 Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 50 55 60 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro 65 70 75 80 Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 85 90 95 Ser Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln 100 105 110 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser 115 120 125 Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly 130 135 140 Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr 145 150 155 160 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 165 170 175 Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 180 185 190 Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Tyr 195 200 205 Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln 210 215 220 Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Ser 225 230 235 240 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala 245 250 255 Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala 260 265 270 Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro 275 280 285 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Asn Gly Pro 290 295 300 Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala 305 310 315 320 Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala 325 330 335 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 340 345 350 Gly Gln Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly 355 360 365 Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly 370 375 380 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala 385 390 395 400 Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro 405 410 415 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln 420 425 430 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 435 440 445 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala 450 455 460 Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro 465 470 475 480 Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly 485 490 495 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln 500 505 510 Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala 515 520 525 Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Ala 530 535 540 Ala Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro 545 550 555 560 Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr 565 570 575 Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 580 585 590 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 595 600 605 <210> 14 <211> 576 <212> PRT <213> Artificial sequence <220> <223> PRT468 <400> 14 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gln 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala 20 25 30 Gly Ser Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Gly 35 40 45 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 50 55 60 Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly 65 70 75 80 Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro 85 90 95 Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly 100 105 110 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala 115 120 125 Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr 130 135 140 Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr 145 150 155 160 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr Gly 165 170 175 Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala 180 185 190 Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Tyr Ala Ser Ala 195 200 205 Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Gln 210 215 220 Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly Pro Gly 225 230 235 240 Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro 245 250 255 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 260 265 270 Ala Ala Gly Ser Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly 275 280 285 Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro 290 295 300 Gly Gln Gln Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly 305 310 315 320 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 325 330 335 Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr 340 345 350 Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 355 360 365 Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln 370 375 380 Gln Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 385 390 395 400 Gly Ser Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 405 410 415 Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 420 425 430 Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln 435 440 445 Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly 450 455 460 Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser Gly Pro 465 470 475 480 Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 485 490 495 Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Gly Pro 500 505 510 Tyr Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 515 520 525 Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser 530 535 540 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Pro Ser Ala Ala Ala 545 550 555 560 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 565 570 575 <210> 15 <211> 2375 <212> PRT <213> Artificial sequence <220> <223> PRT799 <400> 15 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gln 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln 20 25 30 Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Gly Gln Tyr 35 40 45 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala 50 55 60 Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 65 70 75 80 Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly 85 90 95 Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 100 105 110 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser 115 120 125 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly 130 135 140 Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 145 150 155 160 Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala 165 170 175 Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro 180 185 190 Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly 195 200 205 Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly 210 215 220 Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro 225 230 235 240 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 245 250 255 Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 260 265 270 Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln 275 280 285 Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln 290 295 300 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 305 310 315 320 Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly 325 330 335 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala 340 345 350 Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln 355 360 365 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln 370 375 380 Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr 385 390 395 400 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly 405 410 415 Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln 420 425 430 Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser 435 440 445 Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala 450 455 460 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 465 470 475 480 Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly 485 490 495 Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln 500 505 510 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 515 520 525 Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 530 535 540 Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln 545 550 555 560 Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly Pro 565 570 575 Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 580 585 590 Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Gln Gly Pro Tyr Gly 595 600 605 Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser 610 615 620 Gly Gln Gln Gly Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln 625 630 635 640 Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly 645 650 655 Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 660 665 670 Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly 675 680 685 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 690 695 700 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln 705 710 715 720 Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln 725 730 735 Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr 740 745 750 Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly 755 760 765 Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Tyr Ala Ser Ala Ala 770 775 780 Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr 785 790 795 800 Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly 805 810 815 Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro 820 825 830 Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr 835 840 845 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn 850 855 860 Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 865 870 875 880 Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro 885 890 895 Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln 900 905 910 Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly 915 920 925 Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly 930 935 940 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala 945 950 955 960 Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro 965 970 975 Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser 980 985 990 Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly 995 1000 1005 Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly 1010 1015 1020 Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser Gly Pro Gly 1025 1030 1035 Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 1040 1045 1050 Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr 1055 1060 1065 Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly 1070 1075 1080 Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly 1085 1090 1095 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala 1100 1105 1110 Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr 1115 1120 1125 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser 1130 1135 1140 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser 1145 1150 1155 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala 