Method for producing protein molded body

Abstract

The purpose of the present invention is to provide a method for advantageously manufacturing a protein molded body, the method making it possible to resolve problems arising due to esterification of hydroxyl groups contained in a protein while maintaining adequate strength. The method for manufacturing a protein molded body according to the present invention comprises a step for hydrolyzing an ester group by bringing the ester group into contact with an acidic or basic medium, while applying tensile force to a raw material molded body including a protein in which hydroxyl groups are esterified.

Description

[Technical field]

[0001] The present invention relates to a method for producing a protein molded article. [Background technology]

[0002] Unlike synthetic fibers derived from petroleum, protein fibers such as silk have high biodegradability, and therefore, in light of the recent increase in environmental concerns, they are expected to be used in various applications as an alternative to synthetic fibers. As a method for producing protein fibers, a spinning method using an acid such as formic acid is widely known. For example, Patent Document 1 discloses a method for treating a biological sample containing a structural polypeptide with an acid. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2004-503204 Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have found that in protein fibers produced using a dope solution (spinning dope) containing a carboxylic acid such as formic acid as a solvent, ester groups are formed during the spinning process by a dehydration condensation reaction between hydroxyl groups in the protein and carboxylic acid. The present inventors have further found that in the protein fibers thus obtained, ester groups added to the protein may be hydrolyzed by a trace amount of carboxylic acid such as formic acid remaining on the surface or inside of the protein as a catalyst, resulting in the liberation of carboxylic acid, and the liberated carboxylic acid may cause bad odors, etc. According to the inventors' investigation into this problem, it has been found that the ester groups that cause bad odors, etc. can be removed or reduced by contacting a protein in which hydroxyl groups have been esterified with an acidic or basic medium to hydrolyze the ester groups.

[0005] As described above, protein molded products such as protein fibers are expected to be used in various applications, and therefore it is desirable for them to have sufficient strength depending on the application. However, it has been found that there is a problem that sufficient strength cannot be obtained by simply contacting a protein in which a hydroxyl group is esterified with an acidic or basic medium to hydrolyze the ester group. The present invention is intended to solve such a problem that has been newly discovered by the present inventors.

[0006] That is, the present invention aims to provide a method for advantageously producing a protein molded article that can solve the problems caused by esterification of hydroxyl groups contained in a protein while maintaining sufficient strength. Another object of the present invention is to provide a method for processing a protein molded article that can solve the problems caused by esterification of hydroxyl groups contained in a protein while maintaining sufficient strength. [Means for solving the problem]

[0007] The present invention relates to, for example, the following inventions. [1] A method for producing a protein molded product, comprising a step of hydrolyzing the ester groups by contacting a raw material molded product containing a protein whose hydroxyl groups have been esterified with an acidic or basic medium while applying a tensile force. [2] The method for producing a protein molded product according to [1], wherein the medium is an aqueous solution. [3] The method for producing a protein molded product according to [2], wherein the aqueous solution is an alkaline aqueous solution having a pH of less than 12. [4] The method for producing a protein shaped product according to [2] or [3], wherein the tensile force is such that the raw material shaped product does not shrink upon contact with the aqueous solution. [5] The method for producing a protein molded product according to any one of [1] to [4], wherein the protein is a structural protein. [6] The method for producing a protein molded body according to [5], wherein the structural protein is fibroin. [7] The method for producing a protein molded body according to [6], wherein the fibroin is spider silk fibroin. [8] The method for producing a protein shaped product according to any one of [1] to [7], wherein the protein having an esterified hydroxyl group contains a formic acid ester. [9] The method for producing a protein molded product according to any one of [1] to [8], wherein the raw material molded product is at least one selected from the group consisting of fibers, heat-pressed molded products, films, porous bodies, gels, and resins.

[10] A method for processing a protein molded product, comprising the step of contacting a protein molded product containing a protein whose hydroxyl groups have been esterified with an acidic or basic medium while applying a tensile force, thereby hydrolyzing the ester groups. Effect of the Invention

[0008] According to the present invention, there is provided a method for advantageously producing a protein molded article that can solve the problems caused by esterification of hydroxyl groups contained in a protein while maintaining sufficient strength. Also, according to the present invention, there is provided a method for processing a protein molded article that can solve the problems caused by esterification of hydroxyl groups contained in a protein while maintaining sufficient strength. [Brief description of the drawings]

[0009] [Figure 1] FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. [Diagram 2] FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. [Diagram 3] FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. [Figure 4] FIG. 1 shows FT-IR spectrum diagrams of protein fibers before and after ester group hydrolysis treatment (1730 cm-1: peak due to C=O of ester). [Diagram 5] FIG. 2 is a graph showing a stress (vertical axis)-elongation (horizontal axis) curve in the evaluation of elongation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, the embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0011] [Method for producing protein molded body] The method for producing a protein molded body according to this embodiment includes a step of hydrolyzing the ester groups by contacting a raw material molded body containing a protein whose hydroxyl groups have been esterified with an acidic or basic medium while applying a tensile force.

[0012] In this embodiment, the raw material molded body may be at least one selected from the group consisting of fibers, hot-press molded bodies, films, porous bodies, gels, and resins.

[0013] In this embodiment, the raw material compact contains a protein having an esterified hydroxyl group. In this specification, the term "protein having an esterified hydroxyl group" refers to a protein having an ester group formed by an ester bond between a hydroxyl group of the protein and a carboxylic acid. The protein having an esterified hydroxyl group may contain a formate ester, an acetate ester, a propionate ester, or the like, and preferably contains a formate ester.

[0014] (protein) The protein according to this embodiment (hereinafter sometimes referred to as the "target protein") may be, for example, a structural protein. A structural protein refers to a protein that forms a biological structure or a protein derived therefrom. That is, the structural protein may be a naturally occurring structural protein, or a modified protein in which a portion of the amino acid sequence (e.g., 10% or less of the amino acid sequence) has been modified based on the amino acid sequence of a naturally occurring structural protein.

[0015] Examples of structural proteins include fibroin, collagen, resilin, elastin, and keratin, as well as proteins derived therefrom. The fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin. The structural protein may be silk fibroin, spider silk fibroin, or a combination thereof.

[0016] Fibroin includes naturally occurring fibroin and modified fibroin. In this specification, "naturally occurring fibroin" means fibroin having the same amino acid sequence as naturally occurring fibroin, and "modified fibroin" means fibroin having an amino acid sequence different from that of naturally occurring fibroin.

[0017] The fibroin is preferably spider silk fibroin. Spider silk fibroin includes natural spider silk fibroin and modified fibroin derived from natural spider silk fibroin. Natural spider silk fibroin includes, for example, spider silk protein produced by spiders.

[0018] Fibroin can be, for example, represented by the formula 1: [(A) n motif-REP1] m Or formula 2: [(A) n motif-REP1] m-(A) n Examples of such proteins include proteins that contain a domain sequence represented by a motif. Fibroin may have further amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminus and C-terminus of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically, but not limited to, regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of about 100 amino acid residues.

[0019] As used herein, the term "domain sequence" refers to a crystalline region specific to fibroin (typically, the (A) n A motif) and an amorphous region (typically corresponding to REP of an amino acid sequence), and the amino acid sequence is represented by the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n The amino acid sequence represented by the motif (A) n The motif shows 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 an integer of 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. n The ratio of the number of alanine residues to the total number of amino acid residues in the motif may be 40% or more, and may 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 the motif is composed of only alanine residues). n At least seven of the motifs may be composed of only 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. m represents an integer of 2 to 300, and may be an integer of 10 to 300. (A) nThe motifs may be identical to each other or different from each other in amino acid sequence, and the multiple REPs may be identical to each other or different from each other in amino acid sequence.

[0020] Modified fibroin can be obtained, for example, by modifying the amino acid sequence of a cloned gene sequence of naturally occurring fibroin by, for example, substituting, deleting, inserting, and / or adding one or more amino acid residues. Substitution, deletion, insertion, and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as site-specific mutagenesis. Specifically, it can be performed according to the methods described in literature, such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0021] Naturally occurring fibroin has the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n A protein containing a domain sequence represented by a motif, specifically, for example, fibroin produced by insects or arachnids.

[0022] Examples of fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, as well as hornet silk proteins excreted by the larvae of the Japanese hornet (Vespa simillima xanthoptera).

[0023] A more specific example of fibroin produced by an insect is, for example, silkworm fibroin L chain (GenBank accession numbers M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

[0024] Examples of fibroin produced by spiders include spiders belonging to the Araneus genus, such as the Japanese squirrel spider, the Japanese garden squirrel spider, the red squirrel spider, the green squirrel spider, and the Japanese bean squirrel spider; spiders belonging to the Neoscona genus, such as the Japanese mountain squirrel spider, the Japanese house squirrel spider, and the Japanese Satsuma squirrel spider; spiders belonging to the Pronus genus, such as the Japanese squirrel spider; spiders belonging to the Cyrtarachne genus, such as the Japanese squirrel spider and the Japanese squirrel spider; spiders of the genus Gasteracantha such as the genus Acanthurus and the genus Siberian siberiana; spiders of the genus Ordgarius such as the genus Orbgarius and the genus Argiope such as the orb-weaver spider, the orb-weaver spider and the genus Arachnura such as the orb-weaver spider; spiders of the genus Acusilas such as the scraping spider; spiders of the genus Centipede such as the orb-weaver spider, the orb-weaver spider and the genus Centipede; spider silk proteins produced by spiders belonging to the genus Poltys such as the Japanese bush spider, the Japanese bush spider, the Japanese bush spider and the Japanese black bush spider, and spiders belonging to the genus Cyclosa such as the Japanese bush spider, the Japanese bush spider, the Japanese black bush spider and the Japanese black bush spider, and spiders belonging to the genus Chorizopes such as the Japanese black bush spider, and spiders belonging to the genus Tetragnatha such as the Japanese long-legged spider, the Japanese long-legged spider, the Japanese bush spider and the Japanese long-legged spider, and spiders belonging to the genus Tetragnatha such as the Japanese long-legged spider, the Japanese long-legged spider, the Japanese black bush spider and the Japanese black bush spider, spiders belonging to the genus Leucauge such as the medium-sized orb-weaver spider and the small orb-weaver spider; spiders belonging to the genus Nephila such as the orb-weaver spider and the giant orb-weaver spider; spiders belonging to the genus Menosira such as the golden spider; spiders belonging to the genus Dyschiriognatha such as the long-legged spider; spiders belonging to the genus Latrodectus such as the black widow spider, the redback spider, the gray widow spider and the three-spotted widow spider;Spider silk proteins include spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), etc.

