Recombinant Structural Protein Multifilament and Method for Producing the Same

Abstract

The present invention aims to provide a method for producing a modified fibroin multifilament with excellent productivity and a multifilament produced thereby. The multifilament of the present invention contains modified fibroin, has 100 or more constituent single yarns, and has a coefficient of variation of elastic modulus of 15% or less, a coefficient of variation of strength of 15% or less, and a coefficient of variation of elongation of less than 33%.

Description

Technical Field

[0001] The present invention relates to a recombinant structural protein multi-filament and a method for producing the same.

Background Art

[0002] Conventionally, as artificial fibroin fibers, silk fibroin fibers that are regenerated silk fibers, spider silk fibroin fibers, etc. are known, and many spinning methods thereof have also been reported.

[0003] For example, a multi-filament of spider silk fibroin derived from a natural-type spider silk fibroin structure composed of 50 single filaments produced using a spinning nozzle having 50 holes has been reported (Patent Document 1).

[0004] However, a recombinant structural protein multi-filament composed of a number of single filaments equal to the number of holes in a spinning nozzle, which is produced on a large scale using a spinning nozzle having a larger number of holes, has not yet been reported.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a number of recombinant structural protein multi-filaments composed of a number of single filaments that do not exist in the world.

Means for Solving the Problems

[0007] That is, the present invention relates to, for example, each of the following inventions. [1] A multifilament that contains modified fibroin, has 100 or more constituent monofilaments, has a coefficient of variation of modulus of elasticity of 15% or less, a coefficient of variation of strength of 15% or less, and a coefficient of variation of elongation of less than 33%. [2] The multifilament according to [1], wherein the coefficient of variation of elongation of the multifilament is 20% or less. [3] The multifilament according to [1] or [2], wherein the coefficient of variation of elongation of the multifilament is 0.01% or more and 20% or less. [4] The multifilament according to any one of [1] to [3], wherein the coefficient of variation of elongation of the multifilament is less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10%, more than 10% to less than 15%, or more than 15% to less than 20%. [5] The multifilament according to any one of [1] to [4], wherein the coefficient of variation of elongation of the multifilament is 15% or less. [6] The multifilament according to any one of [1] to [4], wherein the coefficient of variation of elongation of the multifilament is 10% or less. [7] The multifilament according to any one of [1] to [6], wherein the coefficient of variation of fineness of the multifilament is 20% or less. [8] The multifilament according to any one of [1] to [7], wherein the coefficient of variation of strength of the multifilament is 10% or less and the coefficient of variation of modulus of elasticity of the multifilament is 10% or less. [9] The multifilament according to any one of [1] to [8], wherein the average hydrophobicity index of the modified fibroin is more than -0.8.

[10] The multifilament according to any one of [1] to [9], wherein the modified fibroin is modified spider silk fibroin.

[11] A multifilament according to any one of [1] to

[10] , having a shrinkage history irreversibly shrunk after spinning.

[12] The multifilament according to

[11] , wherein the shrinkage history is a shrinkage history irreversibly shrunk by bringing the multifilament into contact with water or a shrinkage history irreversibly shrunk by heat-relaxing the multifilament.

[13] The multifilament according to

[11] or

[12] , wherein the multifilament having a shrinkage history irreversibly shrunk after spinning has a shrinkage rate defined by the following formula of 5% or less. Shrinkage rate [%] = (1 - (length of the multifilament when dried from the wet state / length of the multifilament when in the wet state)) × 100

[14] The multifilament according to any one of [1] to

[13] , wherein the coefficient of variation of the fineness of the multifilament is 0.01% to 6.5%.

[15] The multifilament according to any one of [1] to

[14] , wherein the coefficient of variation of the strength of the multifilament is 0.01% to 3.8%.

[16] A multifilament containing a recombinant structural protein, having 100 or more constituent monofilaments, and having a coefficient of variation of elongation of less than 33%.

[17] The multifilament according to

[16] , wherein the recombinant structural protein satisfies the following (1) or (2). (1) It has 150 or more amino acid residues, an alanine residue content of 12 to 40%, and a glycine residue content of 11 to 55%. (2) The total content of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine, the alanine residue content, and the glycine residue content is 56% or more.

[18] The multifilament according to

[16] or

[17] , wherein the recombinant structural protein satisfies both of the above (1) and (2).

[19] The recombinant structural protein has a plurality of repetitive sequence units, The multi-filament according to any one of

[16] to

[18] , wherein the number of amino acid residues in the repetitive sequence unit is 6 to 200.

[20] The multi-filament according to any one of

[16] to

[19] , wherein the recombinant structural protein is at least one selected from the group consisting of fibroin, collagen, resilin, elastin and keratin, and proteins derived therefrom.

[21] The recombinant structural protein is (A) n The multi-filament according to any one of

[16] to

[20] , which contains a motif.

[22] A method for producing a multi-filament, comprising the step of discharging a spinning dope containing a recombinant structural protein and a solvent from a spinning nozzle having 100 or more pore numbers and bringing it into contact with a coagulating liquid to coagulate the recombinant structural protein.

[23] The method for producing a multi-filament according to

[22] , wherein the recombinant structural protein satisfies the following (1) or (2). (1) The number of amino acid residues is 150 or more, the alanine residue content is 12 to 40%, and the glycine residue content is 11 to 55%. (2) The total content of at least one amino acid residue selected from the group consisting of serine, threonine and tyrosine, the alanine residue content and the glycine residue content is 56% or more.

[24] The method for producing a multi-filament according to

[22] or

[23] , wherein the recombinant structural protein satisfies both (1) and (2) above.

[25] The recombinant structural protein has a plurality of repetitive sequence units, The method for producing a multi-filament according to any one of

[22] to

[24] , wherein the number of amino acid residues in the repetitive sequence unit is 6 to 200.

[26] The production method according to any one of

[22] to

[25] , wherein the recombinant structural protein is at least one selected from the group consisting of fibroin, collagen, resilin, elastin, keratin, and proteins derived therefrom.

[27] The recombinant structural protein is (A) n A multifilament according to any one of

[22] to

[26] , which contains a motif.

[28] A method for producing a multifilament, comprising a step of discharging a spinning dope containing a modified fibroin and a solvent from a spinning nozzle having 100 or more holes and bringing it into contact with a coagulation liquid to coagulate the modified fibroin.

[29] The production method according to

[28] , wherein the number of holes of the spinning nozzle is 100 or more and 9000 or less.

[30] The production method according to any one of

[28] or

[29] , wherein the average hydrophobicity index of the modified fibroin is more than -0.8.

[31] The production method according to any one of

[28] to

[30] , wherein the modified fibroin is a modified spider silk fibroin.

[32] The production method according to any one of

[28] to

[31] , wherein the solvent is at least one organic solvent selected from the group consisting of formic acid, DMSO, and HFIP.

[33] The production method according to any one of

[28] to

[32] , wherein the coagulation liquid contains at least one component selected from the group consisting of lower alcohols having 1 to 5 carbon atoms, ketones, water, and aqueous solutions having a pH of 0.25 or more and 10.00 or less.

[34] The production method according to

[33] , wherein the content of the above component is 70% by mass or more based on 100% by mass of the total amount of the coagulation liquid.

[35] The production method according to any one of

[28] to

[34] , wherein the coagulation liquid contains at least one selected from the group consisting of methanol, acetone, water, aqueous sodium chloride solution, aqueous sodium sulfate solution, and aqueous formic acid solution.

[36] The production method according to any one of

[28] to

[35] , wherein the coagulating liquid contains at least one selected from the group consisting of methanol, an aqueous sodium sulfate solution, and an aqueous formic acid solution.

[37] The method according to any one of

[28] to

[36] , wherein the coagulating liquid contains an organic solvent, and the content of the organic solvent is 30% by mass or less based on 100% by mass of the total amount of the coagulating liquid. [Advantages of the Invention]

[0008] According to the present invention, it is possible to provide a recombinant protein multifilament composed of a large number of single filaments that do not exist in the world, and a method for producing the same. The recombinant protein multifilament according to the present invention has a smaller elongation, elastic modulus, strength, and / or coefficient of variation of fineness, and particularly has a smaller coefficient of variation of elongation compared to conventional ones (for example, Patent Document 1). That is, the recombinant protein multifilament of the present invention has a relatively small variation in physical properties and is extremely excellent in quality stability. [Brief Description of the Drawings]

[0009]

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Figure 13

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Figure 15

Mode for Carrying Out the Invention

[0010] (Recombinant Structural Protein) A structural protein refers to a protein that forms a biological structure or a protein derived therefrom. A recombinant structural protein is a structural protein produced by genetic recombination technology. The recombinant structural protein may have the amino acid sequence of a naturally occurring structural protein, or may be a modified structural protein in which a part of the amino acid sequence is modified based on the amino acid sequence of a naturally occurring structural protein.

[0011] The recombinant structural protein according to this embodiment may satisfy any one of the following (1) or (2). (1) It has 150 or more amino acid residues, the alanine residue content is 12 - 40%, and the glycine residue content is 11 - 55%. (2) The total content of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine, the alanine residue content, and the glycine residue content is 56% or more.

[0012] In this specification, the "alanine residue content" is a value represented by the following formula. Alanine residue content = (number of alanine residues contained in the recombinant structural protein / total number of amino acid residues of the polypeptide) × 100 (%) Also, the glycine residue content, serine residue content, threonine residue content, and tyrosine residue content are synonymous with those obtained by replacing alanine residues with glycine residues, serine residues, threonine residues, and tyrosine residues, respectively, in the above formula.

[0013] (1) The recombinant structural protein satisfying (1) only needs to have 150 or more amino acid residues. The number of amino acid residues may be, for example, 200 or more, 250 or more, preferably 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more.

[0014] (1) The recombinant structural protein satisfying (1) only needs to have an alanine residue content of 12 - 40%. The alanine residue content may be, for example, 15 - 40%, 18 - 40%, 20 - 40%, or 22 - 40%.

[0015] (1) The recombinant structural protein satisfying (1) only needs to have a glycine residue content of 11 - 55%. The glycine residue content may be, for example, 11% - 55%, 13% - 55%, 15% - 55%, 18% - 55%, 20% - 55%, 22% - 55%, or 25% - 55%.

[0016] The recombinant structural protein satisfying (2) may have a total content (total content) of at least one amino acid residue content selected from the group consisting of serine, threonine, and tyrosine (i.e., serine residue content, threonine residue content, tyrosine residue content, total of serine residue content and threonine residue content, total of serine residue content and tyrosine residue content, total of threonine residue content and tyrosine residue content, total of serine residue content, threonine residue content, and tyrosine residue content), alanine residue content, and glycine residue content of 56% or more. The total content may be, for example, 57% or more, 58% or more, 59% or more, or 60% or more. The upper limit of the total content is not particularly limited, and may be, for example, 90% or less, 85% or less, or 80% or less.

[0017] In one embodiment, the recombinant structural protein satisfying (2) may have a total of serine residue content, threonine residue content, and tyrosine residue content of 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, or 7% or more. The total of serine residue content, threonine residue content, and tyrosine residue content may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0018] The recombinant structural protein according to this embodiment preferably satisfies both of the above (1) and (2). Thereby, the effects according to the present invention are more significantly exhibited.

[0019] The recombinant structural protein according to this embodiment has an average distribution of serine residues, threonine residues, or tyrosine residues, and among any consecutive 20 amino acid residues, the total content of serine residues, threonine residues, and tyrosine residues may be 5% or more, 10% or more, or 15% or more, and may be 50% or less, 40% or less, 30% or less, or 20% or less.

[0020] The recombinant structural protein according to one embodiment may have a repetitive sequence. That is, the recombinant structural protein according to the present embodiment may have a plurality of amino acid sequences (repetitive sequence units) with high sequence identity within the recombinant structural protein. There is no particular limitation on the amino acid sequence of the repetitive sequence unit, and it may be any as long as the whole recombinant structural protein satisfies (1) or (2) described above. The number of amino acid residues in the repetitive sequence unit is preferably 6 to 200. Also, the sequence identity between the repetitive sequence units may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0021] The recombinant structural protein according to one embodiment may contain (A) n motif. As used herein, (A) n motif means an amino acid sequence mainly composed of alanine residues. (A) n The number of amino acid residues in the motif may be 2 to 27, and may be an integer of 2 to 20, 2 to 16, or 2 to 12. Also, (A) 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 composed only of alanine residues).

[0022] In one embodiment, (A) n motif may be included in the repetitive sequence unit. (A) n Since the motif mainly contains alanine residues, it is likely to adopt an α-helix structure or a β-sheet structure. (A) n When the motif is included in the repetitive sequence unit, the recombinant structural protein according to the present embodiment will have these secondary structures repeatedly. Therefore, when the recombinant structural protein is in the form of a fiber, it is expected to exhibit high strength due to these secondary structures.

[0023] Examples of the recombinant structural protein include any structural protein that is preferably produced on an industrial scale, specifically, structural proteins that can be used industrially, structural proteins that can be used medically, and the like. Specific examples of the structural protein that can be used industrially or medically include fibroin, collagen, resilin, elastin, and keratin, which are insoluble proteins, and proteins derived therefrom, etc., but may also be water-soluble proteins. Fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin.

[0024] The fibroin according to the present embodiment includes naturally-derived fibroin and modified fibroin. In this specification, "naturally-derived fibroin" means fibroin having the same amino acid sequence as naturally-derived fibroin, and "modified fibroin" means fibroin having an amino acid sequence different from that of naturally-derived fibroin. In this specification, "modified fibroin" means artificially produced fibroin (synthetic fibroin). The modified fibroin may be fibroin whose domain sequence is different from the amino acid sequence of naturally-derived fibroin, or may be fibroin having the same amino acid sequence as that of naturally-derived fibroin.

[0025] The fibroin according to the present embodiment is preferably spider silk fibroin (spider silk protein). Spider silk fibroin includes natural spider silk fibroin and modified spider silk fibroin derived from natural spider silk fibroin. Examples of natural spider silk fibroin include spider silk proteins produced by spiders.

[0026] The fibroin according to the present embodiment is, for example, Formula 1: [(A) n Motif-REP] m or Formula 2: [(A) n Motif-REP]m -(A) n It may be a protein containing a domain sequence represented by a motif. The fibroin according to this embodiment may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited thereto, but are typically regions that do not have repeats of amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

[0027] 〔Modified fibroin〕 The modified fibroin used as a raw material is not particularly limited, and may be fibroin produced by microorganisms or the like by genetic recombination technology, or may be fibroin produced by synthesis. However, naturally-derived fibroin is excluded from the modified fibroin. In this embodiment, when the modified fibroin is modified spider silk fibroin, the heat retention property, the hygroscopic heat generation property, and / or the flame retardancy are more excellent.

[0028] The modified fibroin according to this embodiment is of formula 1: [(A) n Motif-REP] m or formula 2: [(A) n Motif-REP] m -(A) n It is a protein containing a domain sequence represented by a motif. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited thereto, but are typically regions that do not have repeats of amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

[0029] As used herein, "modified fibroin" means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be a fibroin whose amino acid sequence is identical to the amino acid sequence of naturally occurring fibroin. The "naturally occurring fibroin" referred to herein is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) n motif.

[0030] The "modified fibroin" may be one that uses the amino acid sequence of naturally occurring fibroin as it is, or one that modifies the amino acid sequence based on the amino acid sequence of naturally occurring fibroin (for example, one that modifies the amino acid sequence by modifying the gene sequence of cloned naturally occurring fibroin), or one that is artificially designed and synthesized without relying on naturally occurring fibroin (for example, one that has a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

[0031] As used herein, the "domain sequence" is an amino acid sequence that gives rise to a crystal region specific to fibroin (typically corresponding to the (A) n motif of the amino acid sequence.) and an amorphous region (typically corresponding to REP of the amino acid sequence), and means an amino acid sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) n motif. Here, the (A) n motif indicates an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27. (A) nThe 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. Also, (A) 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% (which means it is composed only of alanine residues). There may be multiple (A) n At least 7 of the motifs may be composed only of alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues, and may also be an amino acid sequence composed of 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 120, 10 to 140, 10 to 160, or 10 to 180 amino acid residues. m represents an integer of 2 to 300, and may also be an integer of 8 to 300, 10 to 300, 10 to 300, 20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to 160, 20 to 140, or 20 to 120. There may be multiple (A) n The motifs may have the same amino acid sequence as each other, or may have different amino acid sequences. Multiple REPs may have the same amino acid sequence as each other, or may have different amino acid sequences.

[0032] The modified fibroin according to this embodiment can be obtained, for example, by modifying the amino acid sequence corresponding to substituting, deleting, inserting, and / or adding one or more amino acid residues to the cloned gene sequence of naturally derived fibroin. 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 the site-directed mutagenesis method. Specifically, it can be performed according to the methods described in documents such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0033] Naturally occurring fibroin is a protein containing a domain array represented by formula 1: [(A) n motif-REP] m or formula 2: [(A) n motif-REP] m -(A) n motif, and specifically, for example, fibroin produced by insects or spiders can be mentioned.

[0034] Examples of fibroin produced by insects include, for example, silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Samia cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, and hornet silk protein secreted by the larvae of Vespa simillima xanthoptera.

[0035] More specific examples of fibroin produced by insects include, for example, silkworm fibroin light chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).

[0036] Examples of fibroins produced by spiders include spider silk proteins produced by spiders belonging to the genus Araneus such as Araneus ventricosus, Araneus marmoreus, Araneus cingulatus, Araneus sericatus, and Araneus gemmoides; spiders belonging to the genus Neoscona such as Neoscona theisi, Neoscona doenitzi, Neoscona nautica, and Neoscona subfusca; spiders belonging to the genus Pronus such as Pronus emarginatus; spiders belonging to the genus Cyrtarachne such as Cyrtarachne triangulata and Cyrtarachne formosana; spiders belonging to the genus Gasteracantha such as Gasteracantha cancriformis and Gasteracantha kuhli; spiders belonging to the genus Ordgarius such as Ordgarius magnus and Ordgarius spiniger; spiders belonging to the genus Argiope such as Argiope bruennichi, Argiope catenulata, and Argiope nigrofasciata; spiders belonging to the genus Arachnura such as Arachnura higginsi; spiders belonging to the genus Acusilas such as Acusilas coccineus; spiders belonging to the genus Cytophora such as Cytophora exanthematica, Cytophora moluccensis, and Cytophora albovittata; spiders belonging to the genus Poltys such as Poltys sp.; spiders belonging to the genus Cyclosa such as Cyclosa argenteoalba, Cyclosa mulmeinensis, Cyclosa ginnaga, and Cyclosa octotuberculata; and spiders belonging to the genus Chorizopes such as Chorizopes yamatoensis; as well as spiders belonging to the genus Tetragnatha such as Tetragnatha maxillosa, Tetragnatha extensa, Tetragnatha versicolor, and Tetragnatha squamata; spiders belonging to the genus Leucauge such as Leucauge decorata, Leucauge blanda, and Leucauge subnigra; spiders belonging to the genus Nephila such as Nephila clavata and Nephila pilipes; spiders belonging to the genus Menosira such as Menosira annulipes; spiders belonging to the genus Dyschiriognatha such as Dyschiriognatha pygmaea; and spiders belonging to the genus Latrodectus such as Latrodectus hasselti, Latrodectus geometricus, Latrodectus pallidus, and Latrodectus trivittatus.and 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), ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

[0037] More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank accession number AAC04504 (amino acid sequence), U37520 (base sequence)), major ampullate spidroin 1 [from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [from Nephila clavata] (GenBank accession number AAL32472 (amino acid sequence), AF441245 (base sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampullate spidroin 2 [from Euprosthenops australis] (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [from Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [from Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [from Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), etc.

[0038] As more specific examples of naturally-derived fibroin, fibroin for which sequence information is registered in NCBI GenBank can be further cited. For example, among the sequences including INV as DIVISION in the sequence information registered in NCBI GenBank, it can be confirmed by extracting the sequences in which spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION, the character string of a specific product from CDS, and the sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

[0039] The modified fibroin according to this embodiment may be modified silk fibroin (a modification of the amino acid sequence of the silk protein produced by silkworms), or may be modified spider silk fibroin (a modification of the amino acid sequence of the spider silk protein produced by spiders). As the modified fibroin, modified spider silk fibroin is preferable.

[0040] As specific examples of the modified fibroin, modified spider silk fibroin (first modified fibroin) derived from the major ampullate spidroin protein produced in the major ampullate gland of spiders, modified spider silk fibroin having a domain sequence with a reduced glycine residue content (second modified fibroin), (A) n modified spider silk fibroin having a domain sequence with a reduced (A) motif content (third modified fibroin), the glycine residue content, and (A) nModified spider silk fibroin with reduced motif content (fourth modified fibroin), modified spider silk fibroin having a domain sequence containing a region with a locally large hydrophobicity index (fifth modified fibroin), and modified spider silk fibroin having a domain sequence with reduced glutamine residue content (sixth modified fibroin) can be mentioned. These modified fibroins are excellent in flame retardancy, moisture absorption and heat generation properties, and heat retention properties, and are suitable for use in fireproof clothing (e.g., fire fighting suits, rescue suits), fireproof gloves (e.g., laboratory use, industrial use, cooking use), gloves, mufflers, sweaters, cold-proof clothing (cold-proof clothing) such as outerwear and jackets, batting for cold-proof clothing, innerwear, sports wear, shirts, bedding, and batting for bedding, etc.

[0041] As the first modified fibroin, proteins containing a domain sequence represented by Formula 1: [(A) n Motif-REP] m can be mentioned. In the first modified fibroin, the number n of amino acid residues of (A) n Motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, still more preferably an integer of 10 to 20, still 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. In the first modified fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, still more preferably 20 to 100 residues, and still more preferably 20 to 75 residues. The first modified fibroin is 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 is preferably 40% or more, more preferably 60% or more, and still more preferably 70% or more based on the total number of amino acid residues.

[0042] The first modified fibroin is Formula 1: [(A) n Motif-REP] mIt may also be a polypeptide containing a unit of the amino acid sequence represented by , and having a C-terminal sequence that is the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

[0043] The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of the 50 C-terminal residues 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. 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.

[0044] As a more specific example of the first modified fibroin, (1-i) the amino acid sequence represented by 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 represented by SEQ ID NO: 4 can be mentioned. The sequence identity is preferably 95% or more.

[0045] The amino acid sequence represented by SEQ ID NO: 4 is obtained by adding an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, His10 tag, and HRV3C protease (Human rhinovirus 3C protease) recognition site to the N-terminus of the amino acid sequence of ADF3, increasing the 1st to 13th repeat regions approximately twice, and mutating so that translation terminates at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence represented by SEQ ID NO: 4 is identical to the amino acid sequence represented by SEQ ID NO: 3.

[0046] (1-i) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 4.

[0047] The second modified fibroin has an amino acid sequence in which the content of glycine residues in its domain sequence is reduced as compared with naturally-derived fibroin. The second modified fibroin can be said to have an amino acid sequence corresponding to the substitution of at least one or more glycine residues in at least REP with another amino acid residue as compared with naturally-derived fibroin.