1160 1165 1170 Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 1175 1180 1185 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 1190 1195 1200 Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln 1205 1210 1215 Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 1220 1225 1230 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly 1235 1240 1245 Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly 1250 1255 1260 Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Tyr Gly 1265 1270 1275 Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala 1280 1285 1290 Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly 1295 1300 1305 Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln 1310 1315 1320 Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln 1325 1330 1335 Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala 1340 1345 1350 Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Tyr Ala 1355 1360 1365 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln 1370 1375 1380 Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly 1385 1390 1395 Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly 1400 1405 1410 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala 1415 1420 1425 Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr 1430 1435 1440 Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr 1445 1450 1455 Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 1460 1465 1470 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1475 1480 1485 Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 1490 1495 1500 Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly 1505 1510 1515 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly 1520 1525 1530 Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 1535 1540 1545 Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro 1550 1555 1560 Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala 1565 1570 1575 Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln 1580 1585 1590 Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly 1595 1600 1605 Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser Gly 1610 1615 1620 Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala 1625 1630 1635 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly 1640 1645 1650 Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly 1655 1660 1665 Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly 1670 1675 1680 Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala 1685 1690 1695 Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly 1700 1705 1710 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 1715 1720 1725 Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser 1730 1735 1740 Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser 1745 1750 1755 Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 1760 1765 1770 Ala Ser Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1775 1780 1785 Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro 1790 1795 1800 Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 1805 1810 1815 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln 1820 1825 1830 Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala 1835 1840 1845 Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln 1850 1855 1860 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 1865 1870 1875 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln 1880 1885 1890 Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser 1895 1900 1905 Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro 1910 1915 1920 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 1925 1930 1935 Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro 1940 1945 1950 Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro 1955 1960 1965 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln 1970 1975 1980 Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala 1985 1990 1995 Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala 2000 2005 2010 Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly 2015 2020 2025 Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly 2030 2035 2040 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala 2045 2050 2055 Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser 2060 2065 2070 Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 2075 2080 2085 Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro 2090 2095 2100 Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly 2105 2110 2115 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala 2120 2125 2130 Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr 2135 2140 2145 Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro 2150 2155 2160 Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro 2165 2170 2175 Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln 2180 2185 2190 Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln 2195 2200 2205 Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro 2210 2215 2220 Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln 2225 2230 2235 Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly 2240 2245 2250 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly 2255 2260 2265 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 2270 2275 2280 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln 2285 2290 2295 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 2300 2305 2310 Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 2315 2320 2325 Gln Ser Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr 2330 2335 2340 Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly 2345 2350 2355 Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His 2360 2365 2370 His His 2375 <210> 16 <211> 608 <212> PRT <213> Artificial Sequence <220> <223> PRT313 <400> 16 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gly 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly 20 25 30 Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ser Ala Ala Ala 35 40 45 Ala Ala Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro Gly Gln Gln Gly 50 55 60 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro 65 70 75 80 Gly Gly Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 85 90 95 Ser Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly 100 105 110 Tyr Gly Pro Gly Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser 115 120 125 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Ser Gly Pro Gly Gln Gln Gly 130 135 140 Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gly Tyr 145 150 155 160 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 165 170 175 Pro Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro Ser Ala Ser Ala Ala 180 185 190 Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Tyr 195 200 205 Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Ser Gly Pro Gly Gln 210 215 220 Gln Gly Pro Tyr Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala Gly Ser 225 230 235 240 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala 245 250 255 Ala Ala Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala 260 265 270 Ala Ala Ala Ala Gly Gly Tyr Gly Tyr Gly Pro Gly Gly Gln Gly Pro 275 280 285 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly Asn Gly Pro 290 295 300 Gly Ser Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser Ala 305 310 315 320 Ala Ala Ala Ala Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 325 330 335 Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro 340 345 350 Gly Gly Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly 355 360 365 Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly 370 375 380 Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gly Ser Ala 385 390 395 400 Ala Ala Ala Ala Gly Gly Tyr Gln Gln Gly Pro Gly Gly Gln Gly Pro 405 410 415 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gly Gln 420 425 430 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 435 440 445 Gly Tyr Gly Pro Gly Gly Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala 450 455 460 Ala Gly Gly Tyr Gly Ser Gly Pro Gly Gly Tyr Gly Pro Tyr Gly Pro 465 470 475 480 Gly Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly 485 490 495 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly 500 505 510 Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gly Ser Ala Ala 515 520 525 Ala Ala Ala Gly Pro Gly Ser Gly Gly Tyr Gly Pro Gly Ala Ser Ala 530 535 540 Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser Gly Gly Tyr Gly Pro 545 550 555 560 Gly Gln Gln Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala Gly Gly Tyr 565 570 575 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 580 585 590 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 595 600 605 <210> 17 <211> 601 <212> PRT <213> Synthetic Sequence <220> <223> PRT399 <400> 17 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gly 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly 20 25 30 Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ser Gly Gly Tyr 35 40 45 Gly Pro Gly Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala 50 55 60 Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro 65 70 75 80 Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly 85 90 95 Pro Gly Ala Ser Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro Gly Gln 100 105 110 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Ser 115 120 125 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly 130 135 140 Pro Gly Ser Gly Gly Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 145 150 155 160 Gly Pro Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro Ser Ala Ser Ala 165 170 175 Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro 180 185 190 Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Ser Gly Pro Gly 195 200 205 Gln Gln Gly Pro Tyr Gly Pro Gly Gly Ser Gly Ser Gly Gln Gln Gly 210 215 220 Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro 225 230 235 240 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 245 250 255 Gly Gly Tyr Gly Tyr Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly 260 265 270 Ala Ser Gly Gly Asn Gly Pro Gly Ser Gly Gly Tyr Gly Pro Gly Gln 275 280 285 Gln Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Gly Gln 290 295 300 