[0025] More specific examples of spider silk proteins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession numbers AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank accession numbers CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence) and the like.

[0026] More specific examples of naturally derived fibroins include fibroins whose sequence information is registered in NCBI GenBank. For example, from among the sequence information registered in NCBI GenBank, sequences that include INV as DIVISION can be confirmed by extracting sequences with spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" as keywords in DEFINITION, sequences with a specific product character string in CDS, and sequences with a specific character string in TISSUE TYPE in SOURCE.

[0027] The modified fibroin may be modified silk fibroin (silk protein produced by silkworms with modified amino acid sequence), or modified spider silk fibroin (spider silk protein produced by spiders with modified amino acid sequence). The modified fibroin may be a modified spider silk fibroin.

[0028] Specific examples of modified fibroins include a modified fibroin derived from the major ampullate gland protein of spiders (first modified fibroin), a modified fibroin having a domain sequence with a reduced content of glycine residues (second modified fibroin), (A) n A modified fibroin having a domain sequence with a reduced content of motifs (third modified fibroin), the content of glycine residues, and (A) n These include a modified fibroin with a reduced motif content (the fourth modified fibroin), a modified fibroin with a domain sequence that includes a region of high local hydrophobicity index (the fifth modified fibroin), and a modified fibroin with a domain sequence with a reduced content of glutamine residues (the sixth modified fibroin).

[0029] The first modified fibroin is represented by the formula 1: [(A) nMotif-REP] m In the first modified fibroin, a protein having a domain sequence represented by the formula: n The number of amino acid residues in the motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, even more preferably an integer of 10 to 20, even more preferably an integer of 4 to 16, particularly preferably an integer of 8 to 16, and most preferably an integer of 10 to 16. The first modified fibroin has a number of amino acid residues constituting REP in formula 1 of preferably 10 to 200 residues, more preferably 10 to 150 residues, even more preferably 20 to 100 residues, and even more preferably 20 to 75 residues. The first modified fibroin has a number of amino acid residues constituting REP in formula 1: [(A) n Motif-REP] m The total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by the formula (I) is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more, based on the total number of amino acid residues.

[0030] The first modified fibroin has the formula 1: [(A) n Motif-REP] m 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 homology to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.

[0031] The amino acid sequence shown in SEQ ID NO:1 is identical to the amino acid sequence consisting of 50 amino acid residues at the C-terminus of the amino acid sequence of ADF3 (GI:1263287, NCBI), the amino acid sequence shown in SEQ ID NO:2 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:3 is identical to the amino acid sequence obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO:1.

[0032] A more specific example of the first modified fibroin is (1-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. The sequence identity is preferably 95% or more.

[0033] The amino acid sequence shown in SEQ ID NO:4 is an amino acid sequence of ADF3 with an amino acid sequence (SEQ ID NO:5) consisting of an initiation codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus, in which the repeat region at positions 1 to 13 is increased approximately twice and mutated so that translation terminates at amino acid residue 1154. The amino acid sequence at the C-terminus of the amino acid sequence shown in SEQ ID NO:4 is identical to the amino acid sequence shown in SEQ ID NO:3.

[0034] The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO:4.

[0035] The second modified fibroin has an amino acid sequence in which the domain sequence has a reduced content of glycine residues compared to naturally-derived fibroin. It can be said that the second modified fibroin has an amino acid sequence in which at least one or more glycine residues in REP have been replaced with another amino acid residue compared to naturally-derived fibroin.

[0036] The second modified fibroin may have an amino acid sequence in which, compared to a naturally occurring fibroin, at least one glycine residue in at least one 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 is replaced with another amino acid residue in at least one or more of the motif sequences.

[0037] In the second modified fibroin, the proportion of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more relative to the total motif sequence.

[0038] The second modified fibroin has the formula 1: [(A) n Motif-REP] m (A) is located at the most C-terminal side of the domain sequence. n The total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence is z, and the amino acid sequence located at the most C-terminus side from the domain sequence is (A). n When the total number of amino acid residues in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence is w, the z / w ratio may be 30% or more, 40% or more, 50% or more, or 50.9% or more. (A) n The number of alanine residues relative to the total number of amino acid residues in the motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that it is composed only of alanine residues).

[0039] The second modified fibroin is preferably one in which one glycine residue of the GGX motif is replaced with another amino acid residue to increase the content of the amino acid sequence consisting of XGX. The second modified fibroin preferably has a content of the amino acid sequence consisting of GGX in the domain sequence of 30% or less, more preferably 20% or less, even more preferably 10% or less, even more preferably 6% or less, even more preferably 4% or less, and particularly preferably 2% or less. The content of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the calculation method for the content (z / w) of the amino acid sequence consisting of XGX below.

[0040] The method for calculating z / w will be described in more detail. First, the formula 1: [(A) n Motif-REP] m In a fibroin (modified fibroin or naturally-occurring fibroin) containing a domain sequence represented by the formula: n An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. The total number of amino acid residues that make up XGX is z. For example, if 50 amino acid sequences consisting of XGX are extracted (no overlaps), z is 50 x 3 = 150. In addition, for example, in the case of an amino acid sequence consisting of XGXGX, if there are Xs contained in two XGXs (the central X), the overlaps are subtracted from the calculation (in the case of XGXGX, it is 5 amino acid residues). w is the number of amino acid residues (A) located at the most C-terminus side from the domain sequence. n It is the total number of amino acid residues contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. For example, in the case of the domain sequence shown in Figure 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 ((A) located at the most C-terminus side). n (The motif is excluded.) Next, z / w (%) can be calculated by dividing z by w.

[0041] Here, the z / w ratio in naturally derived fibroin will be described. First, as described above, fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method exemplified above, and 663 types of fibroin (of which, 415 types were spider-derived fibroin) were extracted. Of all the extracted fibroins, those with the formula 1: [(A) n Motif-REP] m The z / w ratio was calculated by the above-mentioned calculation method from the amino acid sequence of naturally occurring fibroin, which contains a domain sequence represented by the formula (I) and the content of the amino acid sequence consisting of GGX in the fibroin is 6% or less. As a result, the z / w ratio of the naturally occurring fibroin was all less than 50.9% (the highest was 50.86%).

[0042] In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, even more preferably 70% or more, and even more preferably 80% or more. There is no particular upper limit to z / w, but it may be, for example, 95% or less.

[0043] The second modified fibroin can be obtained, for example, by modifying the gene sequence of a cloned naturally-derived fibroin by substituting at least a part of the base sequence encoding a glycine residue to encode another amino acid residue. In this case, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be substituted so that z / w is 50.9% or more. Alternatively, for example, an amino acid sequence satisfying the above-mentioned aspects may be designed from the amino acid sequence of a naturally-derived fibroin, and a nucleic acid encoding the designed amino acid sequence may be chemically synthesized to obtain the second modified fibroin. In either case, in addition to the modification equivalent to substituting the glycine residue in REP with another amino acid residue from the amino acid sequence of a naturally-derived fibroin, the amino acid sequence may be modified by substituting, deleting, inserting and / or adding one or more amino acid residues.

[0044] The other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but is preferably a hydrophobic amino acid residue such as a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a methionine (M) residue, a proline (P) residue, a phenylalanine (F) residue, or a tryptophan (W) residue, or a hydrophilic amino acid residue such as a glutamine (Q) residue, an asparagine (N) residue, a serine (S) residue, a lysine (K) residue, or a glutamic acid (E) residue, more preferably a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a phenylalanine (F) residue, or a glutamine (Q) residue, and even more preferably a glutamine (Q) residue.

[0045] More specific examples of the second modified fibroin include (2-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0046] The modified fibroin (2-i) will be described. The amino acid sequence shown in SEQ ID NO:6 is the amino acid sequence shown in SEQ ID NO:10 (Met-PRT313) corresponding to naturally-occurring fibroin, in which all GGX in the REP is replaced with GQX. The amino acid sequence shown in SEQ ID NO:7 is the amino acid sequence shown in SEQ ID NO:6. From the amino acid sequence, every third amino acid from the N-terminus to the C-terminus is (A) n The motif was deleted, and the C-terminal sequence was further amplified with [(A) n The amino acid sequence shown in SEQ ID NO:8 is a sequence in which each of (A) of the amino acid sequence shown in SEQ ID NO:7 is inserted. n Two alanine residues were inserted at the C-terminus of the motif, and some glutamine (Q) residues were further replaced with serine (S) residues, with some amino acids deleted at the C-terminus to have approximately the same molecular weight as SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 has a predetermined hinge sequence and His tag sequence added to the C-terminus of a sequence in which the 20 domain sequence regions present in the amino acid sequence shown in SEQ ID NO: 7 (with the exception that several amino acid residues at the C-terminus of said regions have been replaced) are repeated four times.

[0047] The z / w value in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally derived fibroin) is 46.8%. The z / w values ​​in the amino acid sequence shown 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 ​​in the jagged ratio (described later) of 1:1.8 to 11.3 in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 15.0%, 15.0%, 93.4%, 92.7%, and 89.8%, respectively.

[0048] The modified fibroin (2-i) may consist of the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0049] The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) also ... n Motif-REP] m It is a protein comprising a domain sequence represented by the following formula: The sequence identity is preferably 95% or more.

[0050] The modified fibroin (2-ii) preferably has a sequence identity of 90% or more 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 has a z / w ratio of 50.9% or more, where z is the total number of amino acid residues in the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in REP, and w is the total number of amino acid residues in REP in the domain sequence.

[0051] The second modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows for isolation, immobilization, detection, visualization, etc. of the modified fibroin.

[0052] An example of a tag sequence is an affinity tag that utilizes specific affinity (binding ability, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). The His tag is a short peptide consisting of about 4 to 10 histidine residues lined up, and has the property of specifically binding to metal ions such as nickel, and can be used to isolate modified fibroin by metal chelating chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 11 (an amino acid sequence including a His tag sequence and a hinge sequence).

[0053] Furthermore, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione, and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

[0054] Furthermore, an "epitope tag" utilizing an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, and FLAG tag. By using an epitope tag, the modified fibroin can be easily purified with high specificity.