[0048] The second modified fibroin may have an amino acid sequence in which at least one glycine residue in at least one motif sequence selected from GGX and GPGXX (where G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP is substituted with another amino acid residue as compared with naturally-derived fibroin.

[0049] The proportion of the motif sequence in which the above-mentioned glycine residue is substituted with another amino acid residue in the second modified fibroin may be 10% or more with respect to all motif sequences.

[0050] The second modified fibroin has a domain sequence represented by Formula 1: [(A) n Motif-REP] m and when the total number of amino acid residues of 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 (A) n motif located at the most C-terminal side to the C-terminal of the above domain sequence is z, and the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the above domain sequence is w, z / w may be 30% or more, 40% or more, 50% or more, or 50.9% or more. (A) nThe number of alanine residues relative to the total number of amino acid residues in the motif may be 83% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 100% (which means consisting only of alanine residues).

[0051] The second modified fibroin preferably has an increased content ratio of the amino acid sequence consisting of XGX by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, still 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 ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated in the same manner as the calculation method of the content ratio (z / w) of the amino acid sequence consisting of XGX described below.

[0052] The calculation method of z / w will be described in more detail. First, in the fibroin (modified fibroin or naturally-derived fibroin) containing the domain sequence represented by formula 1: [(A) n motif-REP] m all REPs contained in the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence are removed from the domain sequence, and the amino acid sequence consisting of XGX is extracted. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (without duplication), z is 50 × 3 = 150. Also, for example, when there is an X (the central X) contained in two XGXs as in the case of the amino acid sequence consisting of XGXGX, the calculation is performed after excluding the overlapping part (in the case of XGXGX, it is 5 amino acid residues). w is the sequence from the domain sequence from the (A) nIt 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 array. For example, in the case of the domain array shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (the (A) located on the most C-terminal side) n (The motif is excluded.). Next, by dividing z by w, z / w (%) can be calculated.

[0053] Here, z / w in naturally occurring fibroin will be described. First, as described above, when confirmed by the method exemplified using fibroin whose amino acid sequence information is registered in NCBI GenBank, 663 types of fibroin (among which, 415 types are fibroin derived from spiders) were extracted. Among all the extracted fibroins, from the amino acid sequences of naturally occurring fibroins containing the domain array represented by Formula 1: [(A) n motif - REP] m and having a content ratio of the amino acid sequence consisting of GGX in fibroin of 6% or less, z / w was calculated by the above-described calculation method. The results are shown in FIG. 2. The horizontal axis in FIG. 2 indicates z / w (%), and the vertical axis indicates the frequency. As is clear from FIG. 2, z / w in naturally occurring fibroin is all less than 50.9% (the highest is 50.86%).

[0054] In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, still more preferably 70% or more, and still more preferably 80% or more. There is no particular limitation on the upper limit of z / w, but for example, it may be 95% or less.

[0055] The second modified fibroin can be obtained, for example, by modifying at least a part of the base sequence encoding a glycine residue in the gene sequence of cloned naturally-derived fibroin so as to encode another amino acid residue. At this time, as the glycine residue to be modified, one glycine residue in the GGX motif and the GPGXX motif may be selected, or substitution may be made so that z / w becomes 50.9% or more. Further, for example, it can also be obtained by designing an amino acid sequence satisfying the above-described aspect from the amino acid sequence of naturally-derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to substituting a glycine residue in REP with another amino acid residue from the amino acid sequence of naturally-derived fibroin, an amino acid sequence modification corresponding to substituting, deleting, inserting, and / or adding one or more amino acid residues may also be performed.

[0056] The above-mentioned another amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but hydrophobic amino acid residues such as valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine (M) residue, proline (P) residue, phenylalanine (F) residue, and tryptophan (W) residue, hydrophilic amino acid residues such as glutamine (Q) residue, asparagine (N) residue, serine (S) residue, lysine (K) residue, and glutamic acid (E) residue are preferable, valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine (F) residue, and glutamine (Q) residue are more preferable, and glutamine (Q) residue is even more preferable.

[0057] As a more specific example of the second modified fibroin, there can be mentioned a modified fibroin containing the amino acid sequence represented by 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 with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0058] (2-i) modified fibroin will be described. The amino acid sequence shown by SEQ ID NO: 6 is the one obtained by substituting all GGXs in REP of the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-derived fibroin with GQX. The amino acid sequence shown by SEQ ID NO: 7 is obtained by deleting every other (A) n motif from the amino acid sequence shown by SEQ ID NO: 6 from the N-terminal side to the C-terminal side, and further inserting [(A) n motif-REP] one before the C-terminal sequence. The amino acid sequence shown by SEQ ID NO: 8 is obtained by inserting two alanine residues on the C-terminal side of each (A) n motif of the amino acid sequence shown by SEQ ID NO: 7, further substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the C-terminal side so that the molecular weight becomes almost the same as that of SEQ ID NO: 7. The amino acid sequence shown by SEQ ID NO: 9 is obtained by adding a predetermined hinge sequence and His tag sequence to the C-terminal of the sequence obtained by repeating four times the region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted).

[0059] The value of z / w in the amino acid sequence shown by SEQ ID NO: 10 (corresponding to naturally-derived fibroin) is 46.8%. The values of z / w in the amino acid sequences shown by 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. Also, the values of x / y in the zigzag ratios (described later) of 1:1.8 to 11.3 of the amino acid sequences shown by 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.

[0060] (2-i) modified fibroin may consist of the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0061] (2-ii) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n Motif-REP] m . Preferably, the above sequence identity is 95% or more.

[0062] The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and when the total number of amino acid residues of the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in REP is z, and the total number of amino acid residues of REP in the above domain sequence is w, it is preferable that z / w is 50.9% or more.

[0063] The second modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. Thereby, isolation, immobilization, detection, visualization, etc. of the modified fibroin become possible.

[0064] Examples of the tag sequence include affinity tags that utilize specific affinity (binding property, affinity) with other molecules. Specific examples of the affinity tag include a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and since it has the property of specifically binding to metal ions such as nickel, it can be used for the isolation of the modified fibroin by chelating metal chromatography. Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 11 (an amino acid sequence containing a His tag sequence and a hinge sequence).

[0065] In addition, 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.

[0066] Furthermore, an "epitope tag" utilizing antigen-antibody reaction can also be used. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag, etc. By utilizing the epitope tag, modified fibroin can be easily purified with high specificity.

[0067] Furthermore, those in which the tag sequence can be cleaved with a specific protease can also be used. By protease-treating the protein adsorbed via the tag sequence, modified fibroin with the tag sequence cleaved can also be recovered.

[0068] More specific examples of modified fibroin containing a tag sequence include modified fibroin containing the amino acid sequence represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799), or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0069] The amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are those obtained by adding the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0070] (2-iii) The modified fibroin may consist of an amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0071] (2-iv) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) n Motif-REP] m It is preferred that the above sequence identity is 95% or more.

[0072] (2-iv) The modified fibroin has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and when the total number of amino acid residues of the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in REP is z, and the total number of amino acid residues of REP in the above domain sequence is w, it is preferred that z / w is 50.9% or more.

[0073] 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 according to the type of the host.

[0074] The third modified fibroin has an amino acid sequence in which the content of the (A) n motif is reduced as compared with the naturally-derived fibroin. The domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to the deletion of at least one or more (A) n motifs as compared with the naturally-derived fibroin.

[0075] The third modified fibroin is from the naturally-derived fibroin (A) nIt may also have an amino acid sequence corresponding to a deletion of 10 to 40% of the motif.

[0076] The third modified fibroin has a domain sequence in which at least 1 to 3 (A) n per motif n It may also have an amino acid sequence corresponding to a deletion of the motif.

[0077] The third modified fibroin has a domain sequence in which at least 2 consecutive (A) n motif deletions, and 1 (A) n It may also have an amino acid sequence corresponding to the repeated deletion of the motif in this order.

[0078] The third modified fibroin has a domain sequence in which (A) n motifs are deleted every other one

[0079] The third modified fibroin has a domain sequence represented by Formula 1: [(A) n motif - REP] m and includes, from the N - terminal side to the C - terminal side, when the amino acid residue numbers of the REPs of two adjacent [(A) n motif - REP] units are sequentially compared and the amino acid residue number of the REP with fewer amino acid residues is set to 1, the ratio of the amino acid residue number of the other REP is 1.8 to 11.3. When the sum of the amino acid residue numbers of two adjacent [(A) n motif - REP] units is x and the total amino acid residue number of the domain sequence is y, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) nThe number of alanine residues relative to the total number of amino acid residues in the motif may be 83% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 100% (which means consisting only of alanine residues).

[0080] The calculation method of x / y will be described in more detail with reference to FIG. 1. FIG. 1 shows the domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. The domain sequence is 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 - Third REP (10 amino acid residues) - (A) n Motif - Fourth REP (20 amino acid residues) - (A) n Motif - Fifth REP (30 amino acid residues) - (A) n has a sequence called motif.

[0081] Two adjacent [(A) n Motif - REP] units are sequentially selected from the N-terminal side to the C-terminal side so as not to overlap. At this time, there may be [(A) n Motif - REP] units that are not selected. FIG. 1 shows Pattern 1 (comparison between the first REP and the second REP, and comparison between the third REP and the fourth REP), Pattern 2 (comparison between the first REP and the second REP, and comparison between the fourth REP and the fifth REP), Pattern 3 (comparison between the second REP and the third REP, and comparison between the fourth REP and the fifth REP), and Pattern 4 (comparison between the first REP and the second REP). In addition, there are other selection methods.

[0082] Next, for each pattern, the two adjacent [(A) nCompare the number of amino acid residues of each REP in the [Motif-REP] unit. The comparison is made by obtaining the ratio of the number of amino acid residues of the other REP when the one with fewer amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the second REP is 100 / 50 = 2. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the fifth REP is 30 / 20 = 1.5.

[0083] In FIG. 1, when the one with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 [(A) n The set of [Motif-REP] units is indicated by a solid line. In this specification, this ratio is called the zigzag ratio. When the one with fewer amino acid residues is set to 1, if the ratio of the number of amino acid residues of the other REP is less than 1.8 or greater than 11.3 [(A) n The set of [Motif-REP] units is indicated by a dashed line.

[0084] In each pattern, the two adjacent [(A) indicated by a solid line n Add up all the amino acid residues of the [Motif-REP] units (not only the REPs but also the amino acid residues of the (A) n Motif). Then, compare the added total values, and let the total value (the maximum value of the total values) of the pattern with the largest total value be x. In the example shown in FIG. 1, the total value of Pattern 1 is the largest.

[0085] Next, by dividing x by the total number of amino acid residues y in the domain sequence, x / y (%) can be calculated.

[0086] In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, still 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 limitation on the upper limit of x / y, and it may be, for example, 100% or less. When the giza ratio is 1:1.9 to 11.3, x / y is preferably 89.6% or more. When the giza ratio is 1:1.8 to 3.4, x / y is preferably 77.1% or more. When the giza ratio is 1:1.9 to 8.4, x / y is preferably 75.9% or more. When the giza ratio is 1:1.9 to 4.1, x / y is preferably 64.2% or more.

[0087] When there are a plurality of the third modified fibroins in the domain sequence (A) n When the modified fibroin is such that at least 7 of the motifs are composed of only alanine residues, x / y is preferably 46.4% or more, more preferably 50% or more, still 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 limitation on the upper limit of x / y, and it may be 100% or less.

[0088] Here, x / y in naturally-derived fibroin will be described. First, as described above, when confirmed by the method of exemplifying fibroin whose amino acid sequence information is registered in NCBI GenBank, 663 types of fibroin (among which 415 types are fibroin derived from spiders) were extracted. Among all the extracted fibroins, the formula 1: [(A) n motif-REP] mFrom the amino acid sequence of naturally occurring fibroin composed of domain arrays represented by , x / y was calculated by the above-described calculation method. The results for cases where the crimp ratio is 1:1.9 to 4.1 are shown in FIG. 3. The horizontal axis in FIG. 3 indicates x / y (%), and the vertical axis indicates frequency. As is clear from FIG. 3, x / y in naturally occurring fibroin is all less than 64.2% (the highest is 64.14%).

[0089] The third modified fibroin can be obtained, for example, from the gene sequence of cloned naturally occurring fibroin by deleting one or more sequences encoding the [A] n motif so that x / y becomes 64.2% or more. Also, for example, from the amino acid sequence of naturally occurring fibroin, one or more [A] n amino acid sequences corresponding to the deletion of the motif so that x / y becomes 64.2% or more are designed, and it can also be obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to the deletion of the [A] n motif from the amino acid sequence of naturally occurring fibroin, further modification of the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed.

[0090] More specific examples of the third modified fibroin include (3-i) the amino acid sequence represented by 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) a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0091] The modified fibroin of (3-i) will be described. The amino acid sequence represented by SEQ ID NO: 17 is from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin, skipping every other one from the N-terminal side to the C-terminal side [A] nThe motif is deleted, and [(A) n Motif-REP] is inserted once in front of the C-terminal sequence. The amino acid sequences represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 are as described for the second modified fibroin.

[0092] The value of x / y in the ratio of 1:1.8 to 11.3 of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally derived fibroin) is 15.0%. The values of x / y in the amino acid sequences represented by SEQ ID NO: 17 and SEQ ID NO: 7 are both 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences represented by 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.

[0093] (3-i) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0094] (3-ii) The modified fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing the domain sequence represented by the formula 1: [(A) n Motif-REP] m The above sequence identity is preferably 95% or more.

[0095] (3-ii) The modified fibroin has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and, from the N-terminal side to the C-terminal side, two adjacent [(A) nFor the REP of the motif-REP unit, the amino acid residue numbers are sequentially compared. When the amino acid residue number of the REP with fewer amino acid residues is set to 1, the ratio of the amino acid residue number of the other REP is 1.8 to 11.3 (the zigzag ratio is 1:1.8 to 11.3) for two adjacent [(A) n For the motif-REP unit, when the maximum value of the total value obtained by adding up the amino acid residue numbers is x and the total amino acid residue number of the domain sequence is y, it is preferable that x / y is 64.2% or more.

[0096] The third modified fibroin may contain the above-described tag sequence at either or both of the N-terminus and the C-terminus.

[0097] As a more specific example of the modified fibroin containing the tag sequence, there can be mentioned a modified fibroin containing the amino acid sequence represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) in (3-iii), or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 in (3-iv).

[0098] The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are those obtained by adding the amino acid sequence represented by SEQ ID NO: 11 (His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0099] (3-iii) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0100] (3-iv) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) also has the formula 1:[(A) nMotif-REP m It is a protein containing a domain array represented by the above. The above sequence identity is preferably 95% or more.

[0101] (3-iv) The modified fibroin has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and from the N-terminal side to the C-terminal side, two adjacent [(A) n Motif-REP] When sequentially comparing the amino acid residue numbers of the REPs of the units, when the amino acid residue number of the REP with fewer amino acid residues is set to 1, the ratio of the amino acid residue numbers of the other REP is 1.8 to 11.3. Two adjacent [(A) n Motif-REP] When the maximum value of the total value obtained by adding the amino acid residue numbers of the units is x and the total amino acid residue number of the domain array is y, it is preferably that x / y is 64.2% or more.

[0102] 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 according to the type of the host.

[0103] The fourth modified fibroin has an amino acid sequence in which the content of (A) n Motif is reduced, and in addition, the content of glycine residues is reduced. The domain array of the fourth modified fibroin has at least one or more (A) n Motif is deleted, and in addition, it can be said that it has an amino acid sequence corresponding to the substitution of at least one or more glycine residues in REP with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the second modified fibroin and the third modified fibroin described above. Specific embodiments and the like are as described for the second modified fibroin and the third modified fibroin.

[0104] As a more specific example of the fourth modified fibroin, (4-i) an amino acid sequence represented by 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) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 can be mentioned as the modified fibroin. Specific embodiments of the modified fibroin containing the amino acid sequence represented by 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.

[0105] The fifth modified fibroin may have an amino acid sequence containing a region with a locally large hydrophobicity index, where its domain sequence has one or more amino acid residues in the REP replaced with amino acid residues having a large hydrophobicity index compared to naturally occurring fibroin, and / or one or more amino acid residues with a large hydrophobicity index inserted into the REP.

[0106] The region with a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

[0107] The amino acid residues with a large hydrophobicity index mentioned above are more preferably amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A).

[0108] The fifth modified fibroin, compared with naturally-derived fibroin, in addition to the modification corresponding to substitution of one or more amino acid residues in the REP with amino acid residues having a large hydrophobicity index and / or insertion of one or more amino acid residues having a large hydrophobicity index into the REP, may further have an amino acid sequence modification corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues.

[0109] The fifth modified fibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP with hydrophobic amino acid residues (for example, amino acid residues having a positive hydrophobicity index) from the gene sequence of cloned naturally-derived fibroin, and / or inserting one or more hydrophobic amino acid residues into the REP. Also, for example, it can be obtained by designing an amino acid sequence corresponding to substitution of one or more hydrophilic amino acid residues in the REP with hydrophobic amino acid residues and / or insertion of one or more hydrophobic amino acid residues into the REP from the amino acid sequence of naturally-derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to substitution of one or more hydrophilic amino acid residues in the REP with hydrophobic amino acid residues and / or insertion of one or more hydrophobic amino acid residues into the REP from the amino acid sequence of naturally-derived fibroin, an amino acid sequence modification corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be further performed.

[0110] The fifth modified fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m and in all REPs contained in the sequence obtained by removing the sequence from the C-terminal most (A) n motif to the C-terminal of the above domain sequence from the above domain sequence, when the total number of amino acid residues contained in a region where the average value of the hydrophobicity indices of four consecutive amino acid residues is 2.6 or more is p, and the C-terminal most (A) nWhen the total number of amino acid residues contained in the sequence obtained by removing the sequence from the motif to the C-terminus of the above domain sequence from the above domain sequence is defined as q, it may have an amino acid sequence in which p / q is 6.2% or more.

[0111] Regarding the hydrophobicity index of amino acid residues, 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) is used. Specifically, the hydrophobicity index (hydrophathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

[0112]

Table 1

[0113] The calculation method of p / q will be explained in more detail. For the calculation, formula 1: [(A) n motif - REP] m From the domain sequence represented by, the sequence (hereinafter referred to as “sequence A”) obtained by removing the sequence from the (A) n motif at the most C-terminal side to the C-terminus of the domain sequence is used. First, for all REPs contained in sequence A, the average value of the hydrophobicity indices of consecutive 4 amino acid residues is calculated. The average value of the hydrophobicity indices is obtained by dividing the sum of the HIs of each amino acid residue contained in the consecutive 4 amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity indices is obtained for all consecutive 4 amino acid residues (each amino acid residue is used 1 - 4 times for calculating the average value). Next, a region where the average value of the hydrophobicity indices of consecutive 4 amino acid residues is 2.6 or more is specified. Even if an amino acid residue belongs to a plurality of “consecutive 4 amino acid residues where the average value of the hydrophobicity indices is 2.6 or more”, it is included as 1 amino acid residue in the region. And the total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

[0114] For example, when 20 "consecutive 4 - amino - acid residues with an average hydrophobicity index of 2.6 or more" are extracted (without duplication), in the region where the average hydrophobicity index of consecutive 4 - amino - acid residues is 2.6 or more, there are 20 consecutive 4 - amino - acid residues (without duplication), and p is 20×4 = 80. Also, for example, when two "consecutive 4 - amino - acid residues with an average hydrophobicity index of 2.6 or more" exist with only 1 amino - acid residue overlapping, in the region where the average hydrophobicity index of consecutive 4 - amino - acid residues is 2.6 or more, 7 amino - acid residues are included (p = 2×4−1 = 7. The "-1" is to subtract the overlapping part). For example, in the case of the domain sequence shown in FIG. 4, since there are 7 "consecutive 4 - amino - acid residues with an average hydrophobicity index of 2.6 or more" without overlap, p is 7×4 = 28. Also, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50+4 + 40+4 + 10+4 + 20+4 + 30 = 170 (not including the (A) motif present at the end on the C - terminal side). n Next, by dividing p by q, p / q(%) can be calculated. In the case of FIG. 4, it is 28 / 170 = 16.47%.

[0115] In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, still 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 for example, it may be 45% or less.

[0116] The fifth modified fibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (for example, amino acid residues with a negative hydrophobicity index) in the REP with hydrophobic amino acid residues (for example, amino acid residues with a positive hydrophobicity index) so as to satisfy the above p / q conditions in the amino acid sequence of the cloned naturally-derived fibroin, and / or inserting one or more hydrophobic amino acid residues into the REP, thereby modifying it into an amino acid sequence containing a region with a locally large hydrophobicity index. Further, for example, it can also be obtained by designing an amino acid sequence that satisfies the above p / q conditions from the amino acid sequence of naturally-derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to the substitution of one or more amino acid residues in the REP with amino acid residues having a large hydrophobicity index and / or the insertion of one or more amino acid residues having a large hydrophobicity index into the REP as compared with naturally-derived fibroin, modifications corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further performed.

[0117] The amino acid residues with a large hydrophobicity index are not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are preferable, and valine (V), leucine (L) and isoleucine (I) are more preferable.

[0118] More specific examples of the fifth modified fibroin include (5-i) the amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), or (5-ii) a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0119] The modified fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 19 is obtained by inserting, at every other REP, an amino acid sequence (VLI) consisting of 3 amino acid residues at two positions, excluding the domain sequence at the terminal on the C-terminal side, with respect to the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410), further substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the C-terminal side. The amino acid sequence shown in SEQ ID NO: 20 is obtained by inserting, at every other REP, an amino acid sequence (VLI) consisting of 3 amino acid residues at one position, with respect to 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, at every other REP, an amino acid sequence (VLI) consisting of 3 amino acid residues at two positions, with respect to the amino acid sequence shown in SEQ ID NO: 8.

[0120] The modified fibroin of (5-i) may consist of the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0121] 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) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m Preferably, the above sequence identity is 95% or more.

[0122] The modified fibroin of (5-ii) has 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, and in all REPs contained in the sequence obtained by removing the sequence from the (A) n motif at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence, when the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in the region containing the total number of amino acid residues p, and the (A) nWhen the total number of amino acid residues contained in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence is defined as q, it is preferable that p / q is 6.2% or more.

[0123] The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.

[0124] More specific examples of the modified fibroin containing a tag sequence include (5-iii) an amino acid sequence represented by 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 with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0125] The amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 are obtained by adding the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0126] (5-iii) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0127] (5-iv) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. (5-iv) The modified fibroin is also a protein containing a domain sequence represented by the formula 1: [(A) n Motif-REP] m The above sequence identity is preferably 95% or more.

[0128] (5-iv) The modified fibroin has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and the (A) located on the most C-terminal siden In all REPs contained in the sequence obtained by removing 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 where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the most C-terminal (A) n When the total number of amino acid residues contained in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence is defined as q, it is preferable that p / q is 6.2% or more.

[0129] 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 according to the type of the host.

[0130] The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues as compared with naturally-derived fibroin.