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr 305 310 315 320 Gly Pro Gly Gly Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ser Gly 325 330 335 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala 340 345 350 Ala Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gly 355 360 365 Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gln Gln Gly Pro Gly Gly Gln 370 375 380 Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gly Gln Gly Pro Tyr 385 390 395 400 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly 405 410 415 Pro Gly Gly Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gly 420 425 430 Tyr Gly Ser Gly Pro Gly Gly Tyr Gly Pro Tyr Gly Pro Gly Gly Ser 435 440 445 Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala 450 455 460 Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro 465 470 475 480 Tyr Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly 485 490 495 Gly Tyr Gly Pro Gly Ala Ser Gly Gly Asn Gly Pro Gly Ser Gly Gly 500 505 510 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala 515 520 525 Gly Gly Tyr Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly 530 535 540 Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Ser Gly Pro Gly Gln 545 550 555 560 Gln Gly Pro Tyr Gly Pro Gly Gly Ser Gly Ser Gly Gln Gln Gly Pro 565 570 575 Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 580 585 590 Ser Gly Gln Gln Gly Pro Gly Ala Ser 595 600 <210> 18 <211> 590 <212> PRT <213> Artificial Sequence <220> <223> Met-PRT399 <400> 18 Met Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 20 25 30 Gly Ser Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro Gly Gln Gln Gly 35 40 45 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro 50 55 60 Gly Gly Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 65 70 75 80 Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gly Tyr Gly Pro Gly Gly 85 90 95 Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 100 105 110 Gly Gly Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala 115 120 125 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gly Tyr Gly Gln Gly Pro Tyr 130 135 140 Gly Pro Gly Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Gln Gly 145 150 155 160 Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro 165 170 175 Gly Gly Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr 180 185 190 Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gly Ser Gly 195 200 205 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala 210 215 220 Ala Ala Ala Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser 225 230 235 240 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Tyr Gly Pro Gly Gly Gln Gly 245 250 255 Pro Tyr Gly Pro Gly Ala Ser Gly Gly Asn Gly Pro Gly Ser Gly Gly 260 265 270 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala 275 280 285 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 290 295 300 Ala Ala Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro Gly Gly Tyr Gly 305 310 315 320 Pro Gly Ser Ser Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser 325 330 335 Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro 340 345 350 Tyr Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gln Gln 355 360 365 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly 370 375 380 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 385 390 395 400 Pro Gly Gly Tyr Gly Pro Gly Gly Gln Gly Pro Ser Ala Ser Ala Ala 405 410 415 Ala Ala Ala Gly Gly Tyr Gly Ser Gly Pro Gly Gly Tyr Gly Pro Tyr 420 425 430 Gly Pro Gly Gly Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro 435 440 445 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 450 455 460 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala 465 470 475 480 Gly Pro Gly Ser Gly Gly Tyr Gly Pro Gly Ala Ser Gly Gly Asn Gly 485 490 495 Pro Gly Ser Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser 500 505 510 Ala Ala Ala Ala Ala Gly Gly Tyr Gln Gln Gly Pro Gly Gly Gln Gly 515 520 525 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr Gly 530 535 540 Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gly Ser Gly Ser 545 550 555 560 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala 565 570 575 Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 580 585 590 <210> 19 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Met-PRT720 <400> 19 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 20 25 30 Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 35 40 45 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Val Leu 50 55 60 Ile Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Ser Ala Ser Ala Ala 65 70 75 80 Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly 85 90 95 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 100 105 110 Ser Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Val Leu Ile Gly Pro 115 120 125 Gly Gln Gln Val Leu Ile Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala 130 135 140 Gly Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala 145 150 155 160 Ser Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser 165 170 175 Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Val Leu Ile Gly Pro Gly 180 185 190 Gln Tyr Val Leu Ile Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly 195 200 205 Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln 210 215 220 Ser Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser 225 230 235 240 Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr 245 250 255 Val Leu Ile Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr 260 265 270 Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly 275 280 285 Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 290 295 300 Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Val Leu Ile 305 310 315 320 Gly Pro Tyr Val Leu Ile Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 325 330 335 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Gly 340 345 350 Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala 355 360 365 Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Val Leu Ile Gly 370 375 380 Pro Tyr Val Leu Ile Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Gly 385 390 395 400 Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala 405 410 415 Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 420 425 430 Ala Ala Ala Gly Pro Gly Gln Tyr Val Leu Ile Gly Pro Gly Gln Gln 435 440 445 Val Leu Ile Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 450 455 460 Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser Gly 465 470 475 480 Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 485 490 495 Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Val Leu Ile 500 505 510 Gly Pro Tyr Val Leu Ile Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala 515 520 525 Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly 530 535 540 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser 545 550 555 560 Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Val Leu Ile Gly Pro Gly 565 570 575 Gln Gln Gly Pro Tyr Val Leu Ile Gly Pro Gly Ala Ser Ala Ala Ala 580 585 590 Ala Ala Gly Pro Gly Ser Gly Gln Gln Val Leu Ile Gly Pro Gly Ala 595 600 605 Ser Val Leu Ile 610 <210> 20 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> Met-PRT665 <400> 20 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Gly Ser Asn Gly Pro Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 35 40 45 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 50 55 60 Gln Tyr Val Leu Ile Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala 65 70 75 80 Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 85 90 95 Ala Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 100 105 110 Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser 115 120 125 Val Leu Ile Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala 130 135 140 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr 145 150 155 160 Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly 165 170 175 Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln 180 185 190 Val Leu Ile Gly Pro Gly Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala 195 200 205 Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro 210 215 220 Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln 225 230 235 240 Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln 245 250 255 Gln Val Leu Ile Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala 260 265 270 Ala Ala Ala Gly Ser Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr 275 280 285 Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly 290 295 300 Pro Gly Gln Gln Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 305 310 315 320 Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Gly Pro Gly Ala Ser 325 330 335 Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Gly 340 345 350 Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro 355 360 365 Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr 370 375 380 Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Gly Pro Gly Pro Ser 385 390 395 400 Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gln Gln Gly Pro Gly Gln 405 410 415 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro 420 425 430 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 435 440 445 Gln Tyr Val Leu Ile Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala 450 455 460 Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Tyr 465 470 475 480 Gly Pro Tyr Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly 485 490 495 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 500 505 510 Gly Ser Tyr Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Gly Pro 515 520 525 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln 530 535 540 Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr 545 550 555 560 Gly Pro Gly Gln Gln Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala 565 570 575 Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Val Leu Ile 580 585 590 <210> 21 <211> 619 <212> PRT <213> Synthetic Sequence <220> <223> Met-PRT666 <400> 21 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Gly Ser Asn Gly Pro Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 35 40 45 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 50 55 60 Gln Tyr Val Leu