[0055] Furthermore, a tag sequence that can be cleaved with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with the protease, the modified fibroin from which the tag sequence has been cleaved can be recovered.

[0056] More specific examples of modified fibroins containing a tag sequence include (2-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (2-iv) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0057] The amino acid sequences shown in SEQ ID NO:16 (PRT313), SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0058] The modified fibroin (2-iii) may consist of the amino acid sequence shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0059] The modified fibroin of (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (2-iv) also ... n Motif-REP] m It is a protein comprising a domain sequence represented by the following formula: The sequence identity is preferably 95% or more.

[0060] The modified fibroin (2-iv) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and preferably has a z / w ratio of 50.9% or more, where z is the total number of amino acid residues in the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in REP, and w is the total number of amino acid residues in REP in the domain sequence.

[0061] The second modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of the host.

[0062] The third modified fibroin has a domain sequence different from that of naturally occurring fibroin, as follows: (A) n The third modified fibroin domain sequence has an amino acid sequence with a reduced content of motifs compared to naturally occurring fibroin. n It can be said that it has an amino acid sequence corresponding to the deletion of the motif.

[0063] The third modified fibroin was derived from naturally occurring fibroin (A). n It may have an amino acid sequence corresponding to a 10 to 40% deletion of the motif.

[0064] The third modified fibroin has a domain sequence that is different from that of naturally-occurring fibroin in that it has at least one to three (A) n One (A) per motif n It may have an amino acid sequence corresponding to the deletion of the motif.

[0065] The third modified fibroin has a domain sequence that is at least two consecutive domains from the N-terminus to the C-terminus compared to naturally-occurring fibroin (A). n Deletion of the motif and one (A) n It may have an amino acid sequence corresponding to repeated deletion of the motif in this order.

[0066] The third modified fibroin has a domain sequence in which at least every third domain from the N-terminus to the C-terminus is (A) n It may have an amino acid sequence corresponding to the deletion of the motif.

[0067] The third modified fibroin has the formula 1: [(A) n Motif-REP] m The domain sequence is represented by the following two adjacent sequences from the N-terminus to the C-terminus: n The number of amino acid residues in the REP of the motif-REP] unit is compared in sequence. When the number of amino acid residues in the REP with the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 in two adjacent [(A) n When the maximum sum of the numbers of amino acid residues in the motif-REP units is x and the total number of amino acid residues in the domain sequence is y, the amino acid sequence may have an x / y ratio of 20% or more, 30% or more, 40% or more, or 50% or more. (A) n The number of alanine residues relative to the total number of amino acid residues in the motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that it is composed only of alanine residues).

[0068] The method of calculating x / y will be described in more detail with reference to Fig. 1. Fig. 1 shows the domain sequence of the modified fibroin excluding the N-terminal sequence and the C-terminal sequence. The domain sequence is as follows from the N-terminal side (left side): (A) n Motif-First REP (50 amino acid residues)-(A) n Motif-Second REP (100 amino acid residues)-(A) n Motif-3 REP (10 amino acid residues)-(A) n Motif-4th REP (20 amino acid residues)-(A) n Motif-5 REP (30 amino acid residues)-(A) n It has a sequence called a motif.

[0069] Two adjacent [(A) n The motif-REP] units are selected sequentially from the N-terminus to the C-terminus, without overlapping. nA motif-REP] unit may be present. In FIG. 1, pattern 1 (comparison of the first REP with the second REP, and comparison of the third REP with the fourth REP), pattern 2 (comparison of the first REP with the second REP, and comparison of the fourth REP with the fifth REP), pattern 3 (comparison of the second REP with the third REP, and comparison of the fourth REP with the fifth REP), and pattern 4 (comparison of the first REP with the second REP) are shown. It should be noted that there are other selection methods.

[0070] Next, for each pattern, select two adjacent [(A) n The number of amino acid residues of each REP in the motif-REP unit is compared. The comparison is performed by determining the ratio of the number of amino acid residues of the other REP to the number of amino acid residues of the one with fewer amino acid residues set as 1. For example, when comparing the first REP (50 amino acid residues) with the second REP (100 amino acid residues), the ratio of the number of amino acid residues of the second REP is 100 / 50=2 when the number of amino acid residues of the first REP with fewer amino acid residues is set as 1. Similarly, when comparing the fourth REP (20 amino acid residues) with the fifth REP (30 amino acid residues), the ratio of the number of amino acid residues of the fifth REP is 30 / 20=1.5 when the number of amino acid residues of the fourth REP with fewer amino acid residues is set as 1.

[0071] In FIG. 1, when the number of amino acid residues of the one with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other one is 1.8 to 11.3 [(A) n The combination of motif-REP] units is shown by a solid line. In this specification, this ratio is called the "Jagged ratio." When the one with the fewer amino acid residues is set to 1, the ratio of the other amino acid residue number is less than 1.8 or more than 11.3 [(A) n The motif-REP] unit pairs are indicated by dashed lines.

[0072] In each pattern, the solid lines The two adjacent ones shown in (A) n Add up the number of amino acid residues in all the motif-REP units (not just REP, but (A) nThe number of amino acid residues in the motif is also x.) Then, the sums are compared, and the sum of the pattern with the largest sum (the maximum sum) is taken as x. In the example shown in Figure 1, the sum of pattern 1 is the largest.

[0073] Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues, y, in the domain sequence.

[0074] In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, even more preferably 65% ​​or more, even more preferably 70% or more, even more preferably 75% or more, and particularly preferably 80% or more. There is no particular upper limit for x / y, and it may be, for example, 100% or less. When the knurling ratio is 1:1.9 to 11.3, x / y is preferably 89.6% or more, when the knurling ratio is 1:1.8 to 3.4, x / y is preferably 77.1% or more, when the knurling ratio is 1:1.9 to 8.4, x / y is preferably 75.9% or more, and when the knurling ratio is 1:1.9 to 4.1, x / y is preferably 64.2% or more.

[0075] The third modified fibroin is present multiple times in the domain sequence (A). n In the case of a modified fibroin in which at least seven of the motifs are composed only of alanine residues, x / y is preferably 46.4% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and particularly preferably 80% or more. There is no particular upper limit to x / y, and it is sufficient as long as it is 100% or less.

[0076] Here, the x / y ratio in naturally derived fibroin will be described. First, as described above, fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method exemplified above, and 663 types of fibroin (of which, 415 types were spider-derived fibroin) were extracted. Of all the extracted fibroins, those with the formula 1: [(A) n Motif-REP] m The ratio x / y was calculated by the above-mentioned calculation method from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the following formula: As a result, the ratio x / y in all naturally occurring fibroins was less than 64.2% (the highest was 64.14%).

[0077] The third modified fibroin is, for example, (A) such that x / y is 64.2% or more based on the gene sequence of a cloned naturally occurring fibroin. n For example, one or more of the sequences encoding the motifs (A) may be deleted from the amino acid sequence of naturally occurring fibroin such that x / y is 64.2% or more. n Alternatively, the amino acid sequence corresponding to the deletion of the motif may be designed and a nucleic acid encoding the designed amino acid sequence may be chemically synthesized. In either case, the amino acid sequence of the naturally occurring fibroin may be selected from (A) n In addition to modifications corresponding to the deletion of a motif, further modifications to the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be made.

[0078] More specific examples of the third modified fibroin include (3-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0079] The modified fibroin (3-i) will be described. The amino acid sequence shown in SEQ ID NO: 17 is a sequence of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin, which is obtained by subtracting every third amino acid from the N-terminus to the C-terminus. n The motif was deleted, and the C-terminal sequence was further amplified with [(A) n The amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for the second modified fibroin.

[0080] The value of x / y in the jagged ratio of 1:1.8-11.3 of the amino acid sequence shown in SEQ ID NO:10 (corresponding to naturally derived fibroin) is 15.0%. The values ​​of x / y in the amino acid sequence shown in SEQ ID NO:17 and the amino acid sequence shown in SEQ ID NO:7 are both 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO:8 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO:9 is 89.8%. The values ​​of z / w in the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 are 46.8%, 56.2%, 70.1%, 66.1% and 70.0%, respectively.

[0081] The modified fibroin (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0082] The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) also ... n Motif-REP] m It is a protein comprising a domain sequence represented by the following formula: The sequence identity is preferably 95% or more.

[0083] The modified fibroin (3-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and has two adjacent amino acids [(A) n The number of amino acid residues in the REP of the motif-REP] unit is compared in sequence. When the number of amino acid residues in the REP with the fewer amino acid residues is set to 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 (the jagged ratio is 1:1.8 to 11.3) between two adjacent [(A) n When the maximum sum of the numbers of amino acid residues in the motif-REP units is x and the total number of amino acid residues in the domain sequence is y, it is preferable that x / y is 64.2% or more.

[0084] The third modified fibroin may contain the above-mentioned tag sequence at either or both of the N-terminus and C-terminus.

[0085] More specific examples of modified fibroins containing a tag sequence include (3-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (3-iv) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0086] The amino acid sequences shown in SEQ ID NO:18, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO:17, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0087] The modified fibroin (3-iii) may consist of the amino acid sequence shown in SEQ ID NO:18, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0088] The modified fibroin of (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (3-iv) also ... n Motif-REP] m It is a protein comprising a domain sequence represented by the following formula: The sequence identity is preferably 95% or more.

[0089] The modified fibroin (3-iv) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and has two adjacent amino acids [(A) n The number of amino acid residues in the REP of the motif-REP] unit is compared in sequence. When the number of amino acid residues in the REP with the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 in two adjacent [(A) n When the maximum sum of the numbers of amino acid residues in the motif-REP units is x and the total number of amino acid residues in the domain sequence is y, it is preferable that x / y is 64.2% or more.

[0090] The third modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of the host.

[0091] The fourth modified fibroin has a domain sequence different from that of naturally occurring fibroin, as follows: (A) n In addition to the reduced content of the motif, the fourth modified fibroin has an amino acid sequence with a reduced content of glycine residues. The fourth modified fibroin domain sequence has at least one or more (A) nIn addition to the deletion of the motif, the fourth modified fibroin has an amino acid sequence in which at least one or more glycine residues in REP have been replaced with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having both the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects are as described for the second modified fibroin and the third modified fibroin.