[0131] The sixth modified fibroin preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

[0132] When the sixth modified fibroin contains the GPGXX motif in REP, the GPGXX motif content rate is usually 1% or more, may be 5% or more, and is preferably 10% or more. There is no particular limitation on the upper limit of the GPGXX motif content rate, which may be 50% or less, or may be 30% or less.

[0133] In the present specification, the "GPGXX motif content rate" is a value calculated by the following method. Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) nIn fibroin (modified fibroin or fibroin derived from nature) containing a domain array represented by a motif, the one located on the most C-terminal side (A) n In all REPs included in the array obtained by removing the array from the motif to the C-terminus of the domain array from the domain array, let s be the number obtained by multiplying the total number of GPGXX motifs contained in that region by 3 (i.e., corresponding to the total number of G and P in the GPGXX motif), and the one located on the most C-terminal side (A) n Remove the array from the motif to the C-terminus of the domain array from the domain array, and further (A) n When the total number of amino acid residues of all REPs excluding the motif is t, the GPGXX motif content rate is calculated as s / t.

[0134] In the calculation of the GPGXX motif content rate, the "array obtained by removing the array from the motif to the C-terminus of the domain array from the domain array" for the "one located on the most C-terminal side (A) n is targeted because the "array from the motif located on the most C-terminal side to the C-terminus of the domain array" (the array corresponding to REP) may contain an array with low correlation with the characteristic array of fibroin, and when m is small (that is, when the domain array is short), it affects the calculation result of the GPGXX motif content rate. Therefore, this influence is excluded. In addition, when the "GPGXX motif" is located at the C-terminus of REP, even if "XX" is, for example, "AA", it is treated as the "GPGXX motif". n Figure 5 is a schematic diagram showing the domain array of modified fibroin. The method for calculating the GPGXX motif content rate will be specifically described with reference to Figure 5. First, in the domain array of the modified fibroin shown in Figure 5 (the type of "[(A)

[0135] motif-REP] n -(A) m motif"). All REPs are the ones located on the most C-terminal side (A) n n ​Since it is included in the "sequence obtained by removing the sequence from the motif to the C-terminus of the domain array from the domain array" (the sequence indicated by "Region A" in Fig. 5), the number of GPGXX motifs for calculating s is 7, and s becomes 7 × 3 = 21. Similarly, for all REPs, "the one located closest to the C-terminus (A)" n Since it is included in the "sequence obtained by removing the sequence from the motif to the C-terminus of the domain array from the domain array" (the sequence indicated by "Region A" in Fig. 5), further from this sequence (A) n When the total number t of amino acid residues of all REPs excluding the (A) motif is 50 + 40 + 10 + 20 + 30 = 150. Next, by dividing s by t, s / t(%) can be calculated, and in the case of the modified fibroin in Fig. 5, it is 21 / 150 = 14.0%.

[0136] The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%.

[0137] In this 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 fibroin (modified fibroin or naturally-derived fibroin) containing a domain array represented by the (A) motif, for all REPs included in the sequence obtained by removing the sequence from the (A) motif located closest to the C-terminus to the C-terminus of the domain array from the domain array (the sequence corresponding to "Region A" in Fig. 5), when the total number of glutamine residues contained in that region is u, and the total number of amino acid residues of all REPs obtained by removing the sequence from the (A) motif located closest to the C-terminus to the C-terminus of the domain array and further removing the (A) n motif is t, the glutamine residue content is calculated as u / t. In the calculation of the glutamine residue content, the "(A) n motif located closest to the C-terminus n When removing the sequence from the (A) motif located closest to the C-terminus to the C-terminus of the domain array from the domain array and further removing the (A) motif, and using t as the total number of amino acid residues of all REPs, the glutamine residue content is calculated as u / t. In the calculation of the glutamine residue content, the "(A) motif located closest to the C-terminus"n The reason for targeting the "sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as the reason described above.

[0138] The sixth modified fibroin may have an amino acid sequence corresponding to the deletion of one or more glutamine residues in the REP or substitution with other amino acid residues in its domain sequence as compared with the naturally-derived fibroin.

[0139] "Other amino acid residues" may be any amino acid residues other than glutamine residues, but are preferably amino acid residues having a larger hydrophobicity index than glutamine residues. The hydrophobicity index of amino acid residues is as shown in Table 1.

[0140] As shown in Table 1, examples of amino acid residues having a larger 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 preferable, and amino acid residues selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) are even more preferable.

[0141] For the sixth modified fibroin, the hydrophobicity degree of the REP is preferably more than -0.8, 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 limitation on the upper limit of the hydrophobicity degree of the REP, and it may be 1.0 or less, or may be 0.7 or less.

[0142] In this specification, the "hydrophobicity degree 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 fibroin (modified fibroin or naturally-derived fibroin) containing a domain sequence represented by a motif, for all REPs contained in the sequence obtained by removing the sequence from the most C-terminal (A) n motif to the C-terminus of the domain sequence from the domain sequence (the sequence corresponding to "Region A" in Fig. 5), when the sum of the hydrophobicity indices of each amino acid residue in that region is v, and for all REPs after removing the sequence from the most C-terminal (A) n motif to the C-terminus of the domain sequence from the domain sequence and further removing the (A) n motif, when the total number of amino acid residues of all REPs is t, the hydrophobicity degree of REP is calculated as v / t. In the calculation of the hydrophobicity degree of REP, the reason for targeting "the sequence obtained by removing the sequence from the most C-terminal (A) n motif to the C-terminus of the domain sequence from the domain sequence" is the same as the reason described above.

[0143] The sixth modified fibroin may have an amino acid sequence modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues in addition to the modification corresponding to deletion of one or more glutamine residues in REP and / or substitution of one or more glutamine residues in REP with other amino acid residues, as compared with naturally-derived fibroin in its domain sequence.

[0144] The sixth modified fibroin can be obtained, for example, by deleting one or more glutamine residues in the REP from the cloned gene sequence of naturally-derived fibroin and / or substituting one or more glutamine residues in the REP with other amino acid residues. Also, for example, it can be obtained by designing an amino acid sequence corresponding to deletion of one or more glutamine residues in the REP and / or substitution of one or more glutamine residues in the REP with other amino acid residues from the amino acid sequence of naturally-derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0145] More specific examples of the sixth modified fibroin include (6-i) a modified fibroin containing an amino acid sequence represented by 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) a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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.

[0146] The modified fibroin of (6-i) will be described. The amino acid sequence represented by SEQ ID NO: 25 is the one in which all QQ in the amino acid sequence (Met-PRT410) represented by SEQ ID NO: 7 are replaced with VL. The amino acid sequence represented by SEQ ID NO: 26 is the one in which all QQ in the amino acid sequence represented by SEQ ID NO: 7 are replaced with TS, and the remaining Qs are replaced with A. The amino acid sequence represented by SEQ ID NO: 27 is the one in which all QQ in the amino acid sequence represented by SEQ ID NO: 7 are replaced with VL, and the remaining Qs are replaced with I. The amino acid sequence represented by SEQ ID NO: 28 is the one in which all QQ in the amino acid sequence represented by SEQ ID NO: 7 are replaced with VI, and the remaining Qs are replaced with L. The amino acid sequence represented by SEQ ID NO: 29 is the one in which all QQ in the amino acid sequence represented by SEQ ID NO: 7 are replaced with VF, and the remaining Qs are replaced with I.

[0147] The amino acid sequence represented by SEQ ID NO: 30 is the one in which all QQ in the amino acid sequence (Met-PRT525) represented by SEQ ID NO: 8 are replaced with VL. The amino acid sequence represented by SEQ ID NO: 31 is the one in which all QQ in the amino acid sequence represented by SEQ ID NO: 8 are replaced with VL, and the remaining Qs are replaced with I.

[0148] The amino acid sequence represented by SEQ ID NO: 32 is the one in which all QQ in the sequence obtained by repeating the region of 20 domain sequences present in the amino acid sequence (Met-PRT410) represented by SEQ ID NO: 7 twice are replaced with VF, and the remaining Qs are replaced with I.

[0149] The amino acid sequence (Met-PRT917) represented by SEQ ID NO: 41 is the one in which all QQ in the amino acid sequence represented by SEQ ID NO: 7 are replaced with LI, and the remaining Qs are replaced with V. The amino acid sequence (Met-PRT1028) represented by SEQ ID NO: 42 is the one in which all QQ in the amino acid sequence represented by SEQ ID NO: 7 are replaced with IF, and the remaining Qs are replaced with T.

[0150] The amino acid sequences represented by 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 rate of 9% or less (Table 2).

[0151]

Table 2

[0152] (6-i) The modified fibroin may consist of the amino acid sequence represented by 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.

[0153] (6-ii) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) n motif. The above sequence identity is preferably 95% or more.

[0154] (6-ii) The modified fibroin preferably has a glutamine residue content rate of 9% or less. Also, the modified fibroin of (6-ii) preferably has a GPGXX motif content rate of 10% or more.

[0155] The sixth modified fibroin may contain a tag sequence at either one or both of the N-terminus and the C-terminus. Thereby, isolation, immobilization, detection, visualization, etc. of the modified fibroin become possible.

[0156] As a more specific example of the sixth modified fibroin containing a tag array, a modified fibroin containing an amino acid sequence represented by 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) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 can be mentioned.

[0157] The amino acid sequences represented by 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 each the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) added to the N-terminus of the amino acid sequences represented by 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. Since only the tag sequence is added to the N-terminus, there is no change in the glutamine residue content rate, and the amino acid sequences represented by 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 rate of 9% or less (Table 3).

[0158]

Table 3

[0159] (6-iii) The modified fibroin may consist of an amino acid sequence represented by 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.

[0160] (6-iv) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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) is also a protein containing a domain sequence represented by Formula 1: [(A) n Motif-REP] m or Formula 2: [(A) n Motif-REP] m -(A) n Motif. The above sequence identity is preferably 95% or more.

[0161] (6-iv) The modified fibroin preferably has a glutamine residue content of 9% or less. Also, the modified fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.

[0162] 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 set according to the type of the host.

[0163] The modified fibroin may be a modified fibroin having at least two or more of the characteristics possessed by 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.

[0164] The modified fibroin may be a hydrophilic modified fibroin or a hydrophobic modified fibroin. The hydrophobic modified fibroin is a modified fibroin in which the sum of the hydrophobicity indices (HI) of all amino acid residues constituting the modified fibroin is determined, and then the value obtained by dividing the sum by the total number of amino acid residues (average HI) is more than -0.8. It is more preferable that the modified fibroin has an average HI of -0.6 or more, more preferably an average HI of -0.4 or more, still more preferably an average HI of -0.2 or more, and particularly preferably an average HI of 0 or more. The hydrophobicity index is as shown in Table 1. The hydrophilic spider silk protein is a modified fibroin having an average HI of -0.8 or less as described above. The average hydrophobicity index of the modified fibroin according to the present embodiment is preferably more than -0.8, preferably -0.7 or more, preferably -0.6 or more, more preferably -0.5 or more, preferably -0.4 or more, preferably -0.3 or more, preferably -0.2 or more, preferably -0.1 or more, more preferably 0 or more, more preferably 0.1 or more, more preferably 0.2 or more, still more preferably 0.3 or more, and particularly preferably 0.4 or more.

[0165] Examples of the hydrophobic modified fibroin include, for example, the above-described sixth modified fibroin. More specific examples of the hydrophobic modified fibroin include amino acid sequences represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43, and modified fibroins containing amino acid sequences represented by 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.

[0166] Examples of the hydrophilic modified fibroin include, for example, the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, and the fifth modified fibroin described above. More specific examples of the hydrophilic modified fibroin include the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15, and modified fibroin containing the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0167] The modified fibroin according to the present embodiment may contain only one type of modified fibroin, or may contain a combination of two or more types of modified fibroin. Further, it may contain a combination of a modified fibroin and a structural protein other than the modified fibroin.

[0168] The structural protein may be a polypeptide derived from the above natural-type structural protein, that is, a recombinant polypeptide.

[0169] Examples of the structural protein derived from collagen include, for example, Formula 3: [REP2] pA protein containing a domain array represented by the following (wherein in Formula 3, p represents an integer of 5 to 300; REP2 represents an amino acid sequence composed of Gly-X-Y, and X and Y represent any amino acid residues other than Gly; a plurality of REP2s may have the same amino acid sequence or different amino acid sequences from each other) can be mentioned. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 45 can be mentioned. The amino acid sequence represented by SEQ ID NO: 45 is the amino acid sequence from the 301st residue to the 540th residue corresponding to the repeat part and motif of a partial sequence of human collagen type 4 (NCBI GenBank accession number: CAA56335.1, GI: 3702452) obtained from the NCBI database, with the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) added to the N-terminus.

[0170] As a structural protein derived from resilin, for example, Formula 4: [REP3] q A protein containing a domain array represented by the following (wherein in Formula 4, q represents an integer of 4 to 300; REP3 represents an amino acid sequence composed of Ser-J-J-Tyr-Gly-U-Pro; J represents any amino acid residue, and is preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr; U represents any amino acid residue, and is preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser; a plurality of REP3s may have the same amino acid sequence or different amino acid sequences from each other) can be mentioned. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 46 can be mentioned. The amino acid sequence represented by SEQ ID NO: 46 is the amino acid sequence from the 19th residue to the 321st residue of the sequence obtained by substituting Thr at the 87th residue with Ser and Asn at the 95th residue with Asp in the amino acid sequence of resilin (NCBI GenBank accession number NP 611157, Gl: 24654243), with the amino acid sequence represented by SEQ ID NO: 49 (tag sequence and hinge sequence) added to the N-terminus.

[0171] Examples of elastin-derived structural proteins include proteins having amino acid sequences such as accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (cow) in NCBI's GenBank. Specifically, proteins containing the amino acid sequence shown in SEQ ID NO: 47 can be mentioned. The amino acid sequence shown in SEQ ID NO: 47 is the amino acid sequence shown in SEQ ID NO: 11 (tag sequence and hinge sequence) added to the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of accession number AAC98395 in NCBI's GenBank.

[0172] Examples of keratin-derived structural proteins include, for example, type I keratin of Capra hircus. Specifically, proteins containing the amino acid sequence shown in SEQ ID NO: 48 (the amino acid sequence of accession number ACY30466 in NCBI's GenBank) can be mentioned.

[0173] Method for producing recombinant structural protein Any of the recombinant structural proteins according to the above embodiments can be produced, for example, by expressing the nucleic acid in a host transformed with an expression vector having a nucleic acid sequence encoding the recombinant structural protein and one or more regulatory sequences operably linked to the nucleic acid sequence. Hereinafter, modified fibroin will be described as an example.

[0174] The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, using the gene encoding natural fibroin, it can be amplified and cloned by polymerase chain reaction (PCR) or the like, and then modified by genetic engineering techniques, or the nucleic acid can be produced by a chemically synthesizing method. The chemical synthesis method of the nucleic acid is also not particularly limited. For example, based on the amino acid sequence information of fibroin obtained from the NCBI web database or the like, the gene can be chemically synthesized by a method of ligating oligonucleotides automatically synthesized by AKTA oligopilot plus 10 / 100 (GE Healthcare Japan Co., Ltd.) or the like by PCR or the like. At this time, in order to facilitate the purification and / or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin consisting of an amino acid sequence with an amino acid sequence consisting of a start codon and a His10 tag added to the N-terminus of the above amino acid sequence may be synthesized.

[0175] The regulatory sequence is a sequence that controls the expression of the modified fibroin in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected according to the type of the host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the modified fibroin may be used. The inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

[0176] The type of the expression vector can be appropriately selected according to the type of the host, such as a plasmid vector, a viral vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, etc. As the expression vector, those that can autonomously replicate in the host cell or can be integrated into the host chromosome and contain a promoter at a position where the nucleic acid encoding the modified fibroin can be transcribed are preferably used.

[0177] As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.

[0178] Preferred examples of the prokaryotic host 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. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

[0179] When using a prokaryote as the host, examples of the vector for introducing the nucleic acid encoding the modified fibroin include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238569), and the like.

[0180] Examples of eukaryotic hosts include yeast and filamentous fungi (such as molds). Examples of yeast include yeast belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces, etc. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, etc.

[0181] When using a eukaryote as the host, examples of vectors for introducing nucleic acids encoding modified fibroin include YEP13 (ATCC37115), YEp24 (ATCC37051), etc. As methods for introducing the expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, methods using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, spheroplast method, protoplast method, lithium acetate method, competent method, etc. can be mentioned.

[0182] As methods for expressing nucleic acids by a host transformed with an expression vector, in addition to direct expression, secretion production, fusion protein expression, etc. can be carried out according to methods described in Molecular Cloning, 2nd Edition, etc.

[0183] Modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, generating and accumulating the modified fibroin in the culture medium, and collecting it from the culture medium. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

[0184] When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, as the culture medium, either a natural medium or a synthetic medium can be used as long as it contains a carbon source, nitrogen source, inorganic salts, etc. that the host can assimilate and can efficiently culture the host.

[0185] As the carbon source, any substance that can be assimilated by the above-mentioned transformed microorganism may be used. 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 can be used. As the nitrogen source, for example, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and their digests can be used. As the inorganic salts, for example, potassium dihydrogen phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

[0186] The culture of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out, for example, under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 °C. The culture time is usually 16 hours to 7 days. It is preferable to maintain the pH of the culture medium during culture at 3.0 to 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0187] Also, during the culture, if necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium. When culturing a microorganism transformed with an expression vector using an inducible promoter as the promoter, an inducer may be added to the medium if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium, and when culturing a microorganism transformed with an expression vector using the trp promoter, indoleacrylic acid or the like may be added to the medium.

[0188] Isolation and purification of the expressed modified fibroin can be carried out by commonly used methods. For example, when the modified fibroin is expressed in a dissolved state in cells, after the completion of the culture, the host cells are recovered by centrifugation, suspended in an aqueous buffer solution, and then the host cells are disrupted by an ultrasonic crusher, a French press, a Manton Gaulin homogenizer, a Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, methods commonly used for protein isolation and purification, namely, solvent extraction method, salting-out method using ammonium sulfate, etc., desalting method, precipitation method using an organic solvent, anion exchange chromatography method using resins such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography method using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography method using resins such as butyl Sepharose, phenyl Sepharose, gel filtration method using a molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing electrophoresis, etc. can be used alone or in combination to obtain a purified preparation.

[0189] In addition, when the modified fibroin is expressed by forming an insoluble body in cells, similarly, after recovering the host cells, disrupting them, and performing centrifugation, the insoluble body of the modified fibroin is recovered as a precipitate fraction. The recovered insoluble body of the modified fibroin can be solubilized with a protein denaturant. After this operation, a purified preparation of the modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.

[0190] 〔Spinning dope〕 The spinning dope according to this embodiment contains a recombinant structural protein (e.g., modified fibroin) and a solvent. Hereinafter, as an example of the recombinant structural protein, the spinning dope containing modified fibroin will be described.

[0191] Any solvent that can dissolve modified fibroin can be used as the solvent of the spinning dope according to this embodiment, and for example, an organic solvent can be mentioned. Examples of the organic solvent include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl-2-imidazolidinone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine N-oxide (NMO), and formic acid. From the viewpoint of better solubility of modified fibroin, HFIP, DMSO, and formic acid are more preferable, and DMSO and formic acid are even more preferable. These organic solvents may contain water. These solvents may be used alone or in combination of two or more.

[0192] When the total amount of the spinning dope is 100% by weight, the concentration of modified fibroin in the spinning dope according to this embodiment is preferably 10 to 50% by weight, more preferably 10 to 40% by weight, more preferably 15 to 40% by weight, more preferably 15 to 35% by weight, more preferably 20 to 35% by weight, more preferably 25 to 35% by weight, even more preferably 27 to 33% by weight, and particularly preferably 28 to 32% by weight. When the concentration of modified fibroin is 10% by weight or more, the productivity is further improved. When the concentration of modified fibroin is 50% by weight or less, the spinning dope can be discharged more stably from the spinneret, and the productivity is improved.

[0193] In the spinning dope according to this embodiment, an inorganic salt may be added as necessary. The inorganic salt can function as a dissolution accelerator for the modified fibroin. Examples of the inorganic salt include alkali metal halides, alkaline earth metal halides, and alkaline earth metal nitrates. Specific examples of the inorganic salt include lithium carbonate, lithium chloride, calcium chloride, calcium nitrate, lithium bromide, barium bromide, calcium bromide, barium chlorate, sodium perchlorate, lithium perchlorate, barium perchlorate, calcium perchlorate, and magnesium perchlorate. At least one of these inorganic salts may be added to the solvent.

[0194] The method for preparing the spinning dope according to this embodiment is not particularly limited, and the modified fibroin and the solvent may be mixed in any order. The spinning dope may be stirred or shaken for a certain period of time to promote dissolution. At that time, the spinning dope may be heated to a temperature at which it can be dissolved according to the modified fibroin and the solvent used as necessary. The spinning dope may be heated to, for example, 30°C or higher, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, or 90°C or higher. From the viewpoint of further preventing the decomposition of the modified fibroin, 40°C is preferable. The upper limit of the heating temperature is, for example, below the boiling point of the solvent.

[0195] The viscosity of the spinning dope according to this embodiment may be appropriately set according to the use of the fiber, the spinning method, etc. For example, at 20°C, it may be 60,000 to 130,000 mPa·sec, and may be 65,000 to 125,000 mPa·sec. Also, for example, at 35°C, it may be 500 to 35,000 mPa·sec, may be 1,000 to 35,000 mPa·sec, may be 3,000 to 30,000 mPa·sec, may be 500 to 20,000 mPa·sec, may be 500 to 15,000 mPa·sec, may be 1,000 to 15,000 mPa·sec, may be 1,000 to 12,000 mPa·sec, may be 1,500 to 12,000 mPa·sec, may be 1,500 to 10,000 mPa·sec, may be 1,500 to 8,000 mPa·sec, etc. Also, for example, at 40°C, it may be 500 to 35,000 mPa·sec, may be 1,000 to 35,000 mPa·sec, may be 5,000 to 35,000 mPa·sec, may be 10,000 to 30,000 mPa·sec, may be 5,000 to 20,000 mPa·sec, may be 8,000 to 20,000 mPa·sec, may be 9,000 to 18,000 mPa·sec, may be 9,000 to 16,000 mPa·sec, may be 10,000 to 15,000 mPa·sec, may be 12,000 to 30,000 mPa·sec, may be 12,000 to 28,000 mPa·sec, may be 12,000 to 18,000 mPa·sec, may be 12,000 to 16,000 mPa·sec, etc. The viscosity of the spinning dope can be measured, for example, using a product named "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

[0196] 〔Coagulating liquid〕 As the coagulating liquid according to this embodiment, any solvent that can remove the solvent may be used. For example, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, ketones such as acetone, water, or an aqueous solution having a pH of 0.25 or more and 10.00 or less can be mentioned. The above solvents may be appropriately combined and used as a mixed solvent.