Ile Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Ser 65 70 75 80 Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln 85 90 95 Gly Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly 100 105 110 Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser 115 120 125 Tyr Gly Ser Val Leu Ile Gly Pro Gly Gln Gln Val Leu Ile Gly Pro 130 135 140 Tyr Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln 145 150 155 160 Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr 165 170 175 Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala 180 185 190 Ala Gly Ser Gly Gln Gln Val Leu Ile Gly Pro Gly Gln Tyr Val Leu 195 200 205 Ile Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr 210 215 220 Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly 225 230 235 240 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala 245 250 255 Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr 260 265 270 Val Leu Ile Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly 275 280 285 Ser Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala 290 295 300 Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln 305 310 315 320 Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln 325 330 335 Gln Val Leu Ile Gly Pro Tyr Val Leu Ile Gly Pro Gly Ala Ser Ala 340 345 350 Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Gly Pro 355 360 365 Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr 370 375 380 Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly 385 390 395 400 Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Val Leu Ile Gly Pro Gly 405 410 415 Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gln Gln Gly Pro 420 425 430 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln 435 440 445 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly 450 455 460 Pro Gly Gln Tyr Val Leu Ile Gly Pro Gly Gln Gln Val Leu Ile Gly 465 470 475 480 Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser 485 490 495 Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser Gly Pro Gly 500 505 510 Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 515 520 525 Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Val Leu Ile 530 535 540 Gly Pro Tyr Val Leu Ile Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala 545 550 555 560 Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln 565 570 575 Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly 580 585 590 Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln 595 600 605 Val Leu Ile Gly Pro Gly Ala Ser Val Leu Ile 610 615 <210> 22 <211> 590 <212> PRT <213> Artificial Sequence <22o> [ <223> Met-PRT410 <400> 22 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 20 25 30 Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 35 40 45 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro 50 55 60 Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 65 70 75 80 Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln 85 90 95 Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 100 105 110 Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala 115 120 125 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr 130 135 140 Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly 145 150 155 160 Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro 165 170 175 Gly Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 180 185 190 Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly 195 200 205 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala 210 215 220 Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser 225 230 235 240 Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly 245 250 255 Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln 260 265 270 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 275 280 285 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 290 295 300 Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly 305 310 315 320 Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser 325 330 335 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro 340 345 350 Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln 355 360 365 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly 370 375 380 Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 385 390 395 400 Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 405 410 415 Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr 420 425 430 Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro 435 440 445 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro 450 455 460 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 465 470 475 480 Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly 485 490 495 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser 500 505 510 Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly 515 520 525 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly 530 535 540 Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser 545 550 555 560 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala 565 570 575 Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 580 585 590 <210> 23 <211> 565 <212> PRT <213> Artificial Sequence <220> <223> Met-PRT468 <400> 23 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Gly Ser Asn Gly Pro Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 35 40 45 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 50 55 60 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala 65 70 75 80 Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly 85 90 95 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 100 105 110 Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly 115 120 125 Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro 130 135 140 Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly 145 150 155 160 Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 165 170 175 Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly 180 185 190 Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser 195 200 205 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly 210 215 220 Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser 245 250 255 Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Tyr Gly Pro Gly Gln 260 265 270 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser 275 280 285 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Pro Ser Ala Ala Ala 290 295 300 Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala 305 310 315 320 Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln 325 330 335 Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly 340 345 350 Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser 355 360 365 Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala 370 375 380 Ala Ala Ala Ala Ala Gly Ser Tyr Gln Gln Gly Pro Gly Gln Gln Gly 385 390 395 400 Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly 405 410 415 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr 420 425 430 Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala 435 440 445 Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro 450 455 460 Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly 465 470 475 480 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro 485 490 495 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala 500 505 510 Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln 515 520 525 Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly 530 535 540 Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln 545 550 555 560 Gly Pro Gly Ala Ser 565 <210> 24 <211> 623 <212> PRT <213> Artificial Sequence <220> <223> PRT468 <400> 24 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gln 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln [[ID=3৮]]20 25 30 Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Gly Gln Tyr 35 40 45 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala 50 55 60 Ala Ala Ala Ala Gly Pro Gly Gln Tyr Val Leu Ile Gly Pro Gly Gln 65 70 75 80 Gln Val Leu Ile Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro 85 90 95 Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly 100 105 110 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala 115 120 125 Ala Gly Ser Tyr Gly Ser Val Leu Ile Gly Pro Gly Gln Gln Val Leu 130 135 140 Ile Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly 145 150 155 160 Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln 165 170 175 Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala 180 185 190 Gly Ser Gly Gln Gln Val Leu Ile Gly Pro Gly Gln Tyr Val Leu Ile 195 200 205 Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly 210 215 220 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln 225 230 235 240 Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala 245 250 255 Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Val Leu Ile Gly Pro 260 265 270 Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly 275 280 285 Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly 290 295 300 Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala 305 310 315 320 Ala Ala Ala Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Val Leu 325 330 335 Ile Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro 340 345 350 Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly 355 360 365 Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly 370 375 380 Ser Tyr Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Val Leu Ile 385 390 395 400 Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly 405 410 415 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln 420 425 430 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro 435 440 445 Gly Gln Tyr Val Leu Ile Gly Pro Gly Gln Gln Val Leu Ile Gly Pro 450 455 460 Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly 465 470 475 480 Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Gln 485 490 495 Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 500 505 510 Gly Ser Tyr Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Val Leu 515 520 525 Ile Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly 530 535 540 Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln 545 550 555 560 Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala 565 570 575 Gly Gln Tyr Gln Gln Val Leu Ile Gly Pro Gly Gln Gln Gly Pro Tyr 580 585 590 