[0092] More specific examples of the fourth modified fibroin include (4-i) modified fibroin containing an amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. Specific embodiments of modified fibroin containing the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

[0093] The fifth modified fibroin may have an amino acid sequence whose domain sequence includes a localized region of high hydrophobicity index, corresponding to the replacement of one or more amino acid residues in REP with amino acid residues with a high hydrophobicity index and / or the insertion of one or more amino acid residues with a high hydrophobicity index into REP, compared to naturally occurring fibroin.

[0094] The region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

[0095] The amino acid residue with a high 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).

[0096] The fifth modified fibroin is a modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues in REP with amino acid residues having a high hydrophobicity index, and / or the insertion of one or more amino acid residues having a high hydrophobicity index into REP, as compared with a naturally occurring fibroin. There may be modifications of the corresponding amino acid sequence.

[0097] The fifth modified fibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in REP from the gene sequence of cloned naturally-derived fibroin with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index) and / or by inserting one or more hydrophobic amino acid residues into REP. Alternatively, for example, it can be obtained by designing an amino acid sequence corresponding to the substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally-derived fibroin and / or the insertion of one or more hydrophobic amino acid residues into REP, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally-derived fibroin and / or the insertion of one or more hydrophobic amino acid residues into REP, the amino acid sequence may be modified by further substituting, deleting, inserting and / or adding one or more amino acid residues.

[0098] The fifth modified fibroin has the formula 1: [(A) n Motif-REP] m and (A) is located at the most C-terminal end. nIn all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region in which the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the region located most C-terminally is defined as (A). n The domain may have an amino acid sequence in which p / q is 6.2% or more, where q is the total number of amino acid residues contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence.

[0099] The hydrophobicity index of amino acid residues is determined by 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 (hereinafter also referred to as "HI") of each amino acid is as shown in Table 1 below.

[0100] [Table 1]

[0101] The method of calculating p / q will be described in more detail. The calculation is carried out using the formula 1: [(A) n Motif-REP] m (A) is located at the C-terminal end of the domain sequence represented by nA sequence excluding the sequence from the motif to the C-terminus of the domain sequence (hereinafter referred to as "sequence A") is used. First, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated for all REPs included in sequence A. The average value of the hydrophobicity index is calculated by dividing the sum of the HI of each amino acid residue included in the four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is calculated for all four consecutive amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of the four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "four consecutive amino acid residues with an average value of the hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. The total number of amino acid residues included in the region is p. The total number of amino acid residues included in sequence A is q.

[0102] For example, if 20 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" are extracted (without overlaps), the region with the average hydrophobicity index of the consecutive 4 amino acid residues of 2.6 or more contains 20 consecutive 4 amino acid residues (without overlaps), and p is 20×4=80. Also, for example, if two "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" overlap by one amino acid residue, the region with the average hydrophobicity index of the consecutive 4 amino acid residues of 2.6 or more contains 7 amino acid residues (p=2×4-1=7, where "-1" is the deduction of the overlap). For example, in the case of the domain sequence shown in FIG. 2, there are 7 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" without overlaps, and p is 7×4=28. For example, in the case of the domain sequence shown in FIG. 2, q is 4+50+4+40+4+10+4+20+4+30=170 ((A) at the end of the C-terminus). n (The motif is not included.) Next, p / q (%) can be calculated by dividing p by q. In the case of Figure 2, it is 28 / 170 = 16.47%.

[0103] In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, even more preferably 20% or more, and even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

[0104] The fifth modified fibroin can be obtained, for example, by modifying the amino acid sequence of a cloned naturally-derived fibroin to include a region with a high hydrophobic index locally by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobic index) in the REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobic index) and / or inserting one or more hydrophobic amino acid residues into the REP so as to satisfy the above-mentioned p / q condition. Alternatively, for example, the fifth modified fibroin can be obtained by designing an amino acid sequence that satisfies the above-mentioned p / q condition from the amino acid sequence of a naturally-derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the substitution of one or more amino acid residues in the REP with amino acid residues with a high hydrophobic index and / or the insertion of one or more amino acid residues with a high hydrophobic index into the REP, modifications corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed.

[0105] The amino acid residues having a high hydrophobicity index are not particularly limited, but are preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A), and more preferably valine (V), leucine (L) and isoleucine (I).

[0106] A more specific example of the fifth modified fibroin includes (5-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), or (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0107] The modified fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 19 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP at two positions, excluding the domain sequence at the C-terminus, and further substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids at the C-terminus, in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410). The amino acid sequence shown in SEQ ID NO: 20 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP at one position, in the amino acid sequence shown in SEQ ID NO: 8 (Met-PRT525). The amino acid sequence shown in SEQ ID NO: 21 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP at two positions, in the amino acid sequence shown in SEQ ID NO: 8.

[0108] The modified fibroin (5-i) may consist of the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.

[0109] The modified fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) also contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of n Motif-REP] m It is a protein comprising a domain sequence represented by the following formula: The sequence identity is preferably 95% or more.

[0110] The modified fibroin (5-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21 and is located at the most C-terminal side (A). n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of amino acid residues contained in the region in which the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the region located most C-terminally is defined as (A). n When the total number of amino acid residues contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence is defined as q, it is preferable that p / q is 6.2% or more.

[0111] The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus.

[0112] More specific examples of modified fibroins containing a tag sequence include (5-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0113] The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are obtained by adding the amino acid sequence shown in SEQ ID NO: 11 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0114] The modified fibroin (5-iii) may consist of the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

[0115] The modified fibroin (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The modified fibroin (5-iv) also ... n Motif-REP] m It is a protein comprising a domain sequence represented by the following formula: The sequence identity is preferably 95% or more.

[0116] The modified fibroin (5-iv) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24 and is located at the most C-terminal side (A). n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of amino acid residues contained in the region in which the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the region located most C-terminally is defined as (A). n When the total number of amino acid residues contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence is defined as q, it is preferable that p / q is 6.2% or more.

[0117] The fifth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of the host.

[0118] The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues compared to naturally occurring fibroin. do.

[0119] It is preferable that the sixth modified fibroin contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

[0120] When the sixth modified fibroin contains a GPGXX motif in the REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and is preferably 10% or more. There is no particular upper limit to the GPGXX motif content, and it may be 50% or less, or may be 30% or less.

[0121] In the present specification, the "GPGXX motif content" is a value calculated by the following method. Formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n In a fibroin (modified fibroin or naturally-occurring fibroin) containing a domain sequence represented by the motif, (A) is located at the most C-terminal side. n For all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of GPGXX motifs contained in that region is multiplied by three (i.e., the total number of G and P in the GPGXX motif), and the number s is the number located at the most C-terminus side (A). n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and further (A) n When the total number of amino acid residues of all REPs excluding the motif is t, the GPGXX motif content is calculated as s / t.

[0122] In calculating the GPGXX motif content, the most C-terminal (A) n The target sequence is the sequence excluding the sequence from the motif to the C-terminus of the domain sequence, which is the sequence located at the most C-terminus (A). nThe "sequence from the motif to the C-terminus of the domain sequence" (sequence corresponding to REP) may contain sequences with low correlation with sequences characteristic of fibroin, and when m is small (i.e., when the domain sequence is short), this affects the calculation results of the GPGXX motif content, so this is to eliminate this influence. Note that when the "GPGXX motif" is located at the C-terminus of REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

[0123] FIG. 3 is a schematic diagram showing the domain sequence of a modified fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. 3. First, the domain sequence of the modified fibroin shown in FIG. n Motif-REP] m -(A) n In the 1990s, all REPs were located at the C-terminus (A) n Since the number of GPGXX motifs is 7, s is 7 × 3 = 21. Similarly, all REPs are included in the "domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence" (the sequence shown as "Region A" in Figure 3). n Since the domain sequence is excluded from the sequence from the motif to the C-terminus of the domain sequence (the sequence shown as "Region A" in Figure 3), the sequence is further divided into (A) n The total number of amino acid residues in all REPs excluding motifs, t, is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t, which is 21 / 150 = 14.0% in the case of the modified fibroin in Figure 3.

[0124] The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%.

[0125] In the present specification, the "glutamine residue content" is a value calculated by the following method. Formula 1: [(A)n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n In a fibroin (modified fibroin or naturally-occurring fibroin) containing a domain sequence represented by the motif, (A) is located at the most C-terminal side. n In the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence (the sequence corresponding to "Region A" in Figure 3), the total number of glutamine residues contained in the region is defined as u, and the number of glutamine residues located at the most C-terminus side (A) is defined as n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and further (A) n When the total number of amino acid residues of all REPs excluding the motif is t, the glutamine residue content is calculated as u / t. n The reason for targeting "the sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as that mentioned above.

[0126] The sixth modified fibroin may have an amino acid sequence whose domain sequence corresponds to the deletion or substitution of one or more glutamine residues in the REP, compared to a naturally occurring fibroin.

[0127] The "other amino acid residue" may be any amino acid residue other than glutamine residue, but is preferably an amino acid residue with a higher hydrophobicity index than glutamine residue. The hydrophobicity indexes of amino acid residues are shown in Table 1.

[0128] As shown in Table 1, examples of amino acid residues having a higher hydrophobicity index than glutamine residues include amino acid residues selected from 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, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A) are more preferred, and amino acid residues selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are even more preferred.

[0129] In the sixth modified fibroin, the hydrophobicity of REP is preferably -0.8 or more, more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or less, or 0.7 or less.

[0130] In the present specification, the "hydrophobicity of REP" is a value calculated by the following method. Formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n In a fibroin (modified fibroin or naturally-occurring fibroin) containing a domain sequence represented by the motif, (A) is located at the most C-terminal side. n For all REPs contained in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence (the sequence corresponding to "Region A" in Figure 3), the sum of the hydrophobicity indexes of each amino acid residue in that region is v, and the REP located at the most C-terminus side (A) n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and further (A) nWhen the total number of amino acid residues of all REPs excluding the motif is t, the hydrophobicity of the REP is calculated as v / t. n The reason for targeting "the sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as that mentioned above.

[0131] The sixth modified fibroin may have a domain sequence that, compared to a naturally occurring fibroin, is modified by the deletion of one or more glutamine residues in the REP and / or the replacement of one or more glutamine residues in the REP with other amino acid residues, and may also have an amino acid sequence modification that is equivalent to the substitution, deletion, insertion and / or addition of one or more amino acid residues.