[0197] The solidifying liquid according to one embodiment mainly contains water or an aqueous solution with a pH of 0.25 or more and 10.00 or less. This makes it possible to provide a method for producing protein fibers with reduced production costs and environmental impact. The aqueous solution may be an aqueous salt solution, an aqueous acid solution, or a mixed solution of an aqueous salt solution and an aqueous acid solution, may be an aqueous salt solution or a mixed solution of an aqueous salt solution and an aqueous acid solution, or may be an aqueous salt solution. Here, the mixed solution of an aqueous salt solution and an aqueous acid solution is not limited to a solution obtained by mixing an aqueous salt solution and an aqueous acid solution, and also includes a solution obtained by mixing an acid into an aqueous salt solution, a solution obtained by mixing a salt into an aqueous acid solution, and a solution obtained by dissolving a salt and an acid in water.

[0198] (Aqueous acid solution) Examples of the aqueous acid solution include aqueous solutions of carboxylic acids and the like. Specific examples of carboxylic acids include formic acid, acetic acid, propionic acid, citric acid, and oxalic acid. These solvents may be used alone or in combination of two or more to form an aqueous solution. For example, the aqueous acid solution may be an aqueous citric acid solution or an aqueous formic acid solution.

[0199] (Aqueous salt solution) Examples of the aqueous salt solution include aqueous salt solutions of organic salts or inorganic salts, and mixed aqueous solutions of organic salts and inorganic salts.

[0200] Examples of organic salts include carboxylates, and specific examples of carboxylates include formates, acetates, propionates, citrates, and oxalates. For example, the organic salt may be a formate, acetate, or citrate.

[0201] Specific examples of formates include, for example, ammonium formate, potassium formate, sodium formate, lithium formate, magnesium formate, and calcium formate.

[0202] Specific examples of acetates include, for example, ammonium acetate, potassium acetate, sodium acetate, lithium acetate, magnesium acetate, and calcium acetate.

[0203] Specific examples of the propionate include, for example, ammonium propionate, potassium propionate, sodium propionate, lithium propionate, magnesium propionate, and calcium propionate, etc.

[0204] Specific examples of the citrate include ammonium citrate, potassium citrate, sodium citrate, lithium citrate, magnesium citrate, and calcium citrate, etc. For example, the citrate may contain at least one selected from the group consisting of ammonium citrate, potassium citrate, sodium citrate, magnesium citrate, and calcium citrate, may contain at least one selected from the group consisting of ammonium citrate, potassium citrate, and sodium citrate, may contain at least one selected from the group consisting of potassium citrate and sodium citrate, and may be sodium citrate.

[0205] Specific examples of the oxalate include ammonium oxalate, potassium oxalate, sodium oxalate, lithium oxalate, magnesium oxalate, and calcium oxalate, etc. As the carboxylate, sodium carboxylate is more preferable, and specific examples of the sodium carboxylate include sodium formate, sodium acetate, sodium propionate, and sodium oxalate, etc.

[0206] Specific examples of the inorganic salt include normal salts, acid salts, and basic salts.

[0207] Specific examples of the normal salt include sulfates, chlorides, nitrates, iodide salts, thiocyanates, and carbonates, etc.

[0208] Specific examples of sulfates include, for example, ammonium sulfate, potassium sulfate, sodium sulfate, lithium sulfate, magnesium sulfate, and calcium sulfate, etc. For example, the sulfate may contain at least one selected from the group consisting of ammonium sulfate, sodium sulfate, magnesium sulfate, and calcium sulfate, may contain at least one selected from the group consisting of ammonium sulfate and sodium sulfate, and may be sodium sulfate.

[0209] Specific examples of chlorides include, for example, ammonium chloride, potassium chloride, sodium chloride, lithium chloride, magnesium chloride, and calcium chloride, etc. For example, the chloride may contain at least one selected from the group consisting of ammonium chloride, potassium chloride, sodium chloride, lithium chloride, calcium chloride, and magnesium chloride, may contain at least one selected from the group consisting of potassium chloride, sodium chloride, and calcium chloride, may contain at least one selected from the group consisting of sodium chloride and calcium chloride, and may be sodium chloride.

[0210] Specific examples of nitrates include, for example, ammonium nitrate, potassium nitrate, sodium nitrate, lithium nitrate, magnesium nitrate, and calcium nitrate, etc.

[0211] Specific examples of iodide salts include, for example, ammonium iodide, potassium iodide, sodium iodide, lithium iodide, magnesium iodide, and calcium iodide, etc.

[0212] Specific examples of thiocyanates include, for example, ammonium thiocyanate, potassium thiocyanate, sodium thiocyanate, lithium thiocyanate, magnesium thiocyanate, calcium thiocyanate, guanidine thiocyanate, etc.

[0213] Specific examples of carbonates include, for example, ammonium carbonate, potassium carbonate, sodium carbonate, lithium carbonate, magnesium carbonate, calcium carbonate, and the like.

[0214] Specific examples of acid salts include bisulfates, hydrogen phosphates, hydrogen carbonates, and the like.

[0215] Specific examples of bisulfates include, for example, ammonium bisulfate, potassium bisulfate, sodium bisulfate, lithium bisulfate, magnesium bisulfate, calcium bisulfate, and the like.

[0216] Specific examples of hydrogen phosphates include, for example, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, diammonium hydrogen phosphate, magnesium dihydrogen phosphate, dimagnesium hydrogen phosphate, calcium dihydrogen phosphate, and dicalcium hydrogen phosphate, and the like.

[0217] Specific examples of hydrogen carbonates include, for example, ammonium hydrogen carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, lithium hydrogen carbonate, lithium hydrogen carbonate, magnesium hydrogen carbonate, and calcium hydrogen carbonate, and the like.

[0218] Specific examples of basic salts include calcium hydroxychloride, magnesium hydroxychloride, and the like.

[0219] The above acids, acid aqueous solutions, salts, and salt aqueous solutions may be used alone or in combination of two or more.

[0220] Examples of the mixed salt aqueous solution obtained by mixing two or more salts or salt aqueous solutions include the mixed aqueous solution of the above organic salts, the mixed aqueous solution of the above inorganic salts, the mixed aqueous solution of the above organic salts and inorganic salts, and the like. From the viewpoint of reducing the production cost, brackish water and seawater are particularly preferred. Brackish water and seawater are known to mainly contain potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, and calcium sulfate.

[0221] The solidifying liquid preferably contains an aqueous salt solution, and more preferably is an aqueous salt solution. By containing salt, the solvent removal rate can be further improved. The salt more preferably contains at least one kind selected from the group consisting of carboxylates, sulfates, chlorides, hydrogen phosphates, and hydrogen carbonates, still more preferably contains at least one kind selected from the group consisting of carboxylates, sulfates, and chlorides, still more preferably contains at least one kind selected from the group consisting of sulfates and chlorides, and particularly preferably contains a sulfate. By containing these salts, the fiber-forming ability can be further improved, and the elongation of the obtained fiber can be further improved.

[0222] As the carboxylate, sodium carboxylate is more preferable, as the sulfate, ammonium sulfate, sodium sulfate, magnesium sulfate, and calcium sulfate are more preferable, as the chloride, potassium chloride, sodium chloride, magnesium chloride, and calcium chloride are more preferable, as the hydrogen carbonate, sodium hydrogen carbonate is more preferable, and as the mixed aqueous solution, brackish water and seawater are particularly preferable. By using these salts and mixed aqueous solutions, in addition to the effect of improving the fiber-forming ability, the manufacturing cost can be further reduced.

[0223] The salt content may be 0.1% by mass or more, 0.3% by mass or more, 0.5% by mass or more, 0.7% by mass or more, 1% by mass or more, 1.3% by mass or more, 1.5% by mass or more, 1.7% by mass or more, 2% by mass or more, 2.3% by mass or more, 2.5% by mass or more, 2.7% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, or 15% by mass or more with respect to the total amount of the coagulating liquid, and the upper limit may be 30% by mass or less, 25% by mass or less, 20% by mass or less, or the content below the solubility. The salt content may be, for example, 0.1% by mass or more and 30% by mass or less, 0.3% by mass or more and 25% by mass or less, 5% by mass or more and 25% by mass or less, 1% by mass or more and 25% by mass or less, 3% by mass or more and 25% by mass or less, 8% by mass or more and 25% by mass or less, 10% by mass or more and 25% by mass or less, 10% by mass or more and 25% by mass or less, 1% by mass or more and 20% by mass or less, 3% by mass or more and 20% by mass or less, 5% by mass or more and 20% by mass or less, 8% by mass or more and 20% by mass or less, 10% by mass or more and 20% by mass or less, 10% by mass or more and 15% by mass or less, 12% by mass or more and 17% by mass or less, 13% by mass or more and 18% by mass or less, 15% by mass or more and 20% by mass or less, or 16% by mass or more and 20% by mass or less with respect to the total amount of the coagulating liquid. The salt content is preferably 0.05 mol / L or more, and may be 0.05 mol / L or more and 5.5 mol / L or less, 0.1 mol / L or more and 5.0 mol / L or less, 0.1 mol / L or more and 4.5 mol / L or less, or 0.1 mol / L or more and 4.0 mol / L or less with respect to the total amount of the coagulating liquid.

[0224] When using sodium chloride, the salt content may be, for example, 0.1 mol / L or more and 5.0 mol / L or less, 0.1 mol / L or more and 4.5 mol / L or less, or 0.1 mol / L or more and 4.0 mol / L or less with respect to the total amount of the coagulating liquid.

[0225] When using sodium sulfate, the salt content may be, for example, 0.1 mol / L or more and 3.4 mol / L or less, 0.1 mol / L or more and 3.0 mol / L or less, 0.1 mol / L or more and 2.5 mol / L or less, or 0.1 mol / L or more and 2.0 mol / L or less with respect to the total amount of the coagulating liquid. Also, for example, it may be 3% by mass or more and 28% by mass or less, 3% by mass or more and 25% by mass or less, 3% by mass or more and 20% by mass or less, 5% by mass or more and 20% by mass or less, or 8% by mass or more and 20% by mass or less with respect to the total amount of the coagulating liquid.

[0226] Moreover, the content of sodium sulfate with respect to the total amount of the coagulating liquid is preferably 10% by mass or more and 20% by mass or less, more preferably 11% by mass or more and 19% by mass or less, still more preferably 11% by mass or more and 18% by mass or less, further preferably 12% by mass or more and 18% by mass or less, still further preferably 12% by mass or more and 17% by mass or less, and particularly preferably 13% by mass or more and 16% by mass or less. When the content of sodium sulfate with respect to the total amount of the coagulating liquid is 10% by mass or more, a sufficient coagulation rate can be obtained, and an increase in cost due to equipment investment can be avoided. When the content of sodium sulfate with respect to the total amount of the coagulating liquid is 20% by mass or less, yarn breakage occurring at the interface between the dope liquid and the coagulated yarn (yarn) due to rapid coagulation of the dope liquid can be avoided.

[0227] Also, from the viewpoint of improving the recovery efficiency of the solvent, the water content in the total amount of the coagulating liquid in the above case is preferably 50% by mass or more and 80% by mass or less, more preferably 60% by mass or more and 80% by mass or less, and even more preferably 60% by mass or more and 70% by mass or less. Further, when using sodium sulfate, the concentration of the aqueous sodium sulfate solution is preferably 10% by mass or more and 22% by mass or less, preferably 10% by mass or more and 20% by mass or less, more preferably 12% by mass or more and 20% by mass or less, even more preferably 14% by mass or more and 20% by mass or less, and particularly preferably 16% by mass or more and 20% by mass or less. When the concentration of the aqueous sodium sulfate solution is 10% by mass or more, a sufficient coagulation rate can be obtained, and an increase in cost due to equipment investment can be avoided. When the concentration of the aqueous sodium sulfate solution is 22% by mass or less, yarn breakage occurring at the interface between the dope liquid and the coagulated yarn (yarn) due to rapid coagulation of the dope liquid can be avoided.

[0228] The aqueous solution contained in the coagulating liquid of the present embodiment may be selected from the group consisting of, for example, an aqueous carboxylic acid solution, an aqueous hydrogen carbonate solution, an aqueous formate solution, an aqueous acetate solution, an aqueous chloride solution, an aqueous sulfate solution, an aqueous hydrogen phosphate solution, an aqueous citrate solution, brackish water, seawater, and a mixed solution thereof. Further, the aqueous solution contained in the coagulating liquid of the present embodiment may be selected from the group consisting of, for example, an aqueous citrate solution, an aqueous formic acid solution, an aqueous sodium hydrogen carbonate solution, an aqueous sodium formate solution, an aqueous sodium acetate solution, an aqueous sodium chloride solution, an aqueous sodium sulfate solution, an aqueous ammonium sulfate solution, an aqueous potassium hydrogen phosphate solution, an aqueous calcium chloride solution, an aqueous sodium citrate solution, brackish water, seawater, and a mixed solution thereof, and may be at least one selected from the group consisting of water, an aqueous formic acid solution, and an aqueous sodium sulfate solution, and may be at least one selected from the group consisting of water and an aqueous sodium sulfate solution.

[0229] The coagulation liquid before contacting with the spinning dope may or may not contain an organic solvent. When the coagulation liquid contains an organic solvent, the organic solvent may be the same as or different from the organic solvent in the spinning dope, but it is preferably the same. Also, even when the coagulation liquid before contacting with the spinning dope does not contain an organic solvent, in the process of contacting the spinning dope with the coagulation liquid, the organic solvent may dissolve from the contacted spinning dope into the coagulation liquid. The content of the organic solvent contained in the coagulation liquid (including the case where it dissolves from the spinning dope contacted with the coagulation liquid into the coagulation liquid) is based on the total amount of the coagulation liquid (when the organic solvent dissolves from the spinning dope into the coagulation liquid, it is the total content of the coagulation liquid before contacting with the spinning dope and the organic solvent dissolved from the spinning dope into the coagulation liquid) being 100% by mass, and may be 0% by mass or more and 30% by mass or less, 5% by mass or more and 30% by mass or less, 5% by mass or more and 25% by mass or less, 0% by mass or more and 20% by mass or less, 5% by mass or more and 20% by mass or less, 5% by mass or more and 15% by mass or less, 10% by mass or more and 20% by mass or less, 0% by mass or more and 10% by mass or less, 0% by mass or more and 5% by mass or less, 0% by mass or more and 2% by mass or less. Preferably, it is 10% by mass or more and 30% by mass or less, more preferably 12% by mass or more and 28% by mass or less, still more preferably 14% by mass or more and 26% by mass or less, and even more preferably 15% by mass or more and 25% by mass or less. When the content of the organic solvent is within the above range, the fiber-forming ability of the layer structure protein is further improved. As the organic solvent, formic acid, DMSO, or HFIP is preferred, formic acid or HFIP is more preferred, and formic acid is even more preferred.

[0230] The pH of the aqueous solution contained in the coagulation liquid may be 0.25 to 10.00, or may be 0.25 to 9.50.

[0231] The pH of the aqueous acid solution in the coagulation liquid may be, for example, 0.25 to less than 7.00, 0.50 to less than 7.00, 1.00 to less than 7.00, 1.50 to less than 7.00, 2.00 to less than 7.00, 3.00 to less than 7.00.

[0232] The pH of the aqueous salt solution in the coagulating liquid may be, for example, from 0.50 to 10.00, or may be from 1.00 to 10.00, or may be from 2.00 to 10.00, or may be from 3.00 to 10.00, or may be from 3.50 to 10.00, or may be from 4.00 to 10.00, or may be from 4.50 to 10.00, or may be from 5.00 to 10.00, or may be from 5.50 to 10.00, or may be from 6.00 to 10.00, or may be from 6.50 to 10.00, or may be from 6.50 to 9.50.

[0233] The content of the above water or aqueous solution in the coagulating liquid may be 60% by mass or more, or may be 65% by mass or more, or may be 68% by mass or more, preferably 70% by mass or more, more preferably 71% by mass or more, more preferably 72% by mass or more, more preferably 73% by mass or more, more preferably 74% by mass or more, more preferably 75% by mass or more, more preferably 76% by mass or more, more preferably 77% by mass or more, more preferably 78% by mass or more, more preferably 79% by mass or more, particularly preferably 80% by mass or more, or may be 85% by mass or more, or may be 90% by mass or more, or may be 95% by mass, based on the total amount of the coagulating liquid. When the content of the above water or aqueous solution is within the above range, the fiber-forming ability of the more hierarchical structure protein is improved. The content of the above water or aqueous solution in the coagulating liquid may be, for example, from 60% by mass or more to 100% by mass or less, or may be from 70% by mass or more to 100% by mass or less, or may be from 75% by mass or more to 100% by mass or less, or may be from 80% by mass or more to 100% by mass or less, or may be from 85% by mass or more to 100% by mass or less, or may be from 90% by mass or more to 100% by mass or less, or may be from 95% by mass or more to 100% by mass or less, or may be from 70% by mass or more to 90% by mass or less, or may be from 75% by mass or more to 85% by mass or less, or may be from 78% by mass or more to 82% by mass or less, based on the total amount of the coagulating liquid.

[0234] The coagulating liquid preferably contains at least one selected from the group consisting of methanol, ethanol, acetone, water and aqueous sulfate solution, and the content of methanol, ethanol, acetone, water and / or aqueous sulfate solution in the coagulating liquid is preferably 70% by mass or more based on 100% by mass of the total amount of the coagulating liquid.

[0235] The temperature of the coagulating liquid may be room temperature, may be 0°C to 90°C, may be 0°C to 80°C, may be 5°C to 80°C, may be 10°C to 80°C, may be 15°C to 80°C, may be 20°C to 80°C, may be 25°C to 80°C, may be 30°C to 80°C, may be 40°C to 80°C, may be 50°C to 80°C, may be 60°C to 80°C, may be 70°C to 80°C, may be 20°C to 70°C, may be 30°C to 70°C, may be 40°C to 70°C, may be 50°C to 70°C, may be 20°C to 60°C, may be 30°C to 60°C, may be 40°C to 60°C, may be 30°C to 50°C, may be 50°C to 60°C. When water or an aqueous solution with a pH of 0.25 or more and 10.00 or less (such as formic acid aqueous solution, sodium sulfate aqueous solution, or a mixed aqueous solution thereof) is used as the coagulating liquid, from the viewpoint of better spinning stability, the temperature of the coagulating liquid is preferably 30°C to 50°C, more preferably 32°C to 48°C, more preferably 33°C to 47°C, more preferably 34°C to 46°C, and even more preferably 35°C to 45°C. The lower limit value of the temperature of the coagulating liquid may be equal to or higher than the melting point of the organic solvent contained in the spinning dope, and the upper limit value of the temperature may be equal to or lower than the boiling point of the organic solvent contained in the spinning dope. By increasing the temperature of the coagulating liquid, the desolvation rate of the spinning dope can be increased.

[0236] The coagulating liquid may further contain a dope solvent. From the viewpoint of improving the solvent recovery efficiency, the content of the dope solvent (e.g., formic acid) in the total amount of the coagulating liquid is preferably 15 to 25% by mass, more preferably 16 to 25% by mass, even more preferably 16 to 24% by mass, and particularly preferably 18 to 24% by mass.

[0237] The solidifying liquid may further contain the above-described dissolution accelerator that can be added to the spinning dope.

[0238] 〔Method for producing recombinant structural protein multi-filament〕 〔Spinning process〕 The method for producing a multi-filament according to this embodiment can be produced by a known wet spinning method, dry spinning method, dry-wet spinning method, melt spinning method, etc. using a spinning nozzle having 100 or more holes, and a multi-filament composed of the same number of single filaments as the number of holes of the spinning nozzle (a multi-filament having 100 or more constituent filaments) can be obtained. The method for producing a multi-filament of this embodiment can be implemented, for example, using the spinning device shown in FIG. 6 or FIG. 7. Preferred spinning methods include wet spinning or dry-wet spinning. Hereinafter, the method for producing a modified fibroin multi-filament will be described as an example.

[0239] FIG. 6 is an explanatory diagram schematically showing an example of a spinning device for producing a modified fibroin multi-filament. The spinning device 10 shown in FIG. 6 is an example of a spinning device for dry-wet spinning, and has an extruder 1, a coagulation bath 20, a washing bath (drawing bath) 21, and a drying device 4 in order from the upstream side.

[0240] The extrusion device 1 has a storage tank 7 where the spinning dope (dope solution) 6 is stored. The coagulation bath 20 stores the coagulation liquid 11. The spinning dope 6 is extruded from the spinneret (nozzle) 9 by a gear pump 8 attached to the lower end of the storage tank 7. The extruded spinning dope 6 is supplied (introduced) into the coagulation liquid 11 in the coagulation bath 20 through the air gap 19. In the coagulation liquid 11, the solvent is removed from the spinning dope and the modified fibroin coagulates to form a fibrous coagulum. Next, the fibrous coagulum is supplied into the cleaning liquid 12 in the cleaning bath 21 and stretched. The stretching ratio is determined by the speed ratio between the first nip roller 13 and the second nip roller 14 installed in the cleaning bath 21. Thereafter, the stretched fibrous coagulum is supplied into the drying device 4, dried in the yarn path 22, and wound up by a winder. In this way, the multifilament is finally obtained as a wound product 5 wound up by the spinning device 10. Note that 18a to 18g are yarn guides.

[0241] When using a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm as the die 9, the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole, and more preferably 1.4 to 4.0 ml / hour. The distance that the solidified modified fibroin passes through the coagulation liquid 11 (substantially the distance from the yarn guide 18a to the yarn guide 18b) only needs to be a length at which desolvation can be efficiently performed, for example, 200 to 500 mm. The take-up speed of the undrawn yarn may be, for example, 1 to 100 m / min, may be 1 to 20 m / min, and is preferably 1 to 3 m / min. When the take-up speed is 1 m / min or more, the productivity can be sufficiently increased. When the take-up speed is 100 m / min or less, significant liquid scattering of the solvent can be avoided. The residence time in the coagulation liquid 11 only needs to be a time during which the solvent is removed from the spinning dope, for example, it may be 0.01 to 3 minutes, and is preferably 0.05 to 0.15 minutes. Also, stretching (pre-stretching) may be performed in the coagulation liquid 11. The coagulation bath 20 may be provided in multiple stages, and stretching may be performed at each stage or at a specific stage as required.

[0242] FIG. 7 is an explanatory diagram schematically showing an example of a spinning apparatus for producing a modified fibroin multifilament. The spinning apparatus 10 shown in FIG. 6 is an example of a spinning apparatus for wet spinning, and is the same as the apparatus of FIG. 6 except that it does not have an air gap 19.

[0243] The die shape, hole shape (hole shape), number of holes (number of holes) of the spinneret are not particularly limited, and can be appropriately selected according to the desired fiber diameter, number of single filaments, etc.

[0244] When the hole shape of the spinneret is circular, the hole diameter can be exemplified as 0.01 mm or more and 0.6 mm or less. When the hole diameter is 0.01 mm or more, the pressure loss can be reduced and the equipment cost can be suppressed. When the hole diameter is 0.6 mm or less, the necessity of the drawing operation for making the fiber diameter thinner can be reduced, and the possibility of drawing breakage between discharge and take-up can be reduced.