Val Leu Ile Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 595 600 605 Ser Gly Gln Gln Val Leu Ile Gly Pro Gly Ala Ser Val Leu Ile 610 615 620 <210> 25 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> PRT665 <400> 25 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gln 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala 20 25 30 Gly Ser Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Gly 35 40 45 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 50 55 60 Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Val Leu Ile 65 70 75 80 Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala 85 90 95 Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Tyr 100 105 110 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala 115 120 125 Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Val Leu Ile Gly Pro 130 135 140 Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly 145 150 155 160 Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 165 170 175 Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala 180 185 190 Ala Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Val Leu Ile Gly Pro 195 200 205 Gly Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly 210 215 220 Ser Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln 225 230 235 240 Ser Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser 245 250 255 Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Val Leu Ile Gly 260 265 270 Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser 275 280 285 Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser 290 295 300 Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly 305 310 315 320 Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln 325 330 335 Val Leu Ile Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 340 345 350 Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly 355 360 365 Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser 370 375 380 Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln 385 390 395 400 Val Leu Ile Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala 405 410 415 Ala Ala Gly Ser Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly 420 425 430 Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala 435 440 445 Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Val Leu Ile 450 455 460 Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala 465 470 475 480 Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro 485 490 495 Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly 500 505 510 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro 515 520 525 Gly Gln Gln Val Leu Ile Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala 530 535 540 Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala 545 550 555 560 Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln 565 570 575 Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser 580 585 590 Gly Gln Gln Gly Pro Gly Ala Ser Val Leu Ile 595 600 <210> 26 <211> 630 <212> PRT <213> Artificial sequence <220> <223> PRT666 <400> 26 Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gln 1 5 10 15 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala 20 25 30 Gly Ser Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Gly 35 40 45 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 50 55 60 Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Val Leu Ile 65 70 75 80 Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Ser Ala Ser Ala Ala Ala 85 90 95 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser 100 105 110 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 115 120 125 Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Val Leu 130 135 140 Ile Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Gly Ser Ala Ala 145 150 155 160 Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro 165 170 175 Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln 180 185 190 Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Gly Gln 195 200 205 Gln Val Leu Ile Gly Pro Gly Gln Tyr Val Leu Ile Gly Pro Tyr Ala 210 215 220 Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly 225 230 235 240 Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly 245 250 255 Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala 260 265 270 Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Val Leu Ile Gly Pro 275 280 285 Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Tyr Gly 290 295 300 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly 305 310 315 320 Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Pro Ser 325 330 335 Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Val Leu Ile Gly 340 345 350 Pro Tyr Val Leu Ile Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 355 360 365 Ala Gly Ser Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro 370 375 380 Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser 385 390 395 400 Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln Val 405 410 415 Leu Ile Gly Pro Tyr Val Leu Ile Gly Pro Gly Pro Ser Ala Ala Ala 420 425 430 Ala Ala Ala Ala Gly Ser Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro 435 440 445 Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro 450 455 460 Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Val 465 470 475 480 Leu Ile Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Ser Ala Ser Ala 485 490 495 Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Tyr 500 505 510 Gly Pro Tyr Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly 515 520 525 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 530 535 540 Gly Ser Tyr Gly Pro Gly Gln Gln Val Leu Ile Gly Pro Tyr Val Leu 545 550 555 560 Ile Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 565 570 575 Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser 580 585 590 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Pro Ser Ala Ala Ala 595 600 605 Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Val Leu Ile Gly Pro 610 615 620 Gly Ala Ser Val Leu Ile 625 630 <210> 27 <211> 593 <212> PRT <213> Artificial Sequence <220> <223> M_PRT888 <400> 27 Met Gly Ser Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala 1 5 10 15 Ser Ala Ala Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Val Leu 20 25 30 Gly Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Gly 35 40 45 Val Leu Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln 50 55 60 Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala 65 70 75 80 Gly Pro Gly Ser Gly Val Leu Gly Pro Gly Ala Ser Gly Gln Tyr Gly 85 90 95 Pro Gly Val Leu Gly Pro Gly Val Leu Gly Pro Gly Ser Ser Ala Ala 100 105 110 Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Val Leu Gly Pro Tyr 115 120 125 Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Gln 130 135 140 Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr Gly Pro Gly 145 150 155 160 Val Leu Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Val 165 170 175 Leu Gly Pro Gly Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala 180 185 190 Gly Gln Tyr Gly Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly 195 200 205 Gln Ser Gly Ser Gly Val Leu Gly Pro Gly Val Leu Gly Pro Tyr Ala 210 215 220 Ser Ala Ala Ala Ala Ala Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro 225 230 235 240 Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly 245 250 255 Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly 260 265 270 Ser Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Gly Gln Ser Ala Ala 275 280 285 Ala Ala Ala Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser 290 295 300 Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Gly 305 310 315 320 Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly 325 330 335 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Val 340 345 350 Leu Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln 355 360 365 Tyr Val Leu Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser 370 375 380 Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 385 390 395 400 Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala 405 410 415 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr 420 425 430 Gly Pro Tyr Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Val Leu Gly 435 440 445 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln 450 455 460 Tyr Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala 465 470 475 480 Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly 485 490 495 Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro 500 505 510 Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Val Leu Gly Pro Gly 515 520 525 Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 530 535 540 Gln Tyr Gly Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Gln 545 550 555 560 Ser Gly Ser Gly Val Leu Gly Pro Gly Val Leu Gly Pro Tyr Ala Ser 5,65 570 575 Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Val Leu Gly Pro Gly Ala 580 585 590 Ser <210> 28 <211> 590 <212> PRT <213> Artificial Sequence <220> <223> M_PRT965 <400> 28 Met Gly Pro Gly Thr Ser Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Gly Ala Asn Gly Pro Gly Ser Gly Thr Ser Gly Pro Gly 20 25 30 Ala Ser Gly Ala Tyr Gly Pro Gly Thr Ser Gly Pro Gly Thr Ser Gly 35 40 45 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Ala Tyr Gly Pro 50 55 60 Gly Thr Ser Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 65 70 75 80 Ser Gly Thr Ser Gly Pro Gly Ala Ser Gly Ala Tyr Gly Pro Gly Thr 85 90 95 Ser Gly Pro Gly Thr Ser Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 100 105 110 Gly Ala Tyr Gly Ser Gly Pro Gly Thr Ser Gly Pro Tyr Gly Ser Ala 115 120 125 Ala Ala Ala Ala Gly Pro Gly Ser Gly Ala Tyr Gly Ala Gly Pro Tyr 130 135 140 Gly Pro Gly Ala Ser Gly Pro Gly Ala Tyr Gly Pro Gly Thr Ser Gly 145 150 155 160 Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Thr Ser Gly Pro 165 170 175 Gly Ala Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Ala Tyr 180 185 190 Gly Ser Gly Pro Gly Thr Ser Gly Pro Tyr Gly Pro Gly Ala Ser Gly 195 200 205 Ser Gly Thr Ser Gly Pro Gly Thr Ser Gly Pro Tyr Ala Ser Ala Ala 210 215 220 Ala Ala Ala Gly Pro Gly Thr Ser Gly Pro Tyr Gly Pro Gly Ser Ser 225 230 235 240 Ala Ala Ala Ala Ala Gly Ala Tyr Gly Tyr Gly Pro Gly Thr Ser Gly 245 250 255 Pro Tyr Gly Pro Gly Ala Ser Gly Ala