[0132] The sixth modified fibroin can be obtained, for example, by deleting one or more glutamine residues in REP from the gene sequence of a cloned naturally-occurring fibroin and / or substituting one or more glutamine residues in REP with other amino acid residues. It can also be obtained, for example, by designing an amino acid sequence corresponding to the deletion of one or more glutamine residues in REP from the amino acid sequence of a naturally-occurring fibroin and / or the substitution of one or more glutamine residues in REP with other amino acid residues, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0133] More specific examples of the sixth modified fibroin include (6-i) modified fibroin having an amino acid sequence shown in SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or SEQ ID NO: 42 (Met-PRT1028), or (6-ii) modified fibroin having an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, 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: 41 or SEQ ID NO: 42.

[0134] The modified fibroin (6-i) will be described. The amino acid sequence shown in SEQ ID NO: 25 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) with VL. The amino acid sequence shown in SEQ ID NO: 26 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with TS, and substituting the remaining Q with A. The amino acid sequence shown in SEQ ID NO: 27 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with VL, and substituting the remaining Q with I. The amino acid sequence shown in SEQ ID NO: 28 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with VI, and substituting the remaining Q with L. The amino acid sequence shown in SEQ ID NO: 29 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with VF, and substituting the remaining Q with I.

[0135] The amino acid sequence shown in SEQ ID NO: 30 is the amino acid sequence shown in SEQ ID NO: 8 (Met-PRT525) in which all QQ are replaced with VL. The amino acid sequence shown in SEQ ID NO: 31 is the amino acid sequence shown in SEQ ID NO: 8 in which all QQ are replaced with VL and the remaining Q is replaced with I.

[0136] The amino acid sequence shown in SEQ ID NO:32 is a sequence in which the 20 domain sequence region present in the amino acid sequence shown in SEQ ID NO:7 (Met-PRT410) is repeated twice, with all QQs replaced with VF and the remaining Qs replaced with I.

[0137] The amino acid sequence shown in SEQ ID NO:41 (Met-PRT917) is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 with LI and the remaining Q with V. The amino acid sequence shown in SEQ ID NO:42 (Met-PRT1028) is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 with IF and the remaining Q with T.

[0138] The amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, 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:41 and SEQ ID NO:42 all have a glutamine residue content of 9% or less (Table 2).

[0139] [Table 2]

[0140] The modified fibroin (6-i) may consist of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, 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: 41 or SEQ ID NO: 42.

[0141] The modified fibroin of (6-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, 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: 41, or SEQ ID NO: 42. The modified fibroin of (6-ii) also contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, 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: 41, or SEQ ID NO: 42. The modified fibroin of (6 n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) nIt is a protein containing a domain sequence represented by the motif. The sequence identity is preferably 95% or more.

[0142] The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less, and the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.

[0143] The sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows the modified fibroin to be isolated, immobilized, detected, visualized, and the like.

[0144] More specific examples of modified fibroins containing a tag sequence include (6-iii) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028), or (6-iv) modified fibroins containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, 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: 43 or SEQ ID NO: 44.

[0145] The amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34, 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:43 and SEQ ID NO:44 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, 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:41 and SEQ ID NO:42, respectively. Since only a tag sequence is added to the N-terminus, there is no change in the glutamine residue content, and the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34, 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:43 and SEQ ID NO:44 all have a glutamine residue content of 9% or less (Table 3).

[0146] [Table 3]

[0147] The modified fibroin (6-iii) may consist of the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, 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: 43 or SEQ ID NO: 44.

[0148] The modified fibroin of (6-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, 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: 43, or SEQ ID NO: 44. The modified fibroin of (6-iv) also ... n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n It is a protein containing a domain sequence represented by the motif. The sequence identity is preferably 95% or more.

[0149] The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less, and the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

[0150] The sixth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately selected depending on the type of the host.

[0151] The modified fibroin may be a modified fibroin having at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin.

[0152] The modified fibroin may be either hydrophilic or hydrophobic. The hydrophobic modified fibroin is a modified fibroin in which the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin is calculated and then the sum is divided by the total number of amino acid residues, resulting in an average HI of 0 or more. The hydrophobicity index is as shown in Table 1. The hydrophilic modified fibroin is a modified fibroin in which the average HI is less than 0.

[0153] An example of the hydrophobic modified fibroin is the modified fibroin No. 6 described above. More specific examples of the hydrophobic modified fibroin include modified fibroins containing 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, 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.

[0154] Examples of the hydrophilic modified fibroin include the above-mentioned first modified fibroin, second modified fibroin, third modified fibroin, fourth modified fibroin, and fifth modified fibroin. More specific examples of the hydrophilic modified fibroin include modified fibroins containing the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0155] (Protein manufacturing method) A protein can be produced, for example, by expressing a nucleic acid by a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence.

[0156] The method for producing a gene encoding a protein is not particularly limited. For example, a gene encoding a natural structural protein can be produced by amplifying and cloning the gene using polymerase chain reaction (PCR) or chemical synthesis. The method for chemically synthesizing a gene is also not particularly limited. For example, a gene can be chemically synthesized by linking oligonucleotides automatically synthesized using AKTA oligopilot plus 10 / 100 (GE Healthcare Japan, Inc.) or the like using PCR based on amino acid sequence information of a structural protein obtained from the NCBI web database or the like. In this case, in order to facilitate purification and identification of the protein, a gene encoding a protein consisting of an amino acid sequence in which an amino acid sequence consisting of an initiation codon and a His10 tag is added to the N-terminus of the above amino acid sequence may be synthesized.

[0157] The regulatory sequence is a sequence (e.g., promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) that controls the expression of a protein in a host, and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in a host cell and can induce the expression of a protein may be used. An inducible promoter is a promoter that can control transcription by physical factors such as the presence of an inducer (expression inducer), the absence of a repressor molecule, or an increase or decrease in temperature, osmotic pressure, or pH value.

[0158] The type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, etc. As the expression vector, one that can autonomously replicate in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a protein can be transcribed is preferably used.

[0159] As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

[0160] Preferred examples of prokaryotes include bacteria belonging to the genera Escherichia, Brevibacillus, 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 Brevibacillus include Brevibacillus agri. 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 divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. An example of the microorganism belonging to the genus Pseudomonas is Pseudomonas putida.

[0161] When a prokaryote is used as a host, examples of vectors for introducing a nucleic acid encoding a recombinant protein include pBTrp2 (Boehringer Mannheim), pGEX (Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (JP Patent Publication No. 2002-238569).

[0162] Examples of eukaryotic hosts include yeasts and filamentous fungi (molds, etc.). Examples of yeasts include yeasts belonging to the genera Saccharomyces, Pichia, and Schizosaccharomyces. Examples of filamentous fungi include filamentous fungi belonging to the genera Aspergillus, Penicillium, and Trichoderma.

[0163] When a eukaryote is used as a host, examples of vectors for introducing a nucleic acid encoding a protein include YEP13 (ATCC37115) and YEp24 (ATCC37051). Any method for introducing an expression vector into the host cell can be used as long as it is a method for introducing DNA into the host cell. Examples of such methods include a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, spheroplast method, protoplast method, lithium acetate method, and competent method.

[0164] Methods for expressing nucleic acids using a host transformed with an expression vector include direct expression, secretory production, fusion protein expression, etc., in accordance with the methods described in Molecular Cloning, 2nd Edition.

[0165] The protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. The method for culturing the host in the culture medium can be performed according to a method commonly used for culturing the host.

[0166] When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium may be either a natural medium or a synthetic medium, so long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host and allows efficient cultivation of the host.

[0167] The carbon source may be any that can be assimilated by the transformed microorganism, and may include, for example, carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. The nitrogen source may include, for example, inorganic acids or ammonium salts of organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation bacteria, and digests thereof. The inorganic salt may include, for example, potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0168] Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions, for example, by shaking culture or submerged aeration and stirring culture. The culture temperature is, for example, 15 to 40°C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

[0169] During the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a trp promoter.

[0170] The expressed protein can be isolated and purified by a commonly used method. For example, when the protein is expressed in a dissolved state within the cells, after the culture is completed, the host cells are collected by centrifugation, suspended in an aqueous buffer solution, and then disrupted by an ultrasonic homogenizer, French press, Manton Gaulin homogenizer, Dyno Mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified specimen can be obtained by using, alone or in combination, methods commonly used for isolating and purifying proteins, namely, solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, and the like.

[0171] In addition, when the protein is expressed by forming an insoluble body within the cells, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble body of the protein as a precipitate fraction. The recovered insoluble body of the protein can be solubilized with a protein denaturant. After this operation, a purified preparation of the protein can be obtained by the same isolation and purification method as above. When the protein is secreted outside the cells, the protein can be recovered from the culture supernatant. That is, the culture is treated by a method such as centrifugation to obtain a culture supernatant, and a purified preparation can be obtained from the culture supernatant by the same isolation and purification method as above.

[0172] [Hydrolysis process] The manufacturing method according to the present embodiment includes a step of hydrolyzing the ester groups by contacting a raw material molded body containing a protein whose hydroxyl groups have been esterified with an acidic or basic medium under a tensile force (hereinafter also referred to as a "hydrolysis step"). It is considered that by carrying out hydrolysis under a tensile force, the penetration of the medium into the raw material molded body is suppressed, so that the ester groups inside the raw material molded body are difficult to remove or reduce, and only the ester groups on the surface of the raw material molded body are easily removed or reduced. Even by only removing or reducing the ester groups on the surface of the raw material molded body, it is expected that the generation of bad odors caused by the hydrolysis of the ester groups in the molded body can be sufficiently suppressed. In addition, it is presumed that the cause of the decrease in strength of the protein molded body is the hydrolysis of the protein main chain inside the raw material molded body. For this reason, it is considered that by carrying out hydrolysis under a tensile force, the protein molded body can solve the problems caused by the esterification of the hydroxyl groups contained in the protein while maintaining sufficient strength. Furthermore, by carrying out hydrolysis under application of a tensile force, the toughness (area of ​​the region enclosed by stress (strength) and elongation in a stress (strength)-elongation curve) of the protein molded article is improved.

[0173] In this embodiment, the tensile force is preferably large enough that the raw material compact does not shrink due to contact with a medium (e.g., an aqueous solution). The tensile force can be applied by, for example, a filament winding (FW) method, and can be adjusted by the applied load, etc.