[0245] The lower limit of the number of holes per cone of the spinning nozzle 1 is 100 or more from the viewpoint of improving productivity, preferably 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, 1000 or more, 1100 or more, 1200 or more, 1300 or more, 1400 or more, 1500 or more, 1600 or more, 1700 or more, 1800 or more, 1900 or more, 2000 or more, 2100 or more, 2200 or more, 2300 or more, 2400 or more, 2500 or more, 2600 or more, 2700 or more, 2800 or more, 2900 or more, 3000 or more, 3100 or more, 3200 or more, 3300 or more, 3400 or more, 3500 or more, 3600 or more, 3700 or more, 3800 or more, 3900 or more, 4000 or more, 4100 or more, 4200 or more, 4300 or more, 4400 or more, 4500 or more, 4600 or more, 4700 or more, 4800 or more, 4900 or more, 5000 or more, 5100 or more, 5200 or more, 5300 or more, 5400 or more, 5500 or more, 5600 or more, 5700 or more, 5800 or more, 5900 or more.

[0246] From the viewpoint of the effect that the productivity can be sufficiently improved, the upper limit value of the number of holes per cone of the spinning nozzle 1 is 9000 or less, and may be 8900 or less, 8800 or less, 8700 or less, 8600 or less, 8500 or less, 8400 or less, 8300 or less, 8200 or less, 8100 or less, 8000 or less, 7900 or less, 7800 or less, 7700 or less, 7600 or less, 7500 or less, 7400 or less, 7300 or less, 7200 or less, 7100 or less, 7000 or less, 6900 or less, 6800 or less, 6700 or less, 6600 or less, 6500 or less, 6400 or less, 6300 or less, 6200 or less, 6100 or less, 6000 or less.

[0247] The number of holes per cone of the spinning nozzle 1 may be, for example, 100 or more and 9,000 or less, 150 to 9,000 or less, 200 to 9,000 or less, 250 to 9,000 or less, 350 to 9,000 or less, 300 to 9,000 or less, 350 to 9,000 or less, 400 to 9,000 or less, 450 to 9,000 or less, 500 to 9,000 or less, 650 to 9,000 or less, 750 to 9,000 or less, 800 to 9,000 or less, 850 to 9,000 or less, 900 to 9,000 or less, 950 to 9,000 or less, or 1,000 to 9,000 or less, for example, 100 or more and 8,000 or less, 150 to 8,000 or less, 200 to 8,000 or less, 250 to 8,000 or less, 350 to 8,000 or less, 300 to 8,000 or less, 350 to 8,000 or less, 400 to 8,000 or less, 450 to 8,000 or less, 500 to 8,000 or less, 650 to 8,000 or less, 750 to 8,000 or less, 800 to 8,000 or less, 850 to 8,000 or less, 900 to 8,000 or less, 950 to 8,000 or less, or 1,000 to 8,000 or less, for example, 100 or more and 7,000 or less, 150 to 7,000 or less, 200 to 7,000 or less, 250 to 7,000 or less, 350 to 7,000 or less, 300 to 7,000 or less, 350 to 7,000 or less, 400 to 7,000 or less, 450 to 7,000 or less, 500 to 7,000 or less, 650 to 7,000 or less, 750 to 7,000 or less, 800 to 7,000 or less, 850 to 7,000 or less, 900 to 7,000 or less, 950 to 7,000 or less, or 1,000 to 7,000 or less, for example, 100 or more and 6,000 or less, 150 to 6,000 or less, 200 to 6,000 or less, 250 to 6,000 or less, 350 to 6,000 or less, 300 to 6,000 or less, 350 to 6,000 or less, 400 to 6,000 or less, 450 to 6,000 or less, 500 to 6,000 or less, 650 to 6,000 or less, 750 to 6,000 or less, 800 to 6,000 or less, 850 to 6,000 or less, 900 to 6,000 or less, 950 to 6,000 or less, or 1,000 to 6,000 or less, for example, 100 or more and 5,500 or less, 150 to 5,500 or less, 200 to 5,500 or less, 250 to 5,500 or less, 350 to 5,Less than 000, less than 300 to 5,500, less than 350 to 5,500, less than 400 to 5,500, less than 450 to 5,500, less than 500 to 5,000, less than 650 to 5,500, less than 750 to 5,500, less than 800 to 5,500, less than 850 to 5,500, less than 900 to 5,500, less than 950 to 5,500 or less than 1,000 to 5,500, and may be, for example, from 100 or more to 5,200 or less, from 150 to 5,200 or less, from 200 to 5,200 or less, from 250 to 5,200 or less, from 350 to 5,200 or less, from 300 to 5,200 or less, from 350 to 5,200 or less, from 400 to 5,200 or less, from 450 to 5,200 or less, from 500 to 5,200 or less, from 650 to 5,200 or less, from 750 to 5,200 or less, from 800 to 5,200 or less, from 850 to 5,200 or less, from 900 to 5,200 or less, from 950 to 5,200 or less or from 1,000 to 5,200 or less, and may be, for example, from 100 or more to 4,800 or less, from 150 to 4,800 or less, from 200 to 4,800 or less, from 250 to 4,800 or less, from 350 to 4,800 or less, from 300 to 4,800 or less, from 350 to 4,800 or less, from 400 to 4,800 or less, from 450 to 4,800 or less, from 500 to 4,800 or less, from 650 to 4,800 or less, from 750 to 4,800 or less, from 800 to 4,800 or less, from 850 to 4,800 or less, from 900 to 4,800 or less, from 950 to 4,800 or less or from 1,000 to 4,800 or less, and may be, for example, from 100 or more to 4,500 or less, from 150 to 4,500 or less, from 200 to 4,500 or less, from 250 to 4,500 or less, from 350 to 4,500 or less, from 300 to 4,500 or less, from 350 to 4,500 or less, from 400 to 4,500 or less, from 450 to 4,500 or less, from 500 to 4,500 or less, from 650 to 4,500 or less, from 750 to 4,500 or less, from 800 to 4,500 or less, from 850 to 4,500 or less, from 900 to 4,500 or less, from 950 to 4,500 or less or from 1,000 to 4,500 or less, and may be, for example, from 100 or more to 4,000 or less, from 150 to 4,500 or less, from 200 to 4,500 or less, from 250 to 4,000 or less, from 350 to 4,500 or less, from 300 to 4,000 or less, from 350 to 4,000 or less, from 400 to 4,000 or less, from 450 to 4,000 or less, from 500 to 4,000 or less, from 650 to 4,000 or less, from 750 to 4,000 or less, from 800 to 4,It may be 000 or less, 850 to 4,500 or less, 900 to 4,000 or less, 950 to 4,000 or less, or 1,000 to 4,000 or less.,

[0248] The number of spindles of the spinning nozzle may be appropriately selected according to the target production amount of the fiber and the like, and is not particularly limited. For example, it may be 1 spindle, 2 spindles, 3 spindles, 4 spindles, 5 spindles, 6 spindles, 7 spindles, 8 spindles, 9 spindles, 10 spindles, 11 spindles, 12 spindles, 13 spindles, 14 spindles, 15 spindles, 16 spindles, 17 spindles, 18 spindles, 19 spindles, 20 spindles, 20 spindles or more, 25 spindles, 30 spindles or more, 35 spindles or more, 40 spindles or more, 45 spindles or more, 50 spindles or more, 55 spindles or more, 60 spindles or more, 65 spindles or more, 70 spindles or more, 75 spindles or more, 80 spindles or more, 85 spindles or more, 90 spindles or more, 95 spindles or more, 100 spindles or more. A plurality of spinning nozzles (a plurality of spindles) having the same number of holes may be combined and used (for example, 12 spinning nozzles with 3,000 holes are combined and used), or a plurality of spinning nozzles having different numbers of holes may be combined and used.

[0249] The temperature of the spinning dope when passing through the spinneret and the temperature of the spinneret are not particularly limited and may be appropriately adjusted according to the concentration and viscosity of the spinning dope used, the type of organic solvent, etc. From the viewpoint of preventing deterioration of the modified fibroin, etc., the temperature is preferably 30°C to 100°C. Also, from the viewpoint of reducing the possibility of pressure increase due to solvent volatilization and blockage in the piping due to solidification of the spinning dope, it is preferable to set the upper limit to a temperature less than the boiling point of the solvent used. This improves process stability.

[0250] The temperature of the coagulating liquid 11 is not particularly limited, and may be 40°C or lower, 30°C or lower, 25°C or lower, 20°C or lower, 10°C or lower, or 5°C or lower. From the viewpoints of workability, cooling cost, etc., it is preferably 0°C or higher. The temperature of the coagulating liquid 11 can be adjusted, for example, by using a spinning device 10 having a coagulation bath 20 provided with a heat exchanger inside and a cooling circulation device. For example, by flowing a medium cooled to a predetermined temperature by a cooling circulation device through a heat exchanger installed in the coagulation bath, the temperature can be adjusted within the above range by heat exchange between the coagulating liquid 11 and the heat exchanger. In this case, more efficient cooling can be achieved by circulating the solvent used for the coagulating liquid 11 as the medium.

[0251] A plurality of coagulation baths for storing the coagulating liquid may be provided.

[0252] After the solidified modified fibroin (fibrous solid) is separated from the coagulation bath or the washing bath, it may be directly wound by a winder, or may pass through a drying device, be dried, and then be wound by a winder.

[0253] The distance that the solidified modified fibroin (fibrous solid) passes through the coagulating liquid may be such that desolvation can be carried out efficiently, and may be determined according to the extrusion speed (discharge speed) of the spinning dope from the nozzle, etc. The residence time of the solidified modified fibroin (or spinning dope) in the coagulating liquid may be determined according to the distance that the solidified modified fibroin passes through the coagulating liquid, the extrusion speed of the spinning dope from the nozzle, etc.

[0254] 〔Stretching step〕 The method for producing the modified fibroin multifilament of the present embodiment may further include a step of stretching the solidified modified fibroin (fibrous solid) (stretching step). Examples of the stretching method include wet heat stretching and dry heat stretching. The stretching step may be carried out, for example, in the coagulation bath 20 or in the washing bath 21. The stretching step can also be carried out in the air.

[0255] The stretching carried out in the washing bath 21 may be so-called wet heat stretching, which is carried out in warm water, a solution obtained by adding an organic solvent or the like to warm water, or the like. The temperature of the wet heat stretching is preferably 50 to 90 °C. When the temperature is 50 °C or higher, the pore diameter of the yarn can be reduced and stabilized. Further, when the temperature is 90 °C or lower, the temperature setting is easy and the spinning stability is improved. The temperature is more preferably 75 to 85 °C.

[0256] The wet heat stretching can be carried out in warm water, a solution obtained by adding an organic solvent or the like to warm water, or during steam heating. The temperature may be, for example, 40 to 200 °C, 50 to 180 °C, 50 to 150 °C, or 75 to 90 °C. The draw ratio in the wet heat stretching may be, for example, 1 to 30 times, 2 to 25 times, 2 to 20 times, 2 to 15 times, 2 to 10 times, 2 to 8 times, 2 to 6 times, or 2 to 4 times with respect to the undrawn yarn (or pre-drawn yarn). However, the draw ratio is not limited as long as the desired fiber thickness, mechanical properties, and other properties can be obtained.

[0257] The dry heat stretching can be carried out by stretching in air using a device equipped with a heat source such as a contact-type hot plate and a non-contact-type furnace, but it is not particularly limited as long as it is a device capable of raising the temperature of the fiber to a predetermined temperature and stretching at a predetermined ratio. The temperature may be, for example, 100 °C to 270 °C, 140 °C to 230 °C, 140 °C to 200 °C, 160 °C to 200 °C, or 160 °C to 180 °C.

[0258] The draw ratio in the dry heat stretching step may be, for example, 1 to 30 times, 2 to 30 times, 2 to 20 times, 3 to 15 times, preferably 3 to 10 times, more preferably 3 to 8 times, and even more preferably 4 to 8 times with respect to the undrawn yarn (or pre-drawn yarn). However, the draw ratio is not limited as long as the desired fiber thickness, mechanical properties, and other properties can be obtained.

[0259] The drawing process may be performed by wet heat drawing and dry heat drawing separately, or may be performed in multiple stages or in combination. That is, as the drawing process, the first-stage drawing may be performed by wet heat drawing and the second-stage drawing may be performed by dry heat drawing, or the first-stage drawing may be performed by wet heat drawing, the second-stage drawing may be performed by wet heat drawing, and the third-stage drawing may be performed by dry heat drawing, etc. The wet heat drawing and dry heat drawing can be appropriately combined.

[0260] The lower limit of the final draw ratio of the multifilament after the drawing process may preferably be any one of 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, or 9 times with respect to the undrawn yarn (or pre-drawn yarn). The upper limit of the final draw ratio of the multifilament after the drawing process may preferably be any one of 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. Also, for example, the final draw ratio may be 3 to 40 times, 3 to 30 times, 5 to 30 times, 5 to 20 times, 5 to 15 times, or 5 to 13 times. However, the draw ratio is not limited as long as the desired fiber thickness, mechanical properties, and other characteristics can be obtained. By adjusting the draw ratio, the fiber diameter of the obtained multifilament can be adjusted to an arbitrary value.

[0261] Before or after drying, if necessary, an oil agent may be applied to the undrawn yarn (or pre-drawn yarn) or drawn yarn for the purpose of imparting antistatic properties, convergence properties, lubricity, etc. The type of the applied oil agent and the amount applied are not particularly limited and can be appropriately adjusted in consideration of the use of the fiber, the handleability of the fiber, etc.

[0262] The manufacturing method according to this embodiment may further include a step of filtering the spinning dope before discharging the spinning dope (filtering step) and / or a step of defoaming the spinning dope before discharging (defoaming step).

[0263] The manufacturing method according to this embodiment may further include a shrinking step before or after the drying step. When performing the shrinking step after the drying step, after winding the multifilament dried after spinning around a bobbin, the multifilament may be unwound from the bobbin to perform the shrinking step.

[0264] 〔Shrinking step〕 The modified fibroin multifilament according to this embodiment may further include a shrinking step of irreversibly shrinking the above-described multifilament. In the shrinking step of irreversibly shrinking the multifilament, the multifilament may be irreversibly shrunk by bringing the multifilament into contact with water, and / or the multifilament may be irreversibly shrunk by heating and relaxing the multifilament. When irreversibly shrinking the multifilament by bringing it into contact with water, the irreversibly shrunk multifilament may be dried to further shrink it.

[0265] 〔Shrinking step (contact step) by contact with water〕 FIG. 8 is a diagram showing an example of the change in the length of a multifilament (fiber containing modified fibroin) due to contact with water. The multifilament (fiber containing modified fibroin) according to this embodiment has the property of shrinking (primary shrinkage) when brought into contact (moistened) with water below the boiling point (the change in length indicated by "primary shrinkage" in FIG. 8). After the primary shrinkage, when dried, it further shrinks (the change in length indicated by "secondary shrinkage" in FIG. 8). After the secondary shrinkage, when brought into contact with water again, it extends to the same or approximately the same length as before the secondary shrinkage, and thereafter, when drying and wetting are repeated, shrinkage and extension are repeated with a width approximately the same as the secondary shrinkage (the width indicated by "expansion / contraction rate (shrinkage rate)" in FIG. 8). That is, the primary shrinkage by bringing the multifilament into contact with water is an irreversible shrinkage. Therefore, in the shrinking step, by bringing the multifilament into contact with water, a modified fibroin multifilament having an irreversible shrinkage history according to this embodiment can be obtained. The step of irreversibly shrinking (primary shrinkage) the multifilament by bringing it into contact with water is hereinafter referred to as the "contact step".

[0266] The irreversible shrinkage of the multifilament (fiber containing modified fibroin) in the contact process (the "primary shrinkage" in FIG. 8) is considered to occur for the following reasons, for example. That is, one reason is considered to be due to the primary structure of the multifilament (fiber containing modified fibroin), and another reason is considered to occur, for example, in the multifilament (fiber containing modified fibroin) having residual stress due to stretching or the like in the manufacturing process, when water penetrates between or within the fibers and the residual stress is relaxed.

[0267] In the contact process, after spinning and before contacting with water, the multifilament is brought into contact with water to make the multifilament in a wet state. The wet state means a state in which at least a part of the multifilament is wetted with water. Thereby, the multifilament can be shrunk without an external force. This shrinkage is irreversible (corresponding to the "primary shrinkage" in FIG. 8).

[0268] The temperature of the water brought into contact with the multifilament in the contact process may be below the boiling point. Thereby, the handleability and the workability of the shrinkage process and the like are improved. Further, from the viewpoint of sufficiently shortening the shrinkage time, the lower limit value of the water temperature is preferably 10°C or higher, more preferably 40°C or higher, and still more preferably 70°C or higher. The upper limit value of the water temperature is preferably 90°C or lower.

[0269] In the contact process, the method of bringing water into contact with the multifilament is not particularly limited. Examples of the method include a method of immersing the multifilament in water, a method of spraying water on the multifilament at room temperature or in a state of heated steam or the like, and a method of exposing the multifilament to a high-humidity environment filled with water vapor. Among these methods, in the contact process, since the shrinkage time can be effectively shortened and the simplification of the processing equipment and the like can be realized, the method of immersing the multifilament in water is preferable.

[0270] In the contact step, when a multifilament is brought into contact with water in a relaxed state, the multifilament may not only simply contract but also contract in a wavy manner. In order to prevent the occurrence of such contraction, for example, the contact step may be carried out in a state where the multifilament is not relaxed, such as by bringing the multifilament into contact with water while pulling it in the fiber axis direction to such an extent that no tension is applied.

[0271] (Drying step) The method for manufacturing a modified fibroin multifilament according to the present embodiment may further include a drying step. The drying step is a step of drying the multifilament that has undergone the contact step (or the modified fibroin multifilament obtained through the contact step) to further contract it (corresponding to "secondary contraction" in FIG. 8). The drying may be, for example, natural drying or forced drying using drying equipment. As the drying equipment, any known contact-type or non-contact-type drying equipment can be used. Also, the drying temperature is not particularly limited as long as it is lower than the temperature at which the modified fibroin contained in the multifilament decomposes or the multifilament suffers thermal damage. Generally, it is a temperature within the range of 20 to 150°C, and preferably within the range of 50 to 100°C. When the temperature is within this range, the fiber can be dried more rapidly and efficiently without causing thermal damage to the fiber or decomposition of the modified fibroin contained in the fiber. The drying time is appropriately set according to the drying temperature and the like. For example, a time that can eliminate the influence on the quality and physical properties of the modified fibroin multifilament due to over-drying as much as possible is adopted.

[0272] FIG. 9 is an explanatory diagram schematically showing an example of a manufacturing apparatus for manufacturing a modified fibroin multifilament. The manufacturing apparatus 40 shown in FIG. 9 includes a feed roller 42 for feeding out a multifilament, a winder 44 for winding up the modified fibroin multifilament 38, a water bath 46 for carrying out the contact step, and a dryer 48 for carrying out the drying step.

[0273] More specifically, the feed roller 42 is configured to be capable of mounting the wound multi-filament 36, and by rotation of an electric motor or the like (not shown), the multi-filament 36 can be continuously and automatically fed out from the wound multi-filament 36. The winder 44 is capable of continuously and automatically winding up the modified fibroin multi-filament 38 manufactured through the contact process and the drying process after being fed out from the feed roller 42 by rotation of an electric motor (not shown). Here, the feeding speed of the multi-filament 36 by the feed roller 42 and the winding speed of the modified fibroin multi-filament 38 by the winder 44 are controllable independently of each other.

[0274] The water bath 46 and the dryer 48 are arranged side by side on the upstream side and the downstream side in the feeding direction of the multi-filament 36 between the feed roller 42 and the winder 44. The manufacturing apparatus 40 shown in FIG. 9 has relay rollers 50 and 52 for relaying the multi-filament 36 before and after the contact process that travels from the feed roller 42 toward the winder 44.

[0275] The water bath 46 has a heater 54, and the water 47 heated by this heater 54 is accommodated in the water bath 46. Further, a tension roller 56 is installed in the water bath 46 in a state of being immersed in the water 47. Thereby, the multi-filament 36 fed out from the feed roller 42 travels toward the winder 44 while being immersed in the water 47 in a state of being wound around the tension roller 56 in the water bath 46. The immersion time of the multi-filament fiber 36 in the water 47 is appropriately controlled according to the traveling speed of the multi-filament 36.

[0276] The dryer 48 has a pair of hot rollers 58. The pair of hot rollers 58 is capable of winding the multifilament 36 that detaches from within the water bath 46 and travels toward the winder 44. Thereby, the multifilament 36 immersed in water 47 within the water bath 46 is heated by the pair of hot rollers 58 within the dryer 48, dried, and then further fed out toward the winder 44.

[0277] When manufacturing the modified fibroin multifilament 38 using the manufacturing apparatus 40 having such a structure, first, for example, a wound product of the multifilament 36 spun using the spinning apparatus 10 shown in FIG. 11 is attached to the feed roller 42. Next, the multifilament 36 is continuously fed out from the feed roller 42 and immersed in water 47 within the water bath 46. At this time, for example, the winding speed of the winder 44 is made slower than the feeding speed of the feed roller 42. Thereby, since the multifilament 36 contracts due to contact with water 47 without relaxing between the feed roller 42 and the winder 44, the occurrence of shrinkage can be prevented. The multifilament 36 irreversibly contracts due to contact with water 47 (corresponding to "primary shrinkage" in FIG. 8).

[0278] Next, the multifilament 36 after contact with water 47 (or the modified fibroin multifilament 38 manufactured through contact with water 47) is heated by the pair of hot rollers 58 of the dryer 48. Thereby, the multifilament 36 after contact with water 47 (or the modified fibroin multifilament 38 manufactured through contact with water 47) can be dried and further shrunk (corresponding to "secondary shrinkage" in FIG. 8). At this time, the ratio between the feeding speed of the feed roller 42 and the winding speed of the winder 44 can also be controlled so that the length of the modified fibroin multifilament 38 does not change. Then, the obtained modified fibroin multifilament 38 is wound by the winder 44 to obtain a wound product of the modified fibroin multifilament 38.

[0279] Note that, instead of the pair of hot rollers 58, the multifilament 36 after contacting with water 47 may be dried using a drying facility composed only of a simple heat source such as a hot plate 64 shown in Fig. 10(b). Also in this case, by adjusting the relative speed between the delivery speed of the feed roller 42 and the winding speed of the winder 44 in the same manner as in the case of using the pair of hot rollers 58 as the drying facility, it is also possible not to change the length of the modified fibroin multifilament. Here, the drying means is constituted by the hot plate 64. Also, the dryer 48 is not essential.

[0280] As described above, by using the manufacturing apparatus 40, the target modified fibroin multifilament 38 can be manufactured automatically, continuously, and extremely easily.

[0281] Fig. 10 is an explanatory diagram schematically showing another example of a manufacturing apparatus for manufacturing a modified fibroin multifilament. Fig. 10(a) shows a processing apparatus that performs a contact process (primary shrinkage) provided in the manufacturing apparatus, and Fig. 10(b) shows a drying apparatus that performs a drying process provided in the manufacturing apparatus. The manufacturing apparatus shown in Fig. 10 has a processing apparatus 60 that performs a contact process on the multifilament 36, and a drying apparatus 62 that dries the multifilament 36 after the contact process (or the modified fibroin multifilament 38 manufactured through the contact process), and they have independent structures from each other.