Asn Gly Pro Gly Ser Gly Ala 260 265 270 Tyr Gly Pro Gly Thr Ser Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 275 280 285 Gly Pro Gly Thr Ser Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 290 295 300 Ala Ala Gly Ala Tyr Gly Pro Gly Thr Ser Gly Pro Gly Ala Tyr Gly 305 310 315 320 Pro Gly Ser Ser Gly Pro Gly Thr Ser Gly Pro Tyr Gly Pro Gly Ser 325 330 335 Ser Ala Ala Ala Ala Ala Gly Ala Tyr Gly Pro Gly Thr Ser Gly Pro 340 345 350 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Ala Tyr Thr Ser 355 360 365 Gly Pro Gly Thr Ser Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly 370 375 380 Thr Ser Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 385 390 395 400 Pro Gly Ala Tyr Gly Pro Gly Thr Ser Gly Pro Ser Ala Ser Ala Ala 405 410 415 Ala Ala Ala Gly Ala Tyr Gly Ser Gly Pro Gly Ala Tyr Gly Pro Tyr 420 425 430 Gly Pro Gly Ala Ser Gly Pro Gly Ser Gly Thr Ser Gly Ala Gly Pro 435 440 445 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Ala Tyr Gly Pro 450 455 460 Gly Thr Ser Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 465 470 475 480 Gly Pro Gly Ser Gly Ala Tyr Gly Pro Gly Ala Ser Gly Ala Asn Gly 485 490 495 Pro Gly Ser Gly Ala Tyr Gly Pro Gly Thr Ser Gly Pro Gly Ala Ser 500 505 510 Ala Ala Ala Ala Ala Gly Ala Tyr Thr Ser Gly Pro Gly Thr Ser Gly 515 520 525 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Ala Tyr Gly 530 535 540 Ser Gly Pro Gly Thr Ser Gly Pro Tyr Gly Pro Gly Ala Ser Gly Ser 545 550 555 560 Gly Thr Ser Gly Pro Gly Thr Ser Gly Pro Tyr Ala Ser Ala Ala Ala 565 570 575 Ala Ala Gly Pro Gly Ser Gly Thr Ser Gly Pro Gly Ala Ser 580 585 590 <21?> 29 <??> 593 <??> PRT <??> Artificial Sequence <??> <??> M_PRT889 <400> 29 Met Gly Ser Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala 1 5 10 15 Ser Ala Ala Ala Ala Ala Gly Ile Asn Gly Pro Gly Ser Gly Val Leu 20 25 30 Gly Pro Gly Ile Ser Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro Gly 35 40 45 It should be noted that there are some tags like <21?> and <??> in the original text which seem to be incomplete or incorrect tags. This translation is done as accurately as possible based on the available information.Val Leu Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Ile 50 55 60 Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala 65 70 75 80 Gly Pro Gly Ser Gly Val Leu Gly Pro Gly Ala Ser Gly Ile Tyr Gly 85 90 95 Pro Gly Val Leu Gly Pro Gly Val Leu Gly Pro Gly Ser Ser Ala Ala 100 105 110 Ala Ala Ala Gly Ile Tyr Gly Ser Gly Pro Gly Val Leu Gly Pro Tyr 115 120 125 Gly Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Ile Tyr Gly Ile 130 135 140 Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Ile Tyr Gly Pro Gly 145 150 155 160 Val Leu Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Val 165 170 175 Leu Gly Pro Gly Ile Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala 180 185 190 Gly Ile Tyr Gly Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly 195 200 205 Ile Ser Gly Ser Gly Val Leu Gly Pro Gly Val Leu Gly Pro Tyr Ala 210 215 220 Ser Ala Ala Ala Ala Ala Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro 225 230 235 240 Gly Ser Ser Ala Ala Ala Ala Ala Gly Ile Tyr Gly Tyr Gly Pro Gly 245 250 255 Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser Gly Ile Asn Gly Pro Gly 260 265 270 Ser Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro Gly Ile Ser Ala Ala 275 280 285 Ala Ala Ala Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser 290 295 300 Ala Ala Ala Ala Ala Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro Gly 305 310 315 320 Ile Tyr Gly Pro Gly Ser Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly 325 330 335 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Ile Tyr Gly Pro Gly Val 340 345 350 Leu Gly Pro Tyr Gly Pro Gly Ile Ser Ala Ala Ala Ala Ala Gly Ile 355 360 365 Tyr Val Leu Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser 370 375 380 Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 385 390 395 400 Ala Ala Gly Pro Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala 405 410 415 Ser Ala Ala Ala Ala Ala Gly Ile Tyr Gly Ser Gly Pro Gly Ile Tyr 420 425 430 Gly Pro Tyr Gly Pro Gly Ile Ser Gly Pro Gly Ser Gly Val Leu Gly 435 440 445 Ile Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Ile 450 455 460 Tyr Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ile Ser Ala Ala 465 470 475 480 Ala Ala Ala Gly Pro Gly Ser Gly Ile Tyr Gly Pro Gly Ala Ser Gly 485 490 495 Ile Asn Gly Pro Gly Ser Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro 500 505 510 Gly Ile Ser Ala Ala Ala Ala Ala Gly Ile Tyr Val Leu Gly Pro Gly 515 520 525 Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 530 535 540 Ile Tyr Gly Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ile 545 550 555 560 Ser Gly Ser Gly Val Leu Gly Pro Gly Val Leu Gly Pro Tyr Ala Ser 565 570 575 Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Val Leu Gly Pro Gly Ala 580 585 590 Ser<00,03908><210> 30 <211> 590 <212> PRT <213> Artificial sequence <220> <223> M_PRT916 <400> 30 Met Gly Pro Gly Val Ile Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Gly Leu Asn Gly Pro Gly Ser Gly Val Ile Gly Pro Gly 20 25 30 Leu Ser Gly Leu Tyr Gly Pro Gly Val Ile Gly Pro Gly Val Ile Gly 35 40 45 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Leu Tyr Gly Pro 50 55 60 Gly Val Ile Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 65 70 75 80 Ser Gly Val Ile Gly Pro Gly Ala Ser Gly Leu Tyr Gly Pro Gly Val 85 90 95 Ile Gly Pro Gly Val Ile Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 100 105 110 Gly Leu Tyr Gly Ser Gly Pro Gly Val Ile Gly Pro Tyr Gly Ser Ala 115 120 125 Ala Ala Ala Ala Gly Pro Gly Ser Gly Leu Tyr Gly Leu Gly Pro Tyr 130 135 140 Gly Pro Gly Ala Ser Gly Pro Gly Leu Tyr Gly Pro Gly Val Ile Gly 145 150 155 160 Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Val Ile Gly Pro 165 170 175 Gly Leu Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Leu Tyr 180 185 190 Gly Ser Gly Pro Gly Val Ile Gly Pro Tyr Gly Pro Gly Leu Ser Gly 195 200 205 Ser Gly Val Ile Gly Pro Gly Val Ile Gly Pro Tyr Ala Ser Ala Ala 210 215 220 Ala Ala Ala Gly Pro Gly Val Ile Gly Pro Tyr Gly Pro Gly Ser Ser 225 230 235 240 Ala Ala Ala Ala Ala Gly Leu Tyr Gly Tyr Gly Pro Gly Val Ile Gly 245 250 255 Pro Tyr Gly Pro Gly Ala Ser Gly Leu Asn Gly Pro Gly Ser Gly Leu 260 265 270 Tyr Gly Pro Gly Val Ile Gly Pro Gly Leu Ser Ala Ala Ala Ala Ala 275 280 285 Gly Pro Gly Val Ile Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 290 295 300 Ala Ala Gly Leu Tyr Gly Pro Gly Val Ile Gly Pro Gly Leu Tyr Gly 305 310 315 320 Pro Gly Ser Ser Gly Pro Gly Val Ile Gly Pro Tyr Gly Pro Gly Ser 325 330 335 Ser Ala Ala Ala Ala Ala Gly Leu Tyr Gly Pro Gly Val Ile Gly Pro 340 345 350 Tyr Gly Pro Gly Leu Ser Ala Ala Ala Ala Ala Gly Leu Tyr Val Ile 355 360 365 Gly Pro Gly Val Ile Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly 370 375 380 Val Ile Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 385 390 395 400 Pro Gly Leu Tyr Gly Pro Gly Val Ile Gly Pro Ser Ala Ser Ala Ala 405 410 415 Ala Ala Ala Gly Leu Tyr Gly Ser Gly Pro Gly Leu Tyr Gly Pro Tyr 420 425 430 Gly Pro Gly Leu Ser Gly Pro Gly Ser Gly Val Ile Gly Leu Gly Pro 435 440 445 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Leu Tyr Gly Pro 450 455 460 Gly Val Ile Gly Pro Tyr Gly Pro Gly Leu Ser Ala Ala Ala Ala Ala 465 470 475 480 Gly Pro Gly Ser Gly Leu Tyr Gly Pro Gly Ala Ser Gly Leu Asn Gly 485 490 495 Pro Gly Ser Gly Leu Tyr Gly Pro Gly Val Ile Gly Pro Gly Leu Ser 500 505 510 Ala Ala Ala Ala Ala Gly Leu Tyr Val Ile Gly Pro Gly Val Ile Gly 515 520 525 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Leu Tyr Gly 530 535 540 Ser Gly Pro Gly Val Ile Gly Pro Tyr Gly Pro Gly Leu Ser Gly Ser 545 550 555 560 Gly Val Ile Gly Pro Gly Val Ile Gly Pro Tyr Ala Ser Ala Ala Ala 565 570 575 Ala Ala Gly Pro Gly Ser Gly Val Ile Gly Pro Gly Ala Ser 580 585 590 <210> 31 <211> 590 <212> PRT <213> Artificial Sequence <220> <223> M_ PRT918 <400> 31 Met Gly Pro Gly Val Phe Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Gly Ile Asn Gly Pro Gly Ser Gly Val Phe Gly Pro Gly 20 25 30 Ile Ser Gly Ile Tyr Gly Pro Gly Val Phe Gly Pro Gly Val Phe Gly 35 40 45 Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Ile Tyr Gly Pro 50 55 60 Gly Val Phe Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly 65 70 75 80 Ser Gly Val Phe Gly Pro Gly Ala Ser Gly Ile Tyr Gly Pro Gly Val 85 90 95 Phe Gly Pro Gly Val Phe Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 100 105 110 Gly Ile Tyr Gly Ser Gly Pro Gly Val Phe Gly Pro Tyr Gly Ser Ala 115 120 125 Ala Ala Ala Ala Gly Pro Gly Ser Gly Ile Tyr Gly Ile Gly Pro Tyr 130 135 140 Gly Pro Gly Ala Ser Gly Pro Gly Ile Tyr Gly Pro Gly Val Phe Gly 145 150 155 160 Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Val Phe Gly Pro 165 170 175 Gly Ile Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Ile Tyr 180 185 190 Gly Ser Gly Pro Gly Val Phe Gly Pro Tyr Gly Pro Gly Ile Ser Gly 195 200 205 Ser Gly Val Phe Gly Pro Gly Val Phe Gly Pro Tyr Ala Ser Ala Ala 210 215 220 Ala Ala Ala Gly Pro Gly Val Phe Gly Pro Tyr Gly Pro Gly Ser Ser 225 230 235 240 Ala Ala Ala Ala Ala Gly Ile Tyr Gly Tyr Gly Pro Gly Val Phe Gly 245 250 255 Pro Tyr Gly Pro Gly Ala Ser Gly Ile Asn Gly Pro Gly Ser Gly Ile 260 265 270 Tyr Gly Pro Gly Val Phe Gly Pro Gly Ile Ser Ala Ala Ala Ala Ala 275 280 285 Gly Pro Gly Val Phe Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 290 295 300 Ala Ala Gly Ile Tyr Gly Pro Gly Val Phe Gly Pro Gly Ile Tyr Gly 305 310 315 320 Pro Gly Ser Ser Gly Pro Gly Val Phe Gly Pro Tyr Gly Pro Gly Ser 325 330 335 Ser Ala Ala Ala Ala Ala Gly Ile Tyr Gly Pro Gly Val Phe Gly Pro 340 345 350 Tyr Gly Pro Gly Ile Ser Ala Ala Ala Ala Ala Gly Ile Tyr Val Phe 355 360 365 Gly Pro Gly Val Phe Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly 370 375 380 Val Phe Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly 385 390 395 400 Pro Gly Ile Tyr Gly Pro Gly Val Phe Gly Pro Ser Ala Ser Ala Ala 405 410 415 Ala Ala Ala Gly Ile Tyr Gly Ser Gly Pro Gly Ile Tyr Gly Pro Tyr 420 425 430 Gly Pro Gly Ile Ser Gly Pro Gly Ser Gly Val Phe Gly Ile Gly Pro 435 440 445 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Ile Tyr Gly Pro 450 455 460 Gly Val Phe Gly Pro Tyr Gly Pro Gly