[0174] The tensile force at which the raw material molded body does not shrink upon contact with a medium can be determined, for example, by contacting the raw material molded body with a medium while applying various tensile forces to the raw material molded body, and determining the tensile force at which the rate of change in size of the raw material molded body (e.g., fiber length in the case of fibers, volume in the case of heated and pressed molded bodies, porous bodies and gels, area in the case of films) before and after contact with the medium is 87 to 113%, preferably 90 to 110%, more preferably 93 to 107%, and particularly preferably 95 to 105%.

[0175] For example, when the raw material shaped body is a fiber (raw fiber), the tensile force during treatment is preferably 150 MPa or less, more preferably 125 MPa or less, further preferably 100 MPa or less, and most preferably 75 MPa or less.

[0176] In this embodiment, the medium is an acidic or basic medium. The medium may be an alcohol-based solvent capable of solvolysis of esters, an amine-based organic solvent, or the like, but it is preferable that the medium contains water from the viewpoint of easily carrying out a hydrolysis treatment, which is a type of solvolysis, and is more preferable to be an aqueous solution, water vapor, or the like. The alkaline aqueous solution (medium) may be alkaline, for example, an alkaline aqueous solution having a pH of more than 7, and is preferably an alkaline aqueous solution having a pH of less than 12 from the viewpoint of suppressing hydrolysis of molecular chains and side reactions. The acidic aqueous solution (medium) may be acidic, for example, an acidic aqueous solution having a pH of less than 7, and is preferably an acidic aqueous solution having a pH of 1 or more from the viewpoint of suppressing hydrolysis of molecular chains and side reactions.

[0177] In the present embodiment, the hydrolysis can be carried out, for example, by contacting the raw material compact with an acidic or alkaline aqueous solution. In this case, the amount of the acidic or alkaline substance is preferably 0.1% by mass or more, more preferably 1.0% by mass or more, based on the total amount of the protein solution.

[0178] The acidic substance that can be used for hydrolysis is not particularly limited, and may be either an inorganic acid or an organic acid. Examples of inorganic acids include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and boric acid. Examples of organic acids include carboxylic acids and sulfonic acids. Examples of carboxylic acids include monocarboxylic acids such as formic acid, acetic acid, dichloroacetic acid, trifluoroacetic acid, propionic acid, butanoic acid, isobutyric acid, pentanoic acid, caproic acid, caprylic acid, capric acid, and benzoic acid, saturated aliphatic carboxylic acids such as capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, montanic acid, melissic acid, and ceroplastic acid, and undecylenic acid. Examples of the carboxylic acids include unsaturated aliphatic carboxylic acids such as oleic acid, elaidic acid, cetoleic acid, erucic acid, brassidic acid, sorbic acid, linoleic acid, linolenic acid, arachidonic acid, propiolic acid, and stearic acid, and dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecane acid, brassidic acid, maleic acid, fumaric acid, and glutaconic acid. The carboxylic acids may be in the form of acid anhydrides or acid chlorides. Examples of the sulfonic acids include methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0179] The alkaline substance that can be used for hydrolysis is not particularly limited as long as it is soluble in water, and may be either an inorganic base or an organic base. The inorganic base is not particularly limited as long as it is soluble in water. Examples of the inorganic base include potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, and sodium carbonate. Examples of the organic base include ammonia, alkylamines such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, and triethylamine, and primary to tertiary amines such as aminoethanol, methylaminoethanol, dimethylaminoethanol, ethylaminoethanol, diethylaminoethanol, and diethanolamine.

[0180] The hydrolysis of an ester group is an equilibrium reaction. The same type of acid generated when the ester group dissociates can be used as the acid required for the hydrolysis, but it is preferable not to use such an acid.

[0181] In this embodiment, the hydrolysis proceeds under acidic or alkaline conditions. The pH is preferably an acidic condition of 1 to 6 or an alkaline condition of 8 to 14, and can be adjusted by the acidic or alkaline substance used.

[0182] In this embodiment, when an alkaline aqueous solution is used, the pH is preferably greater than 8 and less than 12 for reasons described below.

[0183] When an alkaline aqueous solution is used, the carboxylic acid dissociates by hydrolysis and then forms a carboxylate anion. At this time, electrophilicity is lost, so that the reverse reaction is less likely to occur. From this point of view, the aqueous solution (medium) is preferably alkaline rather than acidic. The pH of the alkaline aqueous solution is preferably 8 to 14 from the viewpoint of exhibiting high reactivity without heating, and from the viewpoint of the reaction rate of hydrolysis, the pH is more preferably greater than 8. In addition, when the raw material composition is a molded product, the pH is preferably less than 12 from the viewpoint of minimizing dispersion destruction and breakage of molecular chains such as amide hydrolysis in a strongly alkaline environment.

[0184] Since the hydrolysis of the ester group proceeds rapidly in an acidic or alkaline aqueous solution, there is no limitation on the reaction time, but from the viewpoint of sufficiently removing the ester group, a reaction time of more than 1 minute is preferable.

[0185] In this embodiment, the temperature at which hydrolysis is carried out is not particularly limited, and may be, for example, 5° C. or higher, 10° C. or higher, 20° C. or higher, 30° C. or higher, or 80° C. or higher. In addition, the temperature at which hydrolysis is carried out may be, for example, 90° C. or lower, 80° C. or lower, 50° C. or lower, 40° C. or lower, or 35° C. or lower.

[0186] In this embodiment, acidic or alkaline substances remain on the molded product removed from the aqueous solution used for hydrolysis, and the acidic or alkaline substances may cause scission of molecular chains. In order to prevent such scission of molecular chains, the method for producing a protein molded product may further include a step of removing the remaining acidic or alkaline substances. An example of the step of removing the remaining acidic or alkaline substances is washing the molded product with water.

[0187] (Method of manufacturing raw material compact) In this embodiment, the method for producing the raw material compact is not particularly limited, and may be, for example, a method including the following steps.

[0188] [Dissolution process] The dissolving step is a step in which a protein is dissolved in a solvent (eg, a carboxylic acid such as formic acid) to obtain a protein solution.

[0189] In the dissolution step, a purified protein may be used as the protein to be dissolved (hereinafter also referred to as "target protein"), or a protein in a host cell expressing the protein (recombinant protein) may be used. The purified protein may be a protein purified from a host cell expressing a protein. When a protein in a host cell is dissolved as the target protein, the host cell is contacted with a solvent to dissolve the protein in the host cell in the solvent. The host cell may be one that expresses the target protein, and may be, for example, an intact cell or a cell that has been subjected to a treatment such as a disruption treatment. Alternatively, the cell may be a cell that has already been subjected to a simple purification treatment.

[0190] The method for purifying a protein from a host cell expressing the protein is not particularly limited, but for example, the methods described in Japanese Patent Nos. 6077570 and 6077569 can be used.

[0191] When a carboxylic acid such as formic acid is used as a solvent in the dissolution step, a dehydration condensation reaction occurs between a hydroxyl group in the protein and the carboxylic acid to produce an esterified protein. Examples of the carboxylic acid include monocarboxylic acids such as formic acid, acetic acid, dichloroacetic acid, trifluoroacetic acid, propionic acid, butanoic acid, isobutyric acid, pentanoic acid, caproic acid, caprylic acid, capric acid, and benzoic acid, saturated aliphatic carboxylic acids such as capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, montanic acid, melissic acid, and ceroplastic acid, undecylenic acid, Unsaturated aliphatic carboxylic acids such as oleic acid, elaidic acid, cetoleic acid, erucic acid, brassidic acid, sorbic acid, linoleic acid, linolenic acid, arachidonic acid, propiolic acid and stearic acid, as well as dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecane acid, brassidic acid, maleic acid, fumaric acid and glutaconic acid, may be used. The carboxylic acid may be in the form of an acid anhydride or acid chloride.

[0192] The dissolving step may be carried out at room temperature, or may be held at various heating temperatures to dissolve the protein in the solvent. The holding time of the heating temperature is not particularly limited, but may be 10 minutes or more, and in consideration of industrial production, it is preferably 10 to 120 minutes, more preferably 10 to 60 minutes, and even more preferably 10 to 30 minutes. The holding time of the heating temperature may be appropriately set under conditions in which the protein is sufficiently dissolved and little dissolution of impurities (substances other than the target protein) is observed.

[0193] The amount of the solvent added to dissolve the protein is not particularly limited as long as it is an amount that can dissolve the protein.

[0194] When dissolving a purified protein, the amount of solvent added may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times the ratio of the volume (mL) of the solvent to the weight (g) of the protein (dry powder containing the protein) (volume (mL) / weight (g)).

[0195] When dissolving a protein in a host cell in which the protein is expressed, the amount of solvent added may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times the ratio of solvent (mL) to host cell weight (g) (volume (mL) / weight (g)).

[0196] The solvent may contain an inorganic salt. By adding an inorganic salt to the solvent, it is possible to increase the solubility of the protein.

[0197] Examples of inorganic salts that can be added to the solvent include alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, thiocyanates, perchlorates, and the like.

[0198] Examples of the alkali metal halide include potassium bromide, sodium bromide, lithium bromide, potassium chloride, sodium chloride, lithium chloride, sodium fluoride, potassium fluoride, cesium fluoride, potassium iodide, sodium iodide, and lithium iodide.

[0199] Examples of the alkaline earth metal halide include calcium chloride, magnesium chloride, magnesium bromide, calcium bromide, magnesium iodide, and calcium iodide.

[0200] Examples of the alkaline earth metal nitrate include calcium nitrate, magnesium nitrate, strontium nitrate, and barium nitrate.

[0201] Examples of thiocyanates include sodium thiocyanate, ammonium thiocyanate, and guanidinium thiocyanate.

[0202] Examples of perchlorates include ammonium perchlorate, potassium perchlorate, calcium perchlorate, silver perchlorate, sodium perchlorate, and magnesium perchlorate.

[0203] These inorganic salts may be used alone or in combination of two or more.

[0204] Suitable inorganic salts include alkali metal halides and alkaline earth metal halides, and specific examples of suitable inorganic salts include lithium chloride, calcium chloride, etc.

[0205] The amount (content) of the inorganic salt added may be from 0.5% by mass to 10% by mass, or from 0.5% by mass to 5% by mass, based on the total mass of the solvent.