[0282] More specifically, the processing apparatus 60 shown in Fig. 10(a) has a structure in which a feed roller 42, a water bath 46, and a winder 44 are arranged in order from the upstream side to the downstream side in the running direction of the multifilament 36. Such a processing apparatus 60 is configured to immerse the multifilament 36 fed out from the feed roller 42 into the water 47 in the water bath 46 and cause it to contract. Then, the obtained modified fibroin multifilament 38 is wound up by the winder 44. At this time, for example, the winding speed of the winder 44 is set slower than the feeding speed of the feed roller 42. Thereby, since the multifilament 36 contracts due to contact with the water 47 in a relaxed state between the feed roller 42 and the winder 44, it is possible to prevent tension from being applied to the fiber. The multifilament 36 irreversibly contracts due to contact with the water 47 (corresponding to the "primary contraction" in Fig. 8).

[0283] The drying apparatus 62 shown in Fig. 10(b) has a feed roller 42 and a winder 44, and a dry heat plate 64. The dry heat plate 64 is arranged between the feed roller 42 and the winder 44 such that the dry heat surface 66 contacts the modified fibroin multifilament 38 and extends along its running direction. In this drying apparatus 62, as described above, for example, by controlling the ratio between the feeding speed of the feed roller 42 and the winding speed of the winder 44, it is also possible not to change the length of the modified fibroin multifilament 38.

[0284] By using a manufacturing apparatus having such a structure, after the multifilament 36 is contracted by the processing apparatus 60 to obtain a modified fibroin multifilament 38, the modified fibroin multifilament 38 can be dried by the drying apparatus 62.

[0285] Note that the processing device 60 shown in Fig. 10(a) may be configured with only the water bath 46 by omitting the feed roller 42 and the winder 44. When using a manufacturing device having such a processing device, for example, a modified fibroin multifilament will be manufactured in a so-called batch system. Also, the drying device 62 shown in Fig. 10(b) is not essential.

[0286] 〔Shrinking process by heat relaxation〕 The shrinking process of irreversibly shrinking the multifilament may be performed by heat-relaxing the multifilament. Heat relaxation of the multifilament can be performed by heating the multifilament and then relaxing and shrinking the heated multifilament. Hereinafter, in the shrinking by heat relaxation of the multifilament, the process of heating the multifilament is referred to as the "heating process", and the process of relaxing and shrinking the heated multifilament is referred to as the "relaxing and shrinking process". The heating process and the relaxing and shrinking process can be performed, for example, by the high-temperature heat relaxation device 140 shown in Figs. 11 and 12.

[0287] (Heating process) In the heating process, it is preferable that the heating temperature of the multifilament 36 is equal to or higher than the softening temperature of the modified fibroin used for the multifilament 36. The softening temperature of the modified fibroin in this specification is the temperature at which the shrinkage due to stress relaxation of the multifilament 36 starts. In heat relaxation shrinkage at a temperature equal to or higher than the softening temperature of the modified fibroin, the fiber shrinks to an extent that cannot be obtained simply by the release of moisture in the fiber. As a result, the residual stress in the fiber generated by stretching in the spinning process can be removed.

[0288] As the temperature corresponding to the above softening temperature, for example, 180°C can be mentioned. When heat relaxation shrinkage is carried out in a high temperature range of 180°C or higher, the higher the relaxation ratio or the higher the temperature, the more efficiently the residual stress in the multifilament can be removed. Therefore, the heating temperature of the multifilament 36 is preferably 180°C or higher, more preferably 180°C to 280°C, even more preferably 200°C to 240°C, and particularly preferably 220°C to 240°C.

[0289] From the viewpoint of not impairing the elongation of the fiber after heat treatment, the heating time in the heating step, that is, the residence time in the high temperature heating furnace 143, is preferably 60 seconds or less, more preferably 30 seconds or less, and even more preferably 5 seconds or less. The length of this heating time is considered not to have a great influence on the stress. When the heating temperature is 200°C and the heating time is 5 seconds or less, a decrease in the elongation of the fiber after heat treatment can be prevented.

[0290] (Relaxation shrinkage step) In the relaxation shrinkage step, the relaxation ratio is preferably more than 1 time, more preferably 1.4 times or more, even more preferably 1.7 times or more, and particularly preferably 2 times or more. The relaxation ratio is the ratio of the feeding speed to the winding speed of the multifilament 36, and more specifically, it is the ratio of the feeding speed by the feeding roller 141 to the winding speed by the winding roller 142.

[0291] In the heat relaxation method using the high temperature heating relaxation device 140, if the multifilament 36 can be relaxed in a heated state, the heating step and the relaxation shrinkage step may be performed separately. That is, the heating device may be a device separated and independent from the relaxation device. In that case, a relaxation device is provided downstream of the heating device (on the downstream side in the running direction of the multifilament 36) so that the relaxation shrinkage step is performed after the heating step.

[0292] In addition to the manufacturing process of the multifilament, a heat relaxation process for the multifilament may be performed. That is, a device similar to the high-temperature heat relaxation device 140 may be provided as an independent device separate from the spinning device 25. A method of sending out the separately manufactured multifilament 36, setting it on a roller, and then sending it out from there may be adopted. The heat relaxation process may be performed on one multifilament or on a plurality of bundled multifilaments.

[0293] 〔Crosslinking process〕 A crosslinking process may be further performed to chemically crosslink between polypeptide molecules in the fiber on the modified fibroin multifilament having an irreversibly shrunk shrinkage history obtained as described above, or on the multifilament before irreversible shrinkage. Functional groups that can be crosslinked include, for example, amino groups, carboxyl groups, thiol groups, and hydroxy groups. For example, the amino group of the lysine side chain contained in the polypeptide can be crosslinked by an amide bond through dehydration condensation with the carboxyl group of the glutamic acid or aspartic acid side chain. Crosslinking may be performed by carrying out a dehydration condensation reaction under vacuum heating, or may be crosslinked with a dehydration condensing agent such as carbodiimide.

[0294] Crosslinking between polypeptide molecules may be performed using a crosslinking agent such as carbodiimide or glutaraldehyde, or may be performed using an enzyme such as transglutaminase. Carbodiimide has the general formula R 1 N=C=NR 2 (wherein, R 1 and R 2Each independently represents an organic group containing an alkyl group or cycloalkyl group having 1 to 6 carbon atoms. It is a compound represented by (). Specific examples of carbodiimide include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, diisopropylcarbodiimide (DIC), and the like. Among these, EDC and DIC are preferred because they have a high ability to form amide bonds between polypeptide molecules and are prone to cross-linking reactions.

[0295] For the cross-linking treatment, it is preferable to apply a cross-linking agent to the fiber and perform cross-linking by vacuum heating and drying. The cross-linking agent may be applied to the fiber as a pure product, or a product diluted to a concentration of 0.005 to 10% by mass with a lower alcohol having 1 to 5 carbon atoms, a buffer solution, or the like may be applied to the fiber. The cross-linking treatment is preferably carried out at a temperature of 20 to 45°C for 3 to 42 hours. By the cross-linking treatment, higher stress (strength) can be imparted to the fiber.

[0296] [Multifilament] The multifilament according to this embodiment contains a recombinant structural protein, has 100 or more constituent monofilaments, and has a coefficient of variation of elongation of less than 33%. Here, the constituent monofilament means a single filament constituting the multifilament (also referred to as a yarn), and the number of constituents means the number of single filaments (number of single filaments) constituting the multifilament. The multifilament according to this embodiment may contain, for example, modified fibroin, and the number of constituent monofilaments of the multifilament may be 100 or more, the coefficient of variation of the elastic modulus of the multifilament may be 15% or less, the coefficient of variation of the strength of the multifilament may be 15% or less, and the coefficient of variation of the elongation of the multifilament may be less than 33%.

[0297] The lower limit of the number of constituent filaments of the multifilament is 100 or more from the viewpoint of improving productivity. From the viewpoint of more suitably obtaining the said effect, it is preferably 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, 1,000 or more, 1,100 or more, 1,200 or more, 1,300 or more, 1,400 or more, 1,500 or more, 1,600 or more, 1,700 or more, 1,800 or more, 1,900 or more, 2,000 or more, 2,100 or more, 2,200 or more, 2,300 or more, 2,400 or more, 2,500 or more, 2,600 or more, 2,700 or more, 2,800 or more, 2,900 or more, 3,000 or more, 3,100 or more, 3,200 or more, 3,300 or more, 3,400 or more, 3,500 or more, 3,600 or more, 3,700 or more, 3,800 or more, 3,900 or more, 4,000 or more, 4,100 or more, 4,200 or more, 4,300 or more, 4,400 or more, 4,500 or more, 4,600 or more, 4,700 or more, 4,800 or more, 4,900 or more, 5,000 or more, 5,100 or more, 5,200 or more, 5,300 or more, 5,400 or more, 5,500 or more, 5,600 or more, 5,700 or more, 5,800 or more, 5,900 or more, and it may be appropriately selected according to the number of holes of the spinning nozzle used and / or the number of weights of the spinning nozzle.

[0298] The upper limit value of the number of constituent filaments is 900,000 or less, 800,000 or less, 700,000 or less, 675,000 or less, 600,000 or less, 500,000 or less, 450,000 or less, 400,000 or less, 300,000 or less, 250,000 or less, 200,000 or less, 150,000 or less, 100,000 or less, 98,000 or less, 96,000 or less, 95,000 or less, 90,000 or less, 85,000 or less, 80,000 or less, 78,000 or less, 76,000 or less, 75,000 or less, 72,000 or less, 70,000 or less, 68,000 or less, 66,000 or less, 64,000 or less, 62,000 or less, 60,000 or less, 58,000 or less, 56,000 or less, 54,000 or less, 52,000 or less, 50,000 or less, 48,000 or less, 46,000 or less, 45,000 or less, 42,000 or less, 40,000 or less, 38,000 or less, 36,000 or less, 35,000 or less, 32,000 or less, 30,000 or less, 28,000 or less, 26,000 or less, 25,000 or less, 24,000 or less, 22,000 or less, 20,000 or less, 18,000 or less, 16,000 or less, 14,000 or less, 12,000 or less, 10,000 or less, 9,500 or less, 9,000 or less, 8,900 or less, 8,800 or less, 8,700 or less, 8600 or less, 8500 or less, 8400 or less, 8,300 or less, 8,200 or less, 8,100 or less, 8,000 or less, 7,900 or less, 7800 or less, 7,700 or less, 7,600 or less, 7500 or less, 7,400 or less, 7300 or less, 7,200 or less, 7,100 or less, 7,000 or less, 6,900 or less, 6,800 or less, 6,700 or less, 6,600 or less, 6,500 or less, 6,400 or less, 6,300 or less, 6,200 or less, 6,100 or less, or 6,000 or less from the perspective of the effect of sufficiently improving productivity.

[0299] The number of constituent filaments is 100 to 900,000 or less from the viewpoint of the effect of being able to sufficiently improve productivity. For example, it may be 100 to 800,000, 100 to 700,000, 100 to 600,000, 100 to 500,000, 100 to 400,000, 100 to 300,000, 100 to 200,000, 100 to 100,000, 1,000 to 90,000, 950 to 90,000, 900 to 90,000, 800 to 90,000, 700 to 90,000, 600 to 90,000, 550 to 90,000, 500 to 90,000, 450 to 90,000, 400 to 90,000, 350 to 90,000, 300 to 90,000, 250 to 90,000, 200 to 90,000, 150 to 90,000 or 100 to 90,000. It may also be 1,000 to 60,000, 950 to 60,000, 900 to 60,000, 850 to 60,000, 800 to 60,000, 750 to 60,000, 700 to 60,000, 650 to 60,000, 600 to 60,000, 550 to 60,000, 500 to 60,000, 450 to 60,000, 400 to 60,000, 350 to 60,000, 300 to 60,000, 250 to 60,000, 200 to 60,000, 150 to 60,000 or 100 to 60,000. It may also be 1,000 to 48,000, 950 to 48,000, 900 to 48,000, 850 to 48,000, 800 to 48,000, 750 to 48,000, 700 to 48,000, 650 to 48,000, 600 to 48,000, 550 to 48,000, 500 to 48,000, 450 to 48,000, 400 to 48,000, 350 to 48,000, 300 to 48,000, 250 to 48,000, 200 to 48,000, 150 to 48,000 or 100 to 48,000. It may also be 1,000 to 39,000, 950 to 39,000, 900 to 39,000, 850 to 39,000, 800 to 39,000, 750 to 39,000, 700 to 39,000, 650 to 39,000, 600 to 39,000, 550 to 39,000, 500 to 39,000, 450 to 39,000, 400 to 39,It may be 0, 350 to 39,000, 300 to 39,000, 250 to 39,000, 200 to 39,000, 150 to 39,000 or 100 to 39,000; it may be 1,000 to 30,000, 950 to 30,000, 900 to 30,000, 850 to 30,000, 800 to 30,000, 750 to 30,000, 700 to 30,000, 650 to 30,000, 600 to 30,000, 550 to 30,000, 500 to 30,000, 450 to 30,000, 400 to 30,000, 350 to 30,000, 300 to 30,000, 250 to 30,000, 200 to 30,000, 150 to 30,000 or 100 to 30,000; it may be 1,000 to 24,000, 950 to 24,000, 900 to 24,000, 850 to 24,000, 800 to 24,000, 750 to 24,000, 700 to 24,000, 650 to 24,000, 600 to 24,000, 550 to 24,000, 500 to 24,000, 450 to 24,000, 400 to 24,000, 350 to 24,000, 300 to 24,000, 250 to 24,000, 200 to 24,000, 150 to 24,000 or 100 to 24,000; it may be 1,000 to 18,000, 950 to 18,000, 900 to 18,000, 850 to 18,000, 800 to 18,000, 750 to 18,000, 700 to 18,000, 650 to 18,000, 600 to 18,000, 550 to 18,000, 500 to 18,000, 450 to 18,000, 400 to 18,000, 350 to 18,000, 300 to 18,000, 250 to 18,000, 200 to 18,000, 150 to 18,000 or 100 to 18,000; it may be 1,000 to 12,000, 950 to 12,000, 900 to 12,000, 850 to 12,000, 800 to 12,000, 750 to 13,000, 700 to 12,000, 650 to 12,000, 600 to 12,000, 550 to 12,000, 500 to 12,000, 450 to 12,000, 400 to 12,000, 350 to 12,000, 300 to 12,000, 250 to 12,000, 200 to 12,It may be 000, 150 to 12,000, or 100 to 12,000, and may be 1,000 to 100,000, 950 to 10,000, 900 to 10,000, 850 to 10,000, 800 to 10,000, 750 to 10,000, 700 to 10,000, 650 to 10,000, 600 to 10,000, 550 to 10,000, 500 to 10,000, 450 to 10,000, 400 to 10,000, 350 to 10,000, 300 to 10,000, 250 to 10,000, 200 to 10,000, 150 to 10,000, or 100 to 10,000, and may be 1,000 to 9,000, 950 to 9,000, 900 to 9,000, 850 to 9,000, 800 to 9,000, 750 to 9,000, 700 to 9,000, 650 to 9,000, 600 to 9,000, 550 to 9,000, 500 to 4,000, 450 to 9,000, 400 to 9,000, 350 to 9,000, 300 to 9,000, 250 to 9,000, 200 to 9,000, 150 to 9,000, or 100 to 9,000, and may be 1,000 to 6,000, 950 to 6,000, 900 to 6,000, 850 to 6,000, 800 to 6,000, 750 to 6,000, 700 to 6,000, 650 to 6,000, 600 to 6,000, 550 to 6,000, 500 to 6,000, 450 to 6,000, 400 to 6,000, 350 to 6,000, 300 to 6,000, 250 to 6,000, 200 to 6,000, 150 to 6,000, or 100 to 6,000.,

[0300] The coefficient of variation of the elastic modulus, strength, elongation, and fineness of the multifilament can be calculated by measuring the elastic modulus [gf / D] ([gf / den]), strength [g / D] ([g / den]), breaking elongation [%], and fineness [D] ([den]) of the multifilament and obtaining the average value and standard deviation of each physical property. The calculation of each coefficient of variation is performed using the following formula. Note that the smaller the value of each coefficient of variation, the smaller the variation in each physical property value. Coefficient of variation (CV) of elastic modulus [%] = Standard deviation of elastic modulus / Average value of elastic modulus × 100 Coefficient of variation (CV) of strength [%] = Standard deviation of strength / Average value of strength × 100 Coefficient of variation (CV) of elongation [%] = Standard deviation of elongation / Average value of elongation × 100 Coefficient of variation (CV) of fineness [%] = Standard deviation of fineness / Average value of fineness × 100

[0301] The measurement of the elastic modulus [gf / D], strength [g / D], and breaking elongation [%] of multifilaments, and the calculation of each standard deviation can be carried out based on JIS L1013 using a tensile testing machine of the 3345 series manufactured by Instron. The test conditions can be, for example, under an environment of temperature 20°C and relative humidity 65%, with a test length of 300 mm and a test speed of 300 mm / min. The load cell capacity can be appropriately selected according to the fineness of the multifilament (fiber). The measured values can be calculated, for example, as the average value with a sample number n = 5.

[0302] The upper limit value of the coefficient of variation of the elongation of the multifilament is less than 33%. When the coefficient of variation of the elongation is less than 33%, the process passability in post-processes such as twisting, spinning, weaving, knitting, and cutting can be further improved. Also, the lower limit value of the coefficient of variation of the elongation of the multifilament may be 0.01% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, 1% or more, 1.3% or more, 1.4% or more, 1.5% or more, 1.6% or more, 1.7% or more, 1.8% or more, 1.9% or more, 2% or more, 2.1% or more, 2.2% or more, 2.3% or more, 2.4% or more, or 2.5% or more, and it may be appropriately selected in consideration of productivity. The coefficient of variation of the elongation of the multifilament is preferably 32% or less, 31% or less, 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, or 23% or less, more preferably 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, or 11% or less, and even more preferably 10% or less, 9.5% or less, 9% or less, 8.5% or less, 8% or less, 7.5% or less, 7% or less, 6.5% or less, 6% or less, 5.5% or less, or 5% or less.

[0303] Also, the coefficient of variation of the elongation of the multifilament is preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10%, more than 10% to less than 15%, more than 15% to less than 20% or more than 20% to less than 30%, more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10%, more than 10% to less than 15%, more than 15% to less than 20% or more than 20% to less than 25%, more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10%, more than 10% to less than 15% or more than 15% to less than 20%, more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10% or more than 10% to less than 15%, still more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5% or more than 5% to less than 10%, particularly preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1% or more than 1% to less than 5%.

[0304] Also, the coefficient of variation of the elongation of the multifilament is preferably less than 0.01% to less than 33%, 0.01% to 30%, 0.01% to 29%, 0.01% to 28%, 0.01% to 27%, 0.01% to 26%, 0.01% to 25%, 0.01% to 24%, 0.01% to 23%, 0.01% to 22%, 0.01% to 21%, 0.01% to 20%, 0.01% to 19%, 0.01% to 18%, 0.01% to 17%, 0.01% to 16%, 0.01% to 15%, 0.01% to 14%, 0.01% to 13%, 0.01% to 12%, 0.01% to 11%, 0.01% to 10%, 0.01% to 9.5%, 0.01% to 9%, 0.01% to 8.5%, 0.01% to 8%, 0.01% to 7.5%, 0.01% to 7%, 0.01% to 6.5%, 0.01% to 6%, 0.01% to 5.5%, 0.01% to 5%, 0.01% to 4.5%, 0.01% to 4%, 0.01% to 3.5%, 0.01% to 3% or 0.01% to 2.5%.

[0305] In addition, the coefficient of variation of the elongation of the multifilament may be 0.1% to 32%, 0.1% to 31%, 0.1% to 30%, 0.1% to 29%, 0.1% to 28%, 0.1% to 27%, 0.1% to 26%, 0.1% to 25%, 0.1% to 24%, 0.1% to 23%, 0.1% to 22%, 0.1% to 21%, 0.1% to 20%, 0.1% to 19%, 0.1% to 18%, 0.1% to 17%, 0.1% to 16%, 0.1% to 15%, 0.1% to 14.5%, 0.1% to 14%, 0.1% to 13.5%, 0.1% to 13%, 0.1% to 12.5%, 0.1% to 12%, 0.1% to 11.5%, 0.1% to 11%, 0.1% to 10.5%, 0.1% to 10%, 0.1% to 9.5%, 0.1% to 9%, 0.1% to 8.5%, 0.1% to 8%, 0.1% to 7.5%, 0.1% to 7%, 0.1% to 6.5%, 0.1% to 6%, 0.1% to 5.5%, 0.1% to 5%, 0.1% to 4.5%, 0.1% to 4%, 0.1% to 3.5%, 0.1% to 3% or 0.1% to 2.5%, may also be less than 0.5% to 33%, 0.5% to 32%, 0.5% to 31%, 0.5% to 30%, 0.5% to 29%, 0.5% to 28%, 0.5% to 27%, 0.5% to 26%, 0.5% to 25%, 0.5% to 24%, 0.5% to 23%, 0.5% to 22%, 0.5% to 21%, 0.5% to 20%, 0.5% to 19%, 0.5% to 18%, 0.5% to 17%, 0.5% to 16%, 0.5% to 15%, 0.5% to 14.5%, 0.5% to 14%, 0.5% to 13.5%, 0.5% to 13%, 0.5% to 12.5%, 0.5% to 12%, 0.5% to 11.5%, 0.5% to 10%, 0.5% to 9.5%, 0.5% to 9%, 0.5% to 8.5%, 0.5% to 8%, 0.5% to 7.5%, 0.5% to 7%, 0.5% to 6.5%, 0.5% to 6%, 0.5% to 5.5%, 0.5% to 5%, 0.5% to 4.5%, 0.5% to 4%, 0.5% to 3.5%, 0.5% to 3% or 0.5% to 2.5%, may also be less than 0.8% to 33%, 0.8% to 32%, 0.8% to 31%, 0.8% to 30%, 0.8% to 29%, 0.8% to 28%, 0.8% to 27%, 0.8% to 26%, 0.8% to 25%, 0.8% to 24%, 0.8% to 23%, 0.8% to 22%, 0.8% to 21%, 0.8% to 20%, 0.8% to 19%, 0.8% to 18%, 0.8% to 17%, 0.8% to 16%, 0.8% to 15%, 0.It may be 8% to 14.5%, 0.8% to 14%, 0.8% to 13.5%, 0.8% to 13%, 0.8% to 12.5%, 0.8% to 12%, 0.8% to 11.5%, 0.8% to 10%, 0.8% to 9.5%, 0.8% to 9%, 0.8% to 8.5%, 0.8% to 8%, 0.8% to 7.5%, 0.8% to 7%, 0.8% to 6.5%, 0.8% to 6% or 0.8% to 5.5%, and may be less than 1% to 33%, 1% to 32%, 1% to 31%, 1% to 30%, 1% to 29%, 1% to 28%, 1% to 27%, 1% to 26%, 1% to 25%, 1% to 24%, 1% to 23%, 1% to 22%, 1% to 21%, 1% to 20%, 1% to 19%, 1% to 18%, 1% to 17%, 1% to 16%, 1% to 15%, 1% to 14.5%, 1% to 14%, 1% to 13.5%, 1% to 13%, 1% to 12%, 1% to 12%, 1% to 11.5%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6.5%, 1% to 6%, 1% to 5.5%, 1% to 5% or 1% to 4%, and may be less than 1.5% to 33%, 1.5% to 32%, 1.5% to 31%, 1.5% to 30%, 1.5% to 29%, 1.5% to 28%, 1.5% to 27%, 1.5% to 26%, 1.5% to 25%, 1.5% to 24%, 1.5% to 23%, 1.5% to 22%, 1.5% to 21%, 1.5% to 20%, 1.5% to 19%, 1.5% to 18%, 1.5% to 17%, 1.5% to 16%, 1.5% to 15%, 1.5% to 14.5%, 1.5% to 14%, 1.5% to 13.5%, 1.5% to 13%, 1.5% to 12%, 1.5% to 12%, 1.5% to 11.5%, 1.5% to 10%, 1.5% to 9%, 1.5% to 8%, 1.5% to 7%, 1.5% to 6.5%, 1.5% to 6%, 1.5% to 5.5% or 1.5% to 5%, and may be less than 2% to 33%, 2% to 32%, 2% to 31%, 2% to 30%, 2% to 29%, 2% to 28%, 2% to 27%, 2% to 26%, 2% to 25%, 2% to 24%, 2% to 23%, 2% to 22%, 2% to 21%, 2% to 20%, 2% to 19%, 2% to 18%, 2% to 17%, 2% to 16%, 2% to 15%, 2% to 14.5%, 2% to 14%, 2% to 13.5%, 2% to 13%, 2% to 12%, 2% to 12%, 2% to 11.5%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 2% to 6.5%, 2% to 6%, 2% to 5.It may be 5%, 2% to 5% or 2% to 4%, and may also be 2.5% to 10%, 2.5% to 9%, 2.5% to 8%, 2.5% to 7%, 2.5% to 6%, 2.5% to 5.5%, 2.5% to 5%, 2.5% to 4.5% or 2.5% to 4%.