Ile Ser Ala Ala Ala Ala Ala 465 470 475 480 Gly Pro Gly Ser Gly Ile Tyr Gly Pro Gly Ala Ser Gly Ile Asn Gly 485 490 495 Pro Gly Ser Gly Ile Tyr Gly Pro Gly Val Phe Gly Pro Gly Ile Ser 500 505 510 Ala Ala Ala Ala Ala Gly Ile Tyr Val Phe Gly Pro Gly Val Phe Gly 515 520 525 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Ile Tyr Gly 530 535 540 Ser Gly Pro Gly Val Phe Gly Pro Tyr Gly Pro Gly Ile Ser Gly Ser 545 550 555 560 Gly Val Phe Gly Pro Gly Val Phe Gly Pro Tyr Ala Ser Ala Ala Ala 565 570 575 Ala Ala Gly Pro Gly Ser Gly Val Phe Gly Pro Gly Ala Ser 580 585 590 <210> 32 <211> 565 <212> PRT <213> Artificial Sequence <220> <223> M_PRT699 <400> 32 Met Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Gly Ser Asn Gly Pro Gly Ser Gly Val Leu Gly 20 25 30 Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Gly Val 35 40 45 Leu Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly<其代码= 50 55 60 Gln Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala Ala Ala Ala 65 70 75 80 Ala Ala Ala Gly Pro Gly Ser Gly Val Leu Gly Pro Gly Ala Ser Gly 85 90 95 It should be noted that there seems to be an error in the "其代码=0004084" in the original text. It might be a misrepresentation. I've translated it as "<其代码= " as per the requirement to preserve the original form. If this is incorrect, please clarify the original text.Gln Tyr Gly Pro Gly Val Leu Gly Pro Gly Val Leu Gly Pro Gly Ser 100 105 110 Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly 115 120 125 Val Leu Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro 130 135 140 Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly 145 150 155 160 Pro Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala Ala 165 170 175 Ala Ala Ala Ala Ala Gly Ser Gly Val Leu Gly Pro Gly Gln Tyr Gly 180 185 190 Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser 195 200 205 Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly 210 215 220 Val Leu Gly Pro Gly Val Leu Gly Pro Tyr Ala Ser Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ser Ser 245 250 255 Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Tyr Gly Pro Gly Val 260 265 270 Leu Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser 275 280 285 Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Gly Pro Ser Ala Ala Ala 290 295 300 Ala Ala Ala Ala Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala 305 310 315 320 Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Val Leu 325 330 335 Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Val Leu Gly 340 345 350 Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser 355 360 365 Tyr Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala 370 375 380 Ala Ala Ala Ala Ala Gly Ser Tyr Val Leu Gly Pro Gly Val Leu Gly 385 390 395 400 Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly 405 410 415 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr 420 425 430 Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala 435 440 445 Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro 450 455 460 Gly Gln Ser Gly Pro Gly Ser Gly Val Leu Gly Gln Gly Pro Tyr Gly 465 470 475 480 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro 485 490 495 Gly Val Leu Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala 500 505 510 Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln 515 520 525 Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Gly 530 535 540 Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Val Leu 545 550 555 560 Gly Pro Gly Ala Ser 565 <210> 33 <211> 565 <212> PRT <213> Artificial sequence <220> <223> M_PRT698 <400> 33 Met Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Gly Ser Asn Gly Pro Gly Ser Gly Val Leu Gly 20 25 30 Pro Gly Ile Ser Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro Gly Val 35 40 45 Leu Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 50 55 60 Ile Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala Ala Ala Ala 65 70 75 80 Ala Ala Ala Gly Pro Gly Ser Gly Val Leu Gly Pro Gly Ala Ser Gly 85 90 95 Ile Tyr Gly Pro Gly Val Leu Gly Pro Gly Val Leu Gly Pro Gly Ser 100 105 110 Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly 115 120 125 Val Leu Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro 130 135 140 Gly Ser Gly Ile Tyr Gly Ile Gly Pro Tyr Gly Pro Gly Ala Ser Gly 145 150 155 160 Pro Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala Ala 165 170 175 Ala Ala Ala Ala Ala Gly Ser Gly Val Leu Gly Pro Gly Ile Tyr Gly 180 185 190 Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser 195 200 205 Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ile Ser Gly Ser Gly 210 215 220 Val Leu Gly Pro Gly Val Leu Gly Pro Tyr Ala Ser Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ser Ser 245 250 255 Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Tyr Gly Pro Gly Val 260 265 270 Leu Gly Pro Tyr Gly Pro Gly Ala Ser Gly Ile Asn Gly Pro Gly Ser 275 280 285 Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro Gly Pro Ser Ala Ala Ala 290 295 300 Ala Ala Ala Ala Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ala 305 310 315 320 Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Val Leu 325 330 335 Gly Pro Gly Ile Tyr Gly Pro Gly Ser Ser Gly Pro Gly Val Leu Gly 340 345 350 Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser 355 360 365 Tyr Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala 370 375 380 Ala Ala Ala Ala Ala Gly Ser Tyr Val Leu Gly Pro Gly Val Leu Gly 385 390 395 400 Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Val Leu Gly Pro Tyr Gly 405 410 415 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ile Tyr 420 425 430 Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala 435 440 445 Ala Gly Ser Tyr Gly Ser Gly Pro Gly Ile Tyr Gly Pro Tyr Gly Pro 450 455 460 Gly Ile Ser Gly Pro Gly Ser Gly Val Leu Gly Ile Gly Pro Tyr Gly 465 470 475 480 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro 485 490 495 Gly Val Leu Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala 500 505 510 Ala Ala Gly Pro Gly Ser Gly Ile Tyr Gly Pro Gly Ala Ser Gly Ile 515 520 525 Asn Gly Pro Gly Ser Gly Ile Tyr Gly Pro Gly Val Leu Gly Pro Gly �30 535 540 Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Val Leu 545 550 555 560 Gly Pro Gly Ala Ser 565 <210> 34 <211> 565 <212> PRT <213> Artificial Sequence <220> <223> M_PRT525 <400> 34 Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Gly Ser Asn Gly Pro Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln 35 40 45 Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly 50 55 60 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala 65 70 75 80 Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly 85 90 95 Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser 100 105 110 Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser Gly Pro Gly 115 120 125 Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro 130 135 140 Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly 145 150 155 160 Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala 165 170 175 Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly 180 185 190 Pro Tyr Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Ser 195 200 205 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly 210 215 220 Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser 245 250 255 Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Tyr Gly Pro Gly Gln 260 265 270 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser 275 280 285 Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Pro Ser Ala Ala Ala 290 295 300 Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala 305 310 315 320 Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Gly Gln Gln 325 330 335 Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly 340 345 350 Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser 355 360 365 Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala 370 375 380 Ala Ala Ala Ala Ala Gly Ser Tyr Gln Gln Gly Pro Gly Gln Gln Gly 385 390 395 400 Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly 405 410 415 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr 420 425 430 Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Ala 435 440 445 Ala Gly Ser Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro 450 455 460 Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly 465 470 475 480 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro 485 490 495 Gly Gln Gln Gly Pro Tyr Gly Pro Gly Pro Ser Ala Ala Ala Ala Ala 500 505 510 Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln 515 520 525 Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly 530 535 540 Pro Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln 545 550 555 560 Gly Pro Gly Ala Ser 565 <210> 35 <21​​​​​​​​​​​​​​​​​​​​​​​​​50 55 60 Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro 65 70 75 80 Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Val Leu Gly 85 90 95 Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Gly Val 100 105 110 Leu Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser 115 120 125 Gly Pro Gly Val Leu Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly 130 135 140 Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 145 150 155 160 Gly Pro Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Ser Ala Ser Ala 165 170 175 Ala Ala Ala Ala Gly Ser Gly Val Leu Gly Pro Gly Gln Tyr Gly Pro 180 185 190 Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly 195 200 205 Val Leu Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Val Leu Gly 210 215 220 Pro Gly Val Leu Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro 225 230 235 240 Gly Val Leu Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 245 250 255 Gly Gln Tyr Gly Tyr Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly 260 265 270 Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Val 275 280 285 Leu Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Val Leu 290 295 300 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr 305 310 315 320 Gly Pro Gly Val Leu Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly 325 330 335 Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala 340 345 350 Ala Gly Gln Tyr Gly Pro Gly Val Leu Gly Pro Tyr Gly Pro Gly Gln 355 360 365 Ser Ala Ala Ala Ala Ala Gly Gln Tyr Val Le...