[0206] Insoluble matter may be removed from the protein solution as necessary. That is, the method for producing the raw material molded body may include a step of removing insoluble matter from the protein solution as necessary after the dissolving step. Methods for removing insoluble matter from the protein solution include common methods such as centrifugation, filter filtration using a drum filter, press filter, etc. In the case of filter filtration, insoluble matter can be more efficiently removed from the protein solution by using a filter aid such as celite or diatomaceous earth in combination with a precoat agent, etc.

[0207] The protein solution contains a protein and a solvent in which the protein is dissolved (solvent for dissolving). The protein solution may contain impurities that were contained together with the protein in the dissolving step. The protein solution may be a solution for molding a protein molded body.

[0208] The protein content in the protein solution may be 5% by mass or more and 35% by mass or less, or 5% by mass or more and 50% by mass or less, based on the total amount of the protein solution.

[0209] The method for producing a protein having an esterified hydroxyl group is not particularly limited, and may be a method in which a carboxylic acid such as formic acid is used as a solvent in the above-mentioned dissolution step, or a method other than the method in which the protein is produced in the above-mentioned dissolution step. For example, the method may be a method in which a protein having a hydroxyl group, such as serine, tyrosine, or threonine, is reacted with at least one selected from the group consisting of a carboxylic acid, an acid anhydride, and an acid chloride.

[0210] [Molding process] The molding step is a step of molding a raw material molded body using a protein solution. The shape of the raw material molded body is not particularly limited, but examples thereof include fibers, heat-pressure molded bodies, films, porous bodies, gels, resins, and the like.

[0211] The protein concentration and viscosity of the protein solution are preferably adjusted depending on the raw material molded body to be molded.

[0212] Methods for adjusting the protein concentration in a protein solution include, but are not limited to, a method for increasing the protein concentration by volatilizing the solvent by distillation, a method for using a solution with a high protein concentration in the dissolving step, or a method for reducing the amount of solvent added relative to the amount of protein.

[0213] The viscosity suitable for spinning is generally 10 to 50,000 cP (centipoise), and the viscosity can be measured, for example, using an "EMS Viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd. If the viscosity of the protein solution is not within the range of 10 to 10,000 cP (centipoise), the viscosity of the protein solution may be adjusted to a viscosity that allows spinning. The above-mentioned methods can be used to adjust the viscosity. The solvent may contain the suitable inorganic salt exemplified above.

[0214] When the raw material molded body to be formed is a fiber (raw fiber), the protein content (concentration) in the protein solution may be adjusted to a concentration and viscosity that allows spinning, if necessary. The method for adjusting the protein concentration and viscosity is not particularly limited. In addition, the spinning method includes wet spinning and the like. When a protein solution adjusted to a concentration and viscosity suitable for spinning is applied as a dope to a coagulation liquid, the protein coagulates. At this time, by applying the protein solution as a thread-like liquid to the coagulation liquid, the protein coagulates in a thread-like form, and a thread (undrawn thread) can be formed. The formation of the undrawn thread can be performed, for example, according to the method described in Japanese Patent No. 5584932.

[0215] An example of wet spinning will be shown below, but the spinning method is not particularly limited and may be dry-wet spinning or the like.

[0216] Wet spinning - drawing (a) Wet spinning The coagulation liquid may be any solution that can remove the solvent. The coagulation liquid is preferably a lower alcohol having 1 to 5 carbon atoms, such as methanol, ethanol, or 2-propanol, or acetone. The coagulation liquid may contain water. From the viewpoint of spinning stability, the temperature of the coagulation liquid is preferably 5 to 30°C.

[0217] The method of applying the protein solution as a thread-like liquid is not particularly limited, but for example, a method of extruding the protein solution from a spinning nozzle into the coagulation liquid in a desolvation tank can be mentioned. The protein is coagulated to obtain an undrawn thread. The extrusion speed when extruding the protein solution into the coagulation liquid can be appropriately set according to the diameter of the nozzle and the viscosity of the protein solution, etc., but for example, in the case of a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm, the extrusion speed is preferably 0.2 to 6.0 mL / h per hole, and more preferably 1.4 to 4.0 mL / h per hole, from the viewpoint of spinning stability. The length of the desolvation tank (coagulation liquid tank) in which the coagulation liquid is placed is not particularly limited, but may be, for example, 200 to 500 mm. The take-up speed of the undrawn thread formed by the coagulation of the protein may be, for example, 1 to 14 m / min, and the residence time may be, for example, 0.01 to 0.15 min. From the viewpoint of the efficiency of desolvation, the take-up speed of the undrawn thread is preferably 1 to 3 m / min. The undrawn yarn formed by protein coagulation may be further drawn (pre-drawn) in the solid-liquid state; however, from the viewpoint of suppressing evaporation of the lower alcohol used in the coagulation liquid, it is preferable to maintain the coagulation liquid at a low temperature and to withdraw the yarn from the coagulation liquid in the form of an undrawn yarn.

[0218] (b) Stretching The process may further include a step of drawing the undrawn yarn obtained by the above-mentioned method. The drawing may be a single-stage drawing, or a multi-stage drawing of two or more stages. Multi-stage drawing is suitable for producing fibers with high toughness because it allows the molecules to be oriented in multiple stages and the total draw ratio to be increased.

[0219] In wet spinning, the raw fiber may be subjected to a shrink-proofing treatment. Examples of the method for shrink-proofing the raw fiber include a method in which the protein fiber after spinning and before contact with water is brought into contact with water to irreversibly shrink it (water shrinking method), and a method in which the protein fiber after spinning and before contact with water is heated to relax the protein fiber in the heated state to irreversibly shrink it (dry heat shrinking method).

[0220] The irreversible shrinkage of protein fibers is thought to occur for the following reasons, for example: One reason is thought to be due to the secondary or tertiary structure of the protein fibers, and another reason is thought to occur when, for example, in protein fibers having residual stress due to stretching or the like in the production process, the residual stress is relaxed.

[0221] The water shrinking method includes a step (shrinking step) of contacting the protein fibers with water after spinning and before contacting with water to irreversibly shrink them. In the shrinking step, the protein fibers shrink due to contact with water without external force. The water to be contacted may be in either liquid or gaseous state. The method of contacting the protein fibers with water is also not particularly limited, and examples thereof include a method of immersing the protein fibers in water, a method of spraying water at room temperature or in the form of heated steam or the like onto the protein fibers, and a method of exposing the protein fibers to a high humidity environment filled with water vapor. Among these methods, the method of immersing the protein fibers in water is preferred because it can effectively shorten the shrinkage time and can simplify the processing equipment. Specific examples of the method of immersing the protein fibers in water include a method of putting the protein fibers into a container containing water at a predetermined temperature and contacting them with water.

[0222] The temperature of the water brought into contact with the protein fibers is not particularly limited, but is preferably, for example, below the boiling point. Such a temperature improves the handleability and the workability of the shrinkage step. The upper limit of the water temperature is preferably 90°C or lower, and more preferably 80°C or lower. The lower limit of the water temperature is preferably 10°C or higher, more preferably 40°C or higher, and even more preferably 70°C or higher. The temperature of the water brought into contact with the protein fibers can be adjusted depending on the fibers that constitute the protein fibers. During the contact of the water with the protein fibers, the temperature of the water may be constant, or may be varied to a predetermined temperature.

[0223] The time for contacting the protein fibers with water is not particularly limited and may be, for example, 1 minute or more. The time may be 10 minutes or more, 20 minutes or more, or 30 minutes or more. There is no particular upper limit to the time, but from the viewpoint of shortening the production process time and eliminating the risk of hydrolysis of the protein fibers, it may be, for example, 120 minutes or less, 90 minutes or less, or 60 minutes or less.

[0224] The water shrinkage method may further include, following the shrinkage step, a step of contacting the protein fibers with water and then drying them (drying step).

[0225] The drying method in the drying step is not particularly limited, and may be, for example, natural drying or forced drying using drying equipment. The drying temperature is not particularly limited as long as it is lower than the temperature at which the protein is thermally damaged, but is generally within the range of 20 to 150°C, preferably within the range of 40 to 120°C, and more preferably within the range of 60 to 100°C. Within such a temperature range, the protein fiber can be dried more quickly and efficiently without causing thermal damage to the protein. The drying time is appropriately selected depending on the drying temperature, etc., and, for example, a time is adopted that can eliminate the effects of overdrying of the protein fiber on the quality and physical properties of the knitted or woven body.

[0226] The dry heat shrinkage method includes a step of heating the protein fibers after spinning and before they come into contact with water (heating step), and a step of relaxing the protein fibers in the heated state to cause them to irreversibly shrink (relaxation and shrinkage step).

[0227] In the heating step, the heating temperature is preferably equal to or higher than the softening temperature of the protein used in the protein fiber. In this specification, the softening temperature of the protein is the temperature at which the protein fiber starts to shrink due to stress relaxation. In the heat relaxation shrinkage at or higher than the softening temperature of the protein, the fiber shrinks to an extent that cannot be obtained by simply removing water from the fiber, and as a result, the obtained protein fiber is sufficiently suppressed from shrinking due to contact with water, i.e., dimensional change. The heating temperature is preferably 80°C or higher, more preferably 180°C to 280°C, even more preferably 200°C to 240°C, and even more preferably 220°C to 240°C.

[0228] From the viewpoint of the elongation of the fiber after the heat treatment, the heating time in the heating step is preferably 60 seconds or less, more preferably 30 seconds or less, and even more preferably 5 seconds or less. It is considered that the length of this heating time does not have a significant effect on the stress.

[0229] In the relaxation contraction step, the relaxation ratio is preferably more than 1, more preferably 1.4 or more, even more preferably 1.7 or more, and particularly preferably 2 or more. The relaxation ratio is understood as, for example, the ratio of the delivery speed to the winding speed of the protein fiber.

[0230] When the raw material molded body is a hot-pressed molded body (raw material hot-pressed molded body), the method of forming the raw material hot-pressed molded body is not particularly limited. For example, after introducing a dry protein powder into a pressure molding machine, the dry protein powder reaches a required temperature by pressing and heating using a hand press or the like, and a hot-pressed molded body can be obtained. In addition, the raw material hot-pressed molded body can be formed in accordance with the method described in the patent document (Patent Application No. 2017-539869, PCT / JP2016 / 076500).