[0306] The value of the elongation of the multifilament may be appropriately selected according to the application. For example, it may be 1% to 100%, and may also be 3% to 100%, 5% to 100%, 5% to 95%, 5% to 90%, 5% to 85%, 5% to 80%, 5% to 70%, 5% to 65%, 5% to 60%, 5% to 55%, 5% to 50%, 8% to 100%, 8% to 95%, 8% to 90%, 8% to 85%, 8% to 80%, 8% to 75%, 8% to 70%, 8% to 65%, 8% to 60%, 8% to 55%, 8% to 50%, 10% to 100%, 10% to 95%, 10% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55% or 10% to 50%.

[0307] The upper limit value of the coefficient of variation of the strength of the multifilament is more preferably 20% or less. When the coefficient of variation of the strength of the multifilament is 20% or less, the process passability in post-processes such as twisting, spinning, weaving, knitting and cutting can be further improved. Also, the lower limit value of the coefficient of variation of the strength may be 0.01% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more or 0.6% or more, and may be appropriately selected in consideration of productivity. The coefficient of variation of the strength of the multifilament is preferably 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14.5% or less, 14% or less, 13.5% or less, 13% or less, 12.5% or less, 12% or less, 11.5% or less or 11% or less, and more preferably 10.5% or less, 10% or less, 9.5% or less, 9% or less, 8.5% or less, 8% or less, 7.5% or less, 7% or less, 6.5% or less, 6% or less, 5.5% or less, 5% or less, 4.5% or less, 4% or less, 3.5% or less, 3% or less or 2.5% or less.

[0308] Also, it is preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10%, or more than 10% to less than 15%, more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, or more than 5% to less than 10%, more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, or more than 1% to less than 5%, still more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, or more than 1% to 3.8%, particularly preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, or more than 1% to less than 3.5%. Further, the coefficient of variation of the strength of the multifilament is preferably 0.01% to 20%, 0.01% to 19%, 0.01% to 18%, 0.01% to 17%, 0.01% to 16%, 0.01% to 15%, 0.01% to 14.5%, 0.01% to 14%, 0.01% to 13.5%, 0.01% to 13%, 0.01% to 12.5%, 0.01% to 12%, 0.01% to 11.5%, 0.01% to 11%, 0.01% to 10.5%, 0.01% to 10%, 0.01% to 9.5%, 0.01% to 9%, 0.01% to 8.5%, 0.01% to 8%, 0.01% to 7.5%, 0.01% to 7%, 0.01% to 6.5%, 0.01% to 6%, 0.01% to 5.5%, 0.01% to 5%, 0.01% to 4.5%, 0.01% to 4%, 0.01% to 3.8%, 0.01% to 3.5%, 0.01% to 3%, 0.01% to 2.5%, 0.01% to 2%, 0.01% to 1.5%, or 0.01% to 1%, and may be 0.1% to 20%, 0.1% to 19%, 0.1% to 18%, 0.1% to 17%, 0.0.1% to 16%, 0.1% to 15%, 0.1% to 14.5%, 0.1% to 14%, 0.1% to 13.5%, 0.1% to 13%, 0.1% to 12.5%, 0.1% to 12%, 0.1% to 11.5%, 0.1% to 11%, 0.1% to 10.5%, 0.1% to 10%, 0.1% to 9.5%, 0.1% to 9%, 0.1% to 8.5%, 0.1% to 8%, 0.1% to 7.5%, 0.1% to 7%, 0.1% to 6.5%, 0.1% to 6%, 0.1% to 5.5%, 0.1% to 5%, 0.1% to 4.5%, 0.1% to 4%, 0.1% to 3.5%, 0.1% to 3%, 0.1% to 2.5%, 0.1% to 2%, 0.1% to 1.5%, or 0.1% to 1%, and also 0.2% to 20%, 0.2% to 19%, 0.It may be 2% to 18%, 0.2% to 17%, 0.02% to 16%, 0.2% to 15%, 0.2% to 14.5%, 0.2% to 14%, 0.2% to 13.5%, 0.2% to 13%, 0.2% to 12.5%, 0.2% to 12%, 0.2% to 11.5%, 0.2% to 11%, 0.2% to 10.5%, 0.2% to 10%, 0.2% to 9.5%, 0.2% to 9%, 0.2% to 8.5%, 0.2% to 8%, 0.2% to 7.5%, 0.2% to 7%, 0.2% to 6.5%, 0.2% to 6%, 0.2% to 5.5%, 0.2% to 5%, 0.2% to 4.5%, 0.2% to 4%, 0.2% to 3.5%, 0.2% to 3% or 0.2% to 2.5%. Also, it may be 0.3% to 20%, 0.3% to 19%, 0.3% to 18%, 0.3% to 17%, 0.03% to 16%, 0.3% to 15%, 0.3% to 14.5%, 0.3% to 14%, 0.3% to 13.5%, 0.3% to 13%, 0.3% to 12.5%, 0.3% to 12%, 0.3% to 11.5%, 0.3% to 11%, 0.3% to 10.5%, 0.3% to 10%, 0.3% to 9.5%, 0.3% to 9%, 0.3% to 8.5%, 0.3% to 8%, 0.3% to 7.5%, 0.3% to 7%, 0.3% to 6.5%, 0.3% to 6%, 0.3% to 5.5%, 0.3% to 5%, 0.3% to 4.5%, 0.3% to 4%, 0.3% to 3.5%, 0.3% to 3% or 0.3% to 2.5%. Also, it may be 0.4% to 20%, 0.4% to 19%, 0.4% to 18%, 0.4% to 17%, 0.04% to 16%, 0.4% to 15%, 0.4% to 14.5%, 0.4% to 14%, 0.4% to 13.5%, 0.4% to 13%, 0.4% to 12.5%, 0.4% to 12%, 0.4% to 11.5%, 0.4% to 11%, 0.4% to 10.5%, 0.4% to 10%, 0.4% to 9.5%, 0.4% to 9%, 0.4% to 8.5%, 0.4% to 8%, 0.4% to 7.5%, 0.4% to 7%, 0.4% to 6.5%, 0.4% to 6%, 0.4% to 5.5%, 0.4% to 5%, 0.4% to 4.5%, 0.4% to 4%, 0.4% to 3.5%, 0.4% to 3% or 0.4% to 2.5%. Also, it may be 0.5% to 20%, 0.5% to 19%, 0.5% to 18%, 0.5% to 17%, 0.05% to 16%, 0.5% to 15%, 0.5% to 14.5%, 0.5% to 14%, 0.5% to 13.5%, 0.5% to 13%, 0.5% to 12.5%, 0.It may be 5% to 12%, 0.5% to 11.5%, 0.5% to 11%, 0.5% to 10.5%, 0.5% to 10%, 0.5% to 9.5%, 0.5% to 9%, 0.5% to 8.5%, 0.5% to 8%, 0.5% to 7.5%, 0.5% to 7%, 0.5% to 6.5%, 0.5% to 6%, 0.5% to 5.5%, 0.5% to 5%, 0.5% to 4.5%, 0.5% to 4%, 0.5% to 3.5%, 0.5% to 3% or 0.5% to 2.5%.

[0309] The upper limit of the coefficient of variation of the elastic modulus of the modified fibroin multifilament is preferably 20% or less. When the coefficient of variation of the elastic modulus is 20% or less, the process passability in post-processes such as twisting, spinning, weaving, knitting, and cutting can be further improved. Also, the lower limit of the coefficient of variation of the elastic modulus of the multifilament may be 0.01% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, or 1% or more, and it may be appropriately selected in consideration of productivity. The coefficient of variation of the elastic modulus of the multifilament is more preferably 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14.5% or less, 14% or less, 13.5% or less, 13% or less, 12.5% or less, 12% or less, 11.5% or less, or 11% or less, and even more preferably 10.5% or less, 10% or less, less than 10%, 9.5% or less, 9% or less, 8.5% or less, 8% or less, 7.5% or less, 7% or less, 6.5% or less, 6% or less, 5.5% or less, or 5% or less.

[0310] In addition, the coefficient of variation of the elastic modulus of the multifilament is preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10% or more than 10% to less than 15%, more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5% or more than 5% to less than 10%, and even more preferably less than 0.01% to less than 0.1%, more than 0.1% to less than 1% or more than 1% to less than 5%. Also, the coefficient of variation of the elastic modulus of the multifilament is preferably 0.01% to 20%, 0.01% to 19%, 0.01% to 18%, 0.01% to 17%, 0.01% to 16%, 0.01% to 15%, 0.01% to 14.5%, 0.01% to 14%, 0.01% to 13.5%, 0.01% to 13%, 0.01% to 12.5%, 0.01% to 12%, 0.01% to 11.5%, 0.01% to 11%, 0.01% to 10.5%, 0.01% to 10%, 0.01% to 9.5%, 0.01% to 9%, 0.01% to 8.5%, 0.01% to 8%, 0.01% to 7.5%, 0.01% to 7%, 0.01% to 6.5%, 0.01% to 6%, 0.01% to 5.5%, 0.01% to 5%, 0.01% to 4.5%, 0.01% to 4%, 0.01% to 3.5%, 0.01% to 3%, 0.01% to 2.5% or 0.01% to 2%, or 0.1% to 20%, 0.1% to 19%, 0.1% to 18%, 0.1% to 17%, 0.0.1% to 16%, 0.1% to 15%, 0.1% to 14.5%, 0.1% to 14%, 0.1% to 13.5%, 0.1% to 13%, 0.1% to 12.5%, 0.1% to 12%, 0.1% to 11.5%, 0.1% to 11%, 0.1% to 10.5%, 0.1% to 10%, 0.1% to 9.5%, 0.1% to 9%, 0.1% to 8.5%, 0.1% to 8%, 0.1% to 7.5%, 0.1% to 7%, 0.1% to 6.5%, 0.1% to 6%, 0.1% to 5.5%, 0.1% to 5%, 0.1% to 4.5%, 0.1% to 4%, 0.1% to 3.5%, 0.1% to 3%, 0.1% to 2.5%, 0.1% to 2%, and may also be 0.2% to 20%, 0.2% to 19%, 0.2% to 18%, 0.2% to 17%, 0.2% to 16%, 0.2% to 15%, 0.2% to 14.5%, 0.2% to 14%, 0.2% to 13.5%, 0.2% to 13%, 0.2% to 12.5%, 0.2% to 12%, 0.2% to 11.5%, 0.2% to 11%, 0.2% to 10.5, 0.It may be 2% to 10%, 0.2% to 9.5%, 0.2% to 9%, 0.2% to 8.5%, 0.2% to 8%, 0.2% to 7.5%, 0.2% to 7%, 0.2% to 6.5%, 0.2% to 6%, 0.2% to 5.5%, 0.2% to 5%, 0.2% to 4.5%, 0.2% to 4%, 0.2% to 3.5%, 0.2% to 3% or 0.2% to 2.5%. Also, it may be 0.3% to 20%, 0.3% to 19%, 0.3% to 18%, 0.3% to 17%, 0.3% to 16%, 0.3% to 15%, 0.3% to 14.5%, 0.3% to 14%, 0.3% to 13.5%, 0.3% to 13%, 0.3% to 12.5%, 0.3% to 12%, 0.3% to 11.5%, 0.3% to 11%, 0.3% to 10.5%, 0.3% to 10%, 0.3% to 9.5%, 0.3% to 9%, 0.3% to 8.5%, 0.3% to 8%, 0.3% to 7.5%, 0.3% to 7%, 0.3% to 6.5%, 0.3% to 6%, 0.3% to 5.5%, 0.3% to 5%, 0.3% to 4.5%, 0.3% to 4%, 0.3% to 3.5%, 0.3% to 3% or 0.3% to 2.5%. Also, it may be 0.4% to 20%, 0.4% to 19%, 0.4% to 18%, 0.4% to 17%, 0.4% to 16%, 0.4% to 15%, 0.4% to 14.5%, 0.4% to 14%, 0.4% to 13.5%, 0.4% to 13%, 0.4% to 12.5%, 0.4% to 12%, 0.4% to 11.5%, 0.4% to 11%, 0.4% to 10.5%, 0.4% to 10%, 0.4% to 9.5%, 0.4% to 9%, 0.4% to 8.5%, 0.4% to 8%, 0.4% to 7.5%, 0.4% to 7%, 0.4% to 6.5%, 0.4% to 6%, 0.4% to 5.5%, 0.4% to 5%, 0.4% to 4.5%, 0.4% to 4%, 0.4% to 3.5%, 0.4% to 3% or 0.4% to 2.5%. Also, it may be 0.5% to 20%, 0.5% to 19%, 0.5% to 18%, 0.5% to 17%, 0.0.5% to 16%, 0.5% to 15%, 0.5% to 14.5%, 0.5% to 14%, 0.5% to 13.5%, 0.5% to 13%, 0.5% to 12.5%, 0.5% to 12%, 0.5% to 11.5%, 0.5% to 11%, 0.5% to 10.5%, 0.5% to 10%, 0.5% to 9.5%, 0.5% to 9%, 0.5% to 8.5%, 0.5% to 8%, 0.5% to 7.5%, 0.5% to 7%, 0.5% to 6.5%, 0.5% to 6%, 0.5% to 5.5%, 0.It may be 5% to 5%, 0.5% to 4.5%, or 0.5% to 4%, and may also be 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, or 1% to 4%.

[0311] The upper limit value of the coefficient of variation of the fineness of the multifilament is preferably 20% or less. When the coefficient of variation of the fineness is 20% or less, the process passability in subsequent processes such as twisting, spinning, weaving, knitting, and cutting can be further improved. Also, the lower limit value of the coefficient of variation of the fineness of the multifilament may be 0.01% or more, 0.1% or more, 0.2% or more, 0.3% or more, or 0.4% or more, and it may be appropriately selected in consideration of productivity. The coefficient of variation of the fineness of the multifilament is preferably 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, or 11% or less, and more preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less. Also, the coefficient of variation of the fineness of the multifilament is preferably 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10%, more than 10% to less than 15%, or more than 15% to less than 20%, more preferably 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, more than 5% to less than 10%, or more than 10% to less than 15%, more preferably 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, or more than 5% to less than 10%, more preferably 0.01% to less than 0.1%, more than 0.1% to less than 1%, more than 1% to less than 5%, or more than 5% to 6.7%, still more preferably 0.01% to less than 0.1%, more than 0.1% to less than 1%, or more than 1% to less than 5%, and particularly preferably 0.01% to less than 0.1%, more than 0.1% to less than 1%, or more than 1% to less than 3.5%.

[0312] Also, the coefficient of variation of the fineness of the multifilament is preferably 0.01% to 15%, 0.01% to 14%, 0.01% to 13%, 0.01% to 12%, 0.01% to 11%, 0.01% to 10%, 0.01% to 9.5%, 0.01% to 9%, 0.01% to 8.5%, 0.01% to 8%, 0.01% to 7.5%, 0.01% to 7%, 0.01% to 6.7%, 0.01% to 6.5%, 0.01% to 6%, 0.01% to 5.5%, 0.01% to 5%, 0.01% to 4.5%, 0.01% to 4%, 0.01% to 3.5%, 0.01% to 3%, 0.01% to 2.5%, 0.01% to 2%, 0.01% to 1.5%, 0.01% to 1%. Also, the coefficient of variation of the fineness of the multifilament may be 0.1% to 20%, 0.1% to 19%, 0.1% to 18%, 0.1% to 17%, 0.1% to 16%, 0.1% to 15%, 0.1% to 14%, 0.1% to 13%, 0.1% to 12%, 0.1% to 11%, 0.1% to 10%, 0.1% to 9.5%, 0.1% to 9%, 0.1% to 8.5%, 0.1% to 8%, 0.1% to 7.5%, 0.1% to 7%, 0.1% to 6.5%, 0.1% to 6%, 0.1% to 5.5%, 0.1% to 5%, 0.1% to 4.5%, 0.1% to 4%, 0.1% to 3.5%, 0.1% to 3%, 0.1% to 2.5%, 0.1% to 2%, 0.1% to 1.5% or 0.1% to 1%, and may be 0.2% to 20%, 0.2% to 19%, 0.2% to 18%, 0.2% to 17%, 0.2% to 16%, 0.2% to 15%, 0.2% to 14%, 0.2% to 13%, 0.2% to 12%, 0.2% to 11%, 0.2% to 10%, 0.2% to 9.5%, 0.2% to 9%, 0.2% to 8.5%, 0.2% to 8%, 0.2% to 7.5%, 0.2% to 7%, 0.2% to 6.5%, 0.2% to 6%, 0.2% to 5.5%, 0.2% to 5%, 0.2% to 4.5%, 0.2% to 4%, 0.2% to 3.5%, 0.2% to 3% or 0.2% to 2.5%, and may be 0.3% to 20%, 0.3% to 19%, 0.3% to 18%, 0.3% to 17%, 0.3% to 16%, 0.3% to 15%, 0.3% to 14%, 0.3% to 13%, 0.3% to 12%, 0.3% to 11%, 0.3% to 10%, 0.3% to 9.5%, 0.3% to 9%, 0.3% to 8.5%, 0.3% to 8%, 0.3% to 7.5%, 0.3% to 7%, 0.3% to 6.5%, 0.3% to 6%, 0.3% to 5.5%, 0.It may be 3% to 5%, 0.3% to 4.5%, 0.3% to 4%, 0.3% to 3.5%, 0.3% to 3% or 0.3% to 2.5%, and may be 0.4% to 20%, 0.4% to 19%, 0.4% to 18%, 0.4% to 17%, 0.4% to 16%, 0.4% to 15%, 0.5% to 14%, 0.4% to 13%, 0.4% to 12%, 0.4% to 11%, 0.4% to 10%, 0.4% to 9.5%, 0.4% to 9%, 0.4% to 8.5%, 0.4% to 8%, 0.4% to 7.5%, 0.4% to 7%, 0.4% to 6.5%, 0.4% to 6%, 0.4% to 5.5%, 0.4% to 5%, 0.4% to 4.5%, 0.4% to 4%, 0.4% to 3.5% or 0.4% to 3%, 0.4% to 2.5%, and may be 0.5% to 20%, 0.5% to 19%, 0.5% to 18%, 0.5% to 17%, 0.5% to 16%, 0.5% to 15%, 0.5% to 14%, 0.5% to 13%, 0.5% to 12%, 0.5% to 11%, 0.5% to 10%, 0.5% to 9.5%, 0.5% to 9%, 0.5% to 8.5%, 0.5% to 8%, 0.5% to 7.5%, 0.5% to 7%, 0.5% to 6.5%, 0.5% to 6%, 0.5% to 5.5%, 0.5% to 5%, 0.5% to 4.5%, 0.5% to 4%, 0.5% to 3.5%, 0.5% to 3% or 0.5% to 2.5%, and may be 0.3% to 3% or 0.3% to 2.8%.

[0313] The value of the fineness [D] ([den]) of the multifilament may be appropriately selected according to the intended use. For example, the fineness per single filament ([D / filament]) may be 0.7 to 150 D, 0.7 to 140, 0.7 to 130 D, 0.7 to 120 D, 0.7 to 110 D, 0.8 to 100 D, 0.7 to 100 D, 0.7 to 90 D, 0.7 to 80 D, 0.7 to 70 D, 0.7 to 60 D, 0.7 to 50 D, 0.7 to 40 D, 0.7 to 30 D, 0.7 to 20 D, 0.7 to 15 D, 0.7 to 10 D, 0.7 to 9 D, 0.7 to 8 D, 0.7 to 7 D, 0.7 to 6 D, 0.7 to 5 D, 0.7 to 4 D, 0.7 to 3 D, 0.7 to 2.5 D, 0.7 to 2.2 D, 0.7 to 2 D, 0.7 to 1.8 D, 0.7 to 1.6 D, 0.7 to 1.5 D, 0.7 to 1.4 D, 0.7 to 1.3 D, 0.7 to 1.2 D, 0.7 to 1.1 D or 0.7 to 1 D. It may also be 0.8 to 3 D, 0.8 to 2.5 D, 0.8 to 2.2 D, 0.8 to 2 D, 0.8 to 1.8 D, 0.8 to 1.6 D, 0.8 to 1.5 D, 0.8 to 1.4 D, 0.8 to 1.3 D, 0.8 to 1.2 D, 0.8 to 1.1 D or 0.8 to 1 D. It may also be 1 to 3 D, 1 to 2.5 D, 1 to 2.2 D, 1 to 2 D, 1 to 1.9 D, 1 to 1.8 D, 1 to 1.7 D or 1 to 1.6 D. The density of the recombinant structural protein is, for example, 1.3 to 1.4 [g / cm 3 and may also be 1.35 [g / cm 3 or 1.34 [g / cm 3 .