Claims

1. A method for preparing protein fiber, wherein: The method comprises the steps of bringing a spinning solution containing protein and an organic solvent into contact with a coagulation solution and coagulating the protein. Based on the total amount of spinning solution, the protein content in the spinning solution is greater than 10% by mass. The average hydrophobicity index of the protein is greater than -1.3, The coagulation liquid is an aqueous solution having a pH of 0.25 or more and a pH of 10.00 or less, The aqueous solution is a saline solution or a mixed solution of the saline solution and an acid aqueous solution, The salt content in the coagulation liquid is 25% by mass or less based on the total amount of the coagulation liquid. The brine solution is a brine solution selected from the group consisting of sodium citrate aqueous solution, sodium formate aqueous solution, potassium chloride aqueous solution, sodium chloride aqueous solution, calcium chloride aqueous solution, sodium sulfate aqueous solution, potassium hydrogen phosphate aqueous solution, brackish water, seawater, and a mixture thereof, or a mixed solution of the brine solution and a brine solution selected from the group consisting of sodium bicarbonate aqueous solution, sodium acetate aqueous solution, ammonium sulfate aqueous solution, and a mixture thereof.

2. The method according to claim 1, wherein: The content of the aqueous solution in the coagulation liquid is 60 mass % or more based on the total amount of the coagulation liquid.

3. The method according to claim 1, wherein: The aqueous solution is a mixed solution of the saline solution and the acid aqueous solution.

4. The method according to claim 1, wherein: The acid aqueous solution is a carboxylic acid aqueous solution.

5. The method according to any one of claims 1 to 4, wherein The salt content in the coagulation liquid is 0.1 mass % or more and 25 mass % or less based on the total amount of the coagulation liquid.

6. The method according to any one of claims 1 to 4, wherein: The salt water solution includes at least one selected from the group consisting of a sodium sulfate aqueous solution, a sodium chloride aqueous solution, brackish water, and seawater.

7. The method according to any one of claims 1 to 4, wherein: The content of the organic solvent dissolved in the coagulation solution from the spinning solution in contact with the coagulation solution is 40% by mass or less, based on the total content of the saline solution in the coagulation solution and the organic solvent dissolved in the coagulation solution being 100% by mass.

8. The method according to any one of claims 1 to 4, wherein: The protein includes at least one selected from the group consisting of spider silk protein, collagen, elastic protein, elastin, and keratin.

9. The method according to any one of claims 1 to 4, wherein: The protein is silk protein.

10. The method according to any one of claims 1 to 4, wherein: The protein is keratin or spider silk protein.

11. The method according to any one of claims 1 to 4, wherein: The protein is spider silk protein.

12. The method according to any one of claims 1 to 4, wherein: The organic solvent includes at least one selected from the group consisting of formic acid and dimethyl sulfoxide.

13. The method according to any one of claims 1 to 4, wherein: The aqueous solution is an aqueous solution having a pH of 0.25 or more and a pH of 2.50 or less, or a pH of 7.50 or more and a pH of 10.00 or less.

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