[0231] When the raw material molded product is a film (raw material film), the protein solution may be adjusted to a concentration and viscosity that allows film formation, if necessary. Methods for forming a protein film include, but are not limited to, a method in which a protein solution is applied to a solvent-resistant flat plate to a predetermined thickness to form a coating film, and the solvent is removed from the coating film to obtain a film of a predetermined thickness.

[0232] A method for forming a film of a predetermined thickness includes, for example, a casting method. When forming a film by the casting method, a protein solution is cast on a flat plate to a thickness of several microns or more using a tool such as a doctor coater or knife coater to form a cast film, and then the solvent is removed by drying under reduced pressure or immersion in a desolvation tank to obtain a protein film (polypeptide film). The raw film can be formed according to the method described in Japanese Patent No. 5678283.

[0233] When the raw material molded body is a porous body (raw material porous body), it may be adjusted to a concentration and viscosity that can be made porous, if necessary. The method for forming the raw material porous body is not particularly limited. For example, a method of adding an appropriate amount of a foaming agent to a protein solution adjusted to a concentration and viscosity suitable for making porous, and removing the solvent to obtain a porous body, or a method according to the method described in Japanese Patent No. 5796147, etc. may be mentioned.

[0234] When the raw material molded body is a gel (raw material gel), the method of forming the raw material gel is not particularly limited. For example, the raw material gel can be obtained by a solution generation step of dissolving a dried protein in a dissolving solvent to obtain a solution of a polypeptide and a step of replacing the solution generated in the solution generation step with a water-soluble solvent. In this case, a step of pouring into a mold and molding into a predetermined shape can be inserted between the solution generation step and the step of replacing the dissolving solvent with a water-soluble solvent, or the predetermined shape can be obtained by cutting after the step of replacing the dissolving solvent with a water-soluble solvent. The raw material gel can be formed according to the method described in Japanese Patent No. 5782580.

[0235] When the raw material molded body is a resin (raw material resin), the protein solution may be adjusted to a concentration and viscosity that allows conversion to a resin, if necessary. The method for forming the raw material resin is not particularly limited, and it can be produced in accordance with the method described in Japanese Patent No. 5678283, etc.

[0236] [Method for processing protein molded body] The processing method for a protein molded body according to this embodiment includes a step of hydrolyzing the ester groups of a protein molded body containing a protein whose hydroxyl groups have been esterified by contacting the protein molded body with an acidic or basic medium while applying a tensile force.

[0237] In this embodiment, the protein in which the hydroxyl group is esterified, the magnitude of the tensile force, the method of applying the tensile force, the properties of the acidic or basic medium, and the method of hydrolyzing the ester group can be similar to those described in the above-mentioned method for producing a protein shaped body. The protein shaped body may be obtained by a method including a step of hydrolyzing the ester group (hydrolysis step) described in the above-mentioned method for producing a protein shaped body, or may be obtained by a method not including a hydrolysis step.

[0238] According to the processing method of this embodiment, the hydroxyl groups in the protein can be re-exposed, making it possible to provide a processing method for a protein molded body that can resolve problems caused by esterification of the hydroxyl groups contained in the protein while maintaining sufficient strength. EXAMPLES

[0239] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0240] [Production of Proteins] (1) Construction of expression vector Based on the nucleotide sequence and amino acid sequence of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), spider silk fibroin having sequence number 40 (hereinafter also referred to as "PRT966") was designed.

[0241] Next, a nucleic acid encoding the designed protein (spider silk fibroin) was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. The nucleic acid was cloned into a cloning vector (pUC118). After that, the nucleic acid was excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+), respectively, to obtain an expression vector.

[0242] (2) Protein expression The expression vector obtained in (1) was used to transform E. coli BLR(DE3). The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture was transferred to 100 mL of seed culture medium (Table 4) containing ampicillin at OD 200. 600 The culture temperature was kept at 30°C, and the OD 600 The flask culture was continued until the number of cells reached 5 (about 15 hours), and a seed culture solution was obtained.

[0243] [Table 4]

[0244] The seed culture solution was transferred to a jar fermenter containing 500 mL of production medium (Table 5) and the OD 600 The culture temperature was kept at 37°C and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration.

[0245] [Table 5]

[0246] Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455g / 1L, yeast extract 120g / 1L) was added at a rate of 1mL / min. The culture temperature was kept at 37°C and the culture was controlled to a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours. Then, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1mM to induce the expression of the modified fibroin. After 20 hours had passed after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the target protein (spider silk fibroin) was confirmed by the appearance of a band of the target recombinant protein depending on the addition of IPTG.

[0247] (3) Protein purification The cells harvested 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until it reached a high purity. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and stirred with a stirrer at 60°C for 30 minutes to dissolve. After dissolution, dialysis was performed against water using a dialysis tube (Cellulose tube 36 / 32, Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation, the water was removed using a freeze-dryer, and the freeze-dried powder was collected to obtain the protein (spider silk fibroin).

[0248] (4) Manufacturing of raw fibers Dry powder of spider silk fibroin was dissolved in formic acid and filtered through a metal filter with 1 μm mesh to obtain a dope solution (protein concentration in the dope solution: 30% by mass). A known spinning device was used, and the dope solution was discharged into the coagulation solution with a gear pump. The spinning conditions were as shown below. In this way, raw fiber (raw spider silk fibroin fiber) was obtained. (Spinning conditions) Nozzle hole diameter: 0.1 mm Coagulation solution: Methanol Temperature of coagulation solution: 25°C Temperature of water washing bath: 25°C Hot roller temperature: 60°C

[0249] (5) Hydrolysis treatment The raw fiber obtained in (4) was subjected to hydrolysis treatment in the following steps [i] to [iii]. [i] The raw fiber was immersed in an alkaline aqueous solution and stirred. [ii] The raw fiber was washed with tap water until the pH was below 8.0. [iii] The raw fiber was air-dried for two days.

[0250] The treatment conditions in step [i] are shown in Table 6. In Example 1, the raw fiber was wound around a stainless steel plate of 300 x 300 x 2 (mm) with tension (load: 10 N) using a filament winder FWM-1500LF (manufactured by Asahi Kasei Engineering Co., Ltd.) (winding width: 20 cm, number of layers: 20 ply). [Table 6]

[0251] (6) Evaluation of removal of formyl residues (formate ester groups) by FT-IR Each fiber obtained in (5) was measured by the ATR method (attenuated total reflection method) using a Fourier transform infrared spectrophotometer (Nicolet iS50FT-IR, manufactured by Thermo Fisher Scientific Co., Ltd.) to evaluate the removal of formyl residues. For each fiber (Example 1 (n=4) and Comparative Example 1), the IR spectrum showed a peak at a wave number of 1730 cm -1 The height of the peak (attributed to C=O of the formyl residue, indicated by an arrow in Figure 4) was checked, and the presence or absence of the peak was evaluated (Figure 4). If no peak was detected, it was determined that the formyl residue had been removed. Note that a sample that had not been subjected to the above-mentioned (5) hydrolysis treatment was used as a control.

[0252] From FIG. 4, it was confirmed that when the raw fiber was subjected to hydrolysis treatment with an alkaline aqueous solution, the hydrolysis reaction of the formyl residues was promoted and the formyl residues in the protein fiber were removed (Example 1 and Comparative Example 1).

[0253] From the FT-IR spectrum (Figure 4), the peaks were fitted using a Lorentzian function, and the peak whose intensity did not change before and after the hydrolysis treatment (1470 cm -1 A peak near the formate ester was selected as a reference peak, and the ratio (area ratio) of the area of ​​the peak due to the formate ester to the area of ​​the reference peak was calculated. The proportion [%] of formyl residues in Example 1 was calculated by calculating the area ratio in Example 1 relative to the area ratio in the control. The results are shown in Table 7.

[0254] [Table 7]

[0255] Assuming that all hydroxyl groups were formylated before treatment, the amount of non-deformylated portion can be calculated to be 0.52 mmol / g by multiplying the amount of hydroxyl groups in spider silk fibroin (1.88 mmol / g) by the proportion of formyl residues (27.89%). Using this calculation result, the amount of deformylated portion can be calculated to be 1.36 mmol / g (= 1.88 mmol / g - 0.52 mmol / g).

[0256] (7) Evaluation of elongation Each hydrolyzed and dried fiber was fixed to a jig with a fiber length of 20 mm, and stress (strength) and elongation were measured at a tensile speed of 10 mm / min using a Textechno single fiber tensile tester FAVIMAT+ under the following conditions: load cell capacity 2N, pretension 1.25 cmN / tex, temperature 20°C, relative humidity 65%. The results are shown in Figure 5.

[0257] A comparison between Example 1 and Comparative Example 1 showed that the strength of the protein fiber was improved by applying a tensile force during the hydrolysis treatment. Furthermore, a comparison between the control and Example 1 showed that the application of a tensile force during the hydrolysis treatment increased the elongation and improved the toughness (area of ​​the region surrounded by stress (strength) and elongation) of the protein fiber.

Claims

1. The method includes a step of hydrolyzing the ester groups by contacting a raw material molded body containing a protein having an esterified hydroxyl group with an acidic or basic medium while applying a tensile force to the raw material molded body, The method for producing a protein molded article, wherein the protein is a structural protein.

2. The method for producing a protein molded body according to claim 1 , wherein the medium is an aqueous solution.

3. The method for producing a protein molded body according to claim 2 , wherein the aqueous solution is an alkaline aqueous solution having a pH of less than 12.

4. The method for producing a protein shaped body according to claim 2 or 3, wherein the tensile force is of a magnitude such that the raw material shaped body does not shrink upon contact with the aqueous solution.

5. The method for producing a protein molded body according to any one of claims 1 to 4, wherein the structural protein is fibroin.

6. The method for producing a protein molded article according to claim 5 , wherein the fibroin is spider silk fibroin.

7. The method for producing a protein shaped body according to any one of claims 1 to 6, wherein the protein having an esterified hydroxyl group comprises a formic acid ester.

8. The method for producing a protein molded product according to any one of claims 1 to 7, wherein the raw material molded product is at least one selected from the group consisting of a fiber, a heat-pressure molded product, a film, a porous body, a gel, and a resin.

9. The method includes a step of contacting a protein shaped article containing a protein having an esterified hydroxyl group with an acidic or basic medium while applying a tensile force, thereby hydrolyzing the ester group; The method for processing a protein molded article, wherein the protein is a structural protein.

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