[0314] The multifilament according to this embodiment may have a shrinkage history that has irreversibly shrunk after spinning. The shrinkage history is a shrinkage history that has irreversibly shrunk by bringing the multifilament into contact with water, and / or a shrinkage history that has irreversibly shrunk by heat-relaxing the multifilament. Since the multifilament according to this embodiment is obtained, for example, by the above-described manufacturing method, it substantially does not contain residual stress generated by stretching during the spinning process.

[0315] <Shrinkage rate> The multifilament having a shrinkage history that is irreversibly shrunk after spinning according to this embodiment preferably has a shrinkage rate defined by the following formula of 5% or less. Note that the multifilament in the following formula is a multifilament having a shrinkage history that is irreversibly shrunk after spinning. Shrinkage rate [%] = (1 - (length of the multifilament when dried from the wet state / length of the multifilament when in the wet state)) × 100

[0316] The shrinkage property due to contact with moisture in the fiber can be evaluated, for example, using the shrinkage rate obtained by the above formula as an index. The "length of the multifilament when in the wet state" and the "length of the multifilament when dried from the wet state" can be measured, for example, by the following method.

[0317] Cut the multifilament into a length of about 30 cm to form a fiber bundle. Immerse this fiber bundle in water at 40°C for 15 minutes (wet), leave it at room temperature for 2 hours, and then dry it. After drying, measure the length of the fiber bundle. Repeat wetting and drying at least 3 times again, and the average length during wetting can be taken as the "length of the multifilament when in the wet state", and the average length during drying can be taken as the "length of the multifilament when dried from the wet state".

[0318] In the multifilament, it is more preferable that such shrinkage is less. In particular, in products such as fabrics made of multifilaments, it is preferable that this shrinkage is less.

[0319] The shrinkage rate defined by the above formula is preferably 5.0% or less, and may be 4.5% or less, 4% or less, 3.5% or less, 3.2% or less, 3.1% or less, 3.0% or less, 2.9% or less, 2.8% or less, 2.7% or less, 2.6% or less, 2.5% or less, 2.4% or less, 2.3% or less, 2.2% or less, 2.1% or less, 2.0% or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, or 0.3% or less.

[0320] 〔Product〕 The modified fibroin multifilament according to this embodiment can be applied as a fiber or a yarn (such as a spun yarn, a twisted yarn, a false-twisted yarn, a processed yarn, a mixed fiber yarn, or a blended yarn), a woven fabric (cloth), a knitted fabric, a braided fabric, or a non-woven fabric, as well as paper and cotton. It can also be applied to high-strength applications such as ropes, surgical sutures, flexible stoppers for electrical components, and further bioactive materials for transplantation (such as artificial ligaments and aortic bands). These can be manufactured according to known methods.

[0321] The modified fibroin fiber may have a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 29 or more, or 30 or more. The above LOI value is a value measured in accordance with the test method for powdery or low-melting synthetic resins by the Chief of the Dangerous Goods Regulation Section of the Fire and Disaster Management Agency, Fire Hazard No. 50, May 31, 1995.

[0322] The modified fibroin fiber may have a maximum moisture absorption heat generation degree of more than 0.025 °C / g, which may be 0.026 °C / g or more, 0.027 °C / g or more, 0.028 °C / g or more, 0.029 °C / g or more, 0.030 °C / g or more, 0.035 °C / g or more, 0.040 °C / g or more, as determined according to the following formula A. There is no particular limitation on the upper limit of the maximum moisture absorption heat generation degree, but it is usually 0.060 °C / g or less. Formula A: Maximum moisture absorption heat generation degree ={(the maximum value of the sample temperature when the sample is transferred to a high humidity environment after being placed in a low humidity environment until the sample temperature reaches equilibrium)-(the sample temperature when the sample is transferred to a high humidity environment after being placed in a low humidity environment until the sample temperature reaches equilibrium)}(°C) / sample weight (g)

[0323] The modified fibroin fiber preferably has excellent heat retention properties, and the heat retention index determined according to the following formula C may be 0.20 or more. Formula C: Heat retention index = heat retention rate (%) / basis weight of the sample (g / m 2 )

[0324] The heat retention index of the modified fibroin fiber may be 0.22 or more, 0.24 or more, 0.26 or more, 0.28 or more, 0.30 or more, 0.32 or more. There is no particular limitation on the upper limit of the heat retention index, but for example, it may be 0.60 or less, or 0.40 or less.

Example

[0325] 〔Production of modified fibroin〕 (1) Preparation 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), modified spider silk fibroin having SEQ ID NO: 40 (hereinafter also referred to as "PRT966") and modified spider silk fibroin having SEQ ID NO: 15 (hereinafter also referred to as PRT799) were designed. The amino acid sequence represented by SEQ ID NO: 40 has a sequence in which all QQ in the sequence obtained by repeating the region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 twice for the purpose of improving the hydrophobicity are replaced with VF, and the remaining Qs are replaced with I, and further, the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminus. The amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence obtained by performing substitution, insertion, and deletion of amino acid residues on the amino acid sequence of fibroin derived from Nephila clavipes for the purpose of improving productivity, and further, the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminus.

[0326] Next, nucleic acids encoding artificial structural proteins (modified fibroins) PRT966 and PRT799 having the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 15 designed above were synthesized. An NdeI site was added to the 5'-end and an EcoRI site was added downstream of the stop codon to the nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, 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.

[0327] (2) Expression of modified fibroin (1) Escherichia coli BLR(DE3) was transformed with the expression vector obtained in (1). The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that OD 600 became 0.005. The culture solution temperature was maintained at 30 °C, and OD 600Flask culture was carried out until it reached 5 (about 15 hours) to obtain a seed culture solution.

[0328]

Table 4

[0329] The seed culture solution was added to a jar fermenter supplemented with 500 mL of production medium (Table 5) so that the OD 600 became 0.05. The culture solution temperature was maintained at 37°C and cultured under constant control at pH 6.9. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

[0330]

Table 5

[0331] Immediately after the glucose in the production medium was completely consumed, a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The culture solution temperature was maintained at 37°C and cultured under constant control at pH 6.9. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of modified fibroin. 20 hours after the addition of IPTG, the culture solution was centrifuged to collect the cells. 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 modified fibroin was confirmed by the appearance of a band of the target modified fibroin size depending on the addition of IPTG.

[0332] (3) Purification of modified fibroin The cells harvested 2 hours after adding 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 using a high-pressure homogenizer (manufactured by GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity was achieved. 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) at a concentration of 100 mg / mL, stirred with a stirrer at 60 °C for 30 minutes, and dissolved. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36 / 32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, and the moisture was removed using a freeze dryer to recover the freeze-dried powder, thereby obtaining modified fibroin (PRT966 and PRT799).

[0333] [Production of Modified Fibroin Multifilament] (1) Preparation of Spinning Dope 26% by mass of the modified fibroin (PRT966) obtained in the production process of the above-mentioned modified fibroin and 74% by mass of formic acid (manufactured by Asahi Chemical Industry Co., Ltd., purity 98%) as a dissolving solvent were mixed, heated at 40 °C with an aluminum block heater for 1 hour while stirring, and dissolved. It was filtered through a metal filter with a pore size of 1 μm and degassed to obtain a spinning dope.

[0334] (2) Dry-Wet Spinning (Example 1) Spinning was carried out using the spinning apparatus shown in Fig. 6. The prepared spinning dope was filled into a reserve tank (storage tank), and the spinning dope was discharged from a spinning nozzle (spinning die) having 100 holes through an air gap into a coagulation bath using a gear pump to form a raw yarn. Subsequently, the coagulated raw yarn was drawn in a water washing bath. After washing and drawing in the water washing bath, it was dried using a hot plate, and the obtained modified fibroin multifilament was wound up with a winder. The number of filaments constituting the multifilament was 100. The conditions for dry-wet spinning were as follows. Diameter of the holes of the spinning die: 0.08 mm Number of holes of the spinning nozzle: 100 Coagulating liquid: 100% methanol Temperature of the coagulating liquid: 10 °C Temperature of the water washing bath: 40 °C Temperature of the drawing bath: 40 °C Total draw ratio: 4.4 times Drying temperature: 70 °C

[0335] (Example 2) Dry-wet spinning was carried out in the same manner as in Example 1 except that a spinning nozzle having a hole diameter of 0.1 mm and 360 holes was used and the total draw ratio was 5.3 times to produce a modified fibroin multifilament. The number of filaments constituting the multifilament was 360.

[0336] (3) Wet spinning (Example 3) Spinning was carried out using the spinning apparatus shown in Fig. 7. The prepared spinning dope was filled into a reserve tank, and the spinning dope was discharged from a spinning nozzle having 200 holes into a coagulation bath using a gear pump to form a raw yarn (yarn). Subsequently, the coagulated raw yarn was drawn in a water washing bath. After washing and drawing in the water washing bath, it was dried using a hot plate, and the obtained modified fibroin multifilament was wound up with a winder. The number of filaments constituting the multifilament was 200. Further, concave portions extending in the filament axis direction were formed on the surface of the obtained multifilament. The conditions for wet spinning were as follows. Diameter of the holes of the spinning nozzle: 0.05 mm Number of holes in the spinning nozzle: 200 Coagulating liquid: A mixed solution of 14.4% by mass of sodium sulfate and 65.6% by mass of water (80% by mass of 18% by mass sodium sulfate aqueous solution), and 20% by mass of formic acid Temperature of the coagulating liquid: 40°C Temperature of the water washing bath: 40°C Temperature of the stretching bath: 60°C Total draw ratio: 4.3 times Drying temperature: 60°C

[0337] (Example 4) Using a spinning nozzle having a pore diameter of 0.1 mm and 1,000 holes, wet spinning was carried out in the same manner as in Example 3 except that the total draw ratio was 6.2 times to produce a modified fibroin multifilament. The number of filaments constituting the obtained multifilament was 1,000. Further, the obtained multifilament had recesses extending in the filament axis direction formed on the surface (Fig. 13).

[0338] (Example 9) The concentration of the spinning dope was 31% by mass. Using a spinning nozzle having a pore diameter of 0.08 mm and 1,664 holes, wet spinning was carried out in the same manner as in Example 3 except that the total draw ratio was 5.7 times to produce a multifilament. The number of filaments constituting the obtained multifilament was 1,664.

[0339] (Example 10) The concentration of the spinning dope was 31% by mass. Using a spinning nozzle having a pore diameter of 0.1 mm and 2,328 holes, wet spinning was carried out in the same manner as in Example 3 except that the total draw ratio was 5.7 times to produce a multifilament. The number of filaments constituting the obtained multifilament was 2,328.

[0340] (Example 11) The spinning dope concentration was set at 31% by mass. Wet spinning was carried out in the same manner as in Example 3, except that a spinning nozzle with a pore diameter of 0.08 mm and 3,000 holes was used and the total draw ratio was 5.7 times, to produce a multifilament. The number of filaments constituting the obtained multifilament was 3,000. A cross-sectional view of the multifilament is shown in Fig. 14.

[0341] 〔Evaluation of Modified Fibroin Multifilament〕 The elastic modulus [gf / D], strength [g / D], elongation at break [%], and fineness [D] of the modified fibroin multifilaments obtained in Examples 1 to 4 and Examples 9 to 11 were measured. Using the following formulas, the coefficient of variation of the elastic modulus, the coefficient of variation of the strength, the coefficient of variation of the elongation at break, and the coefficient of variation of the fineness were calculated respectively. Coefficient of variation of elastic modulus (CV) [%] = standard deviation of elastic modulus / average value of elastic modulus × 100 Coefficient of variation of strength (CV) [%] = standard deviation of strength / average value of strength × 100 Coefficient of variation of elongation (CV) [%] = standard deviation of elongation / average value of elongation × 100 Coefficient of variation of fineness (CV) [%] = standard deviation of fineness / average value of fineness × 100 In addition, the density [g / cm 3 of the modified fibroin used was 1.34 [g / cm 3 .

[0342] The measurement of the elastic modulus [gf / D], strength [g / D], and elongation at break [%] of the modified fibroin multifilament, and the calculation of each standard deviation were carried out using an Instron 3345 series tensile tester based on JIS L1013. The test conditions were an environment of temperature 20°C and relative humidity 65%, a test length of 300 mm, and a test speed of 300 mm / min. For Examples 1 and 3, a load cell capacity of 10 N was used, and for Examples 2, 4, and 9 to 11, a load cell capacity of 50 N was used for measurement. The calculation of the fineness [D] of the modified fibroin multifilament was carried out by measuring the mass of the multifilament cut to a length of 3 m in an environment of temperature 20°C and relative humidity 65% and converting it to the mass per 9,000 m. The average values of the physical properties of the modified fibroin multifilament in Examples 1 to 4 were calculated as the average values with a sample number n = 5. The average values of the physical properties of the multifilament in Example 9 were calculated as the average values with a sample number n = 10. The average values of the physical properties of the multifilament in Example 10 were calculated as the average values with a sample number n = 15. The average values of the physical properties of the multifilament in Example 11 were calculated as the average values with a sample number n = 30. Using the average values of the physical properties of the measured multifilament (average value of elastic modulus, average value of strength, average value of elongation, and average value of fineness), the values of each coefficient of variation were calculated from the above formula. The evaluation results of each coefficient of variation are shown in Table 6. Also, Table 7 shows the average values and standard deviations of the elongation at break [%] and fineness [D] in Examples 1 to 4 and 9 to 11.

[0343]

Table 6

[0344] As shown in Table 6, by using a spinning nozzle having 100 to 3,000 holes, the productivity could be dramatically improved. Furthermore, the obtained multifilaments (Examples 1 to 4 and Examples 9 to 11) had a coefficient of variation (CV) of elastic modulus of 1.2 to 4.7, a coefficient of variation (CV) of strength of 0.8 to 2.2, a coefficient of variation (CV) of elongation of 2.6 to 5.3, and a coefficient of variation (CV) of fineness of 0.04 to 2.6. The variation in the physical property values of the multifilament was extremely small, and it was significantly excellent in quality stability.

Table 7

[0345] (Examples 5 to 8) (4) Shrinking process and drying process The multifilaments of the modified fibroin obtained in Examples 1 to 4 were cut to a length of about 30 cm, and each multifilament bundle was immersed in water at 40°C for 90 seconds to be shrunk. Then, each multifilament bundle was taken out of the water and dried, and the length of each dried multifilament bundle was measured.

[0346] (5) Evaluation of the shrinkability of multifilaments having an irreversible shrinkage history The shrinkability of the modified fibroin multifilaments obtained in (4) above was evaluated using the shrinkage rate determined by the following method as an index. The number of samples was set to n = 3 and calculated according to the following formula. The calculation results are shown in Table 8. Shrinkage rate [%] = (1 - (length of multifilament when dried from wet state / length of multifilament when made wet state)) × 100

[0347]

Table 8

[0348] As shown in Table 8, the shrinkage rate of the modified fibroin multifilaments after the shrinkage treatment was within ±0.4% (Examples 5 to 8), and they had excellent dimensional stability against moisture. As described above, it was shown that multifilaments having an irreversible shrinkage history are excellent in dimensional stability against moisture, have extremely small relative variations in the physical property values of the multifilaments, and are remarkably excellent in quality stability.

[0349] Test Examples 1 to 3: Peelability test of dope solution (1) Preparation of dope solution 31% by mass of the modified fibroin (PRT966) obtained in the production process of the above-mentioned modified fibroin was mixed with 69% by mass of formic acid (manufactured by Asahi Chemical Industry Co., Ltd., purity 99%) as a solvent for dissolution, and heated at 40 °C with an aluminum block heater for 1 hour while stirring to dissolve it. It was filtered through a metal filter with a pore size of 1 μm and defoamed to prepare a dope solution. (2) Peelability test The peelability of the dope solution with respect to the material of the spinning nozzle (spinning die) was evaluated. The dope solution was dropped on the surfaces of circular test pieces made of different materials within a range of about φ15 mm. These were immersed in the coagulation liquid, and after 5 seconds elapsed, the dope solution (semi-solidified product) was peeled off from the surface of each test piece in the coagulation liquid. The peelability of the dope solution with respect to the spinning nozzle material was evaluated based on the degree of force required to peel off the dope solution (semi-solidified product). The materials of the spinning nozzles used and the evaluation results are shown in Table 9. The coagulation liquid was a mixed solution prepared by mixing an aqueous sodium sulfate solution with a concentration of 18% by mass and formic acid at a ratio of 80% by mass and 20% by mass, respectively. The evaluation index for peelability was as follows. ○: Good peelability (the dope solution (semi-solidified product) can be peeled off with a light force) △: Poor peelability (compared with the material evaluated as ○, a large force is required to peel off the dope solution (semi-solidified product))

Table 9

[0350] As shown in Table 9, compared with the case where the material of the spinning nozzle is Pt / Au, when the material of the spinning nozzle is Hastelloy or SUS316L, it is possible to peel off the dope solution (semi-solidified product) from the surface of the test piece with a lighter force, and it was confirmed that the peelability of the dope solution is excellent. Thus, the recombinant structural protein has the property of being easily adhered (adsorbed) to Pt / Au. In particular, when using a dope solution containing modified fibroin, by using a material (such as Hastelloy or SUS316L) from which the modified fibroin can be easily peeled off as the material of the spinning nozzle, it is possible to further prevent clogging of the spinning nozzle that may occur due to the adhesion of the dope solution to the spinning nozzle, and the spinning stability can be further enhanced. In particular, it is suitable when performing large-scale spinning using a spinning nozzle having more than 100 holes.

[0351] Reference Example 1: Combustibility Test of Modified Fibroin Lyophilized powder of modified fibroin (PRT799) was added to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0% by mass) so that the concentration became 24% by mass, and dissolved by mixing using a shaker for 3 hours. Then, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning dope).

[0352] The obtained spinning dope was heated to 90°C, filtered through a metal filter with an opening of 5 μm, then left standing in a 30 mL stainless steel syringe, defoamed, and then discharged from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The discharge temperature was 90°C. After coagulation, the obtained raw yarn was wound up and naturally dried to obtain modified fibroin fibers (raw material fibers).

[0353] Using a twisted yarn obtained by twisting the raw material fibers, a knitted fabric (thickness: 180 denier, gauge number: 18) was produced by circular knitting using a circular knitting machine. 20 g of the obtained knitted fabric was cut out and used as a test piece.

[0354] The combustion test complied with the "Test Method for Powdery or Low-Melting Synthetic Resins" described in "Fire Hazard No. 50 (dated May 31, 1995)". The test was carried out under the conditions of a temperature of 22°C, a relative humidity of 45%, and an atmospheric pressure of 1021 hPa. The measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)) are shown in Table 10.

Table 10

[0355] As a result of the combustion test, the limiting oxygen index (LOI) value of the fabric knitted with modified fibroin (PRT799) fibers was 27.2. Generally, when the LOI value is 26 or more, it is known to be flame-retardant. It can be seen that modified fibroin is excellent in flame retardancy.

[0356] Reference Example 2: Evaluation of the Moisture Absorption and Heat Generation Properties of Modified Fibroin Lyophilized powder of modified fibroin was added to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0% by mass) to a concentration of 24% by mass, and dissolved by mixing with a shaker for 3 hours. Then, insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning dope).

[0357] The obtained spinning dope was heated to 60°C, filtered through a metal filter with a pore size of 5 μm, then left standing in a 30 mL stainless steel syringe, degassed, and then discharged from a solid nozzle with a needle diameter of 0.2 mm into a 100% methanol coagulation bath. The discharge temperature was 60°C. After coagulation, the obtained raw yarn was wound up and naturally dried to obtain modified fibroin fibers (raw material fibers).

[0358] For comparison, commercially available wool fibers, cotton fibers, tencel fibers, rayon fibers, and polyester fibers were prepared as raw material fibers.

[0359] Using each raw material fiber, knitted fabrics were respectively manufactured by flat knitting using a flat knitting machine. The thickness and gauge number of the knitted fabrics using PRT918 fiber or PRT799 fiber are as shown in Table 11. For the knitted fabrics using other raw material fibers, the thickness and gauge number were adjusted so as to have almost the same cover factor as that of the knitted fabric of the modified fibroin fiber. Specifically, it is as follows.

Table 11

[0360] Two pieces of the knitted fabric cut into 10 cm × 10 cm were combined, and the four sides were sewn together to make a test piece (sample). After leaving the test piece in a low humidity environment (temperature 20 ± 2°C, relative humidity 40 ± 5%) for 4 hours or more, it was transferred to a high humidity environment (temperature 20 ± 2°C, relative humidity 90 ± 5%), and the temperature was measured at 1-minute intervals for 30 minutes using a temperature sensor attached to the center inside the test piece.

[0361] From the measurement results, the maximum moisture absorption heat generation degree was obtained according to the following formula A. Formula A: Maximum moisture absorption heat generation degree = {(the maximum value of the sample temperature when the sample is transferred to a high humidity environment after being placed in a low humidity environment until the sample temperature reaches equilibrium) - (the sample temperature when the sample is transferred to a high humidity environment after being placed in a low humidity environment until the sample temperature reaches equilibrium)} (°C) / sample weight (g)

[0362] Figure 15 is a graph showing an example of the results of the moisture absorption heat generation test. The horizontal axis of the graph represents 0 when the sample is transferred from a low humidity environment to a high humidity environment, and shows the standing time (minutes) in the high humidity environment. The vertical axis of the graph represents the temperature (sample temperature) measured by the temperature sensor. In the graph shown in Figure 15, the point indicated by M corresponds to the maximum value of the sample temperature.

[0363] The calculation results of the maximum moisture absorption heat generation degree of each knitted fabric are shown in Table 12.

Table 12

[0364] As shown in Table 12, it can be seen that the modified fibroins (PRT918 and PRT799) have a higher maximum moisture absorption and heat generation degree and are excellent in moisture absorption and heat generation compared with existing materials.

[0365] Reference Example 3: Evaluation of the heat retention property of modified fibroin Lyophilized powder of modified fibroin was added to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0% by mass) to a concentration of 24% by mass, and dissolved by mixing with a shaker for 3 hours. Then, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning dope).

[0366] The obtained spinning dope was heated to 60 °C, filtered through a metal filter with an aperture of 5 μm, then left standing in a 30 mL stainless steel syringe, degassed, and then discharged from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The discharge temperature was 60 °C. After coagulation, the obtained raw yarn was wound up and naturally dried to obtain modified fibroin fibers (raw material fibers).

[0367] For comparison, commercially available wool fibers, silk fibers, cotton fibers, rayon fibers, and polyester fibers were prepared as raw material fibers.

[0368] Using each raw material fiber, knitted fabrics were respectively produced by flat knitting using a flat knitting machine. The count, twist number, gauge number, and areal density of the knitted fabric using PRT966 fibers or PRT799 fibers are as shown in Table 13. The knitted fabrics using other raw material fibers were adjusted to have almost the same cover factor as the knitted fabric of the modified fibroin fibers. Specifically, it is as follows.

Table 13

[0369] The heat retention property was evaluated using a KES-F7 Thermo Lab II tester manufactured by Kato Tech Co., Ltd. with the dry contact method (a method assuming the situation where the skin and the clothing are in direct contact in a dry state). One piece of knitted fabric cut into a rectangle of 20 cm × 20 cm was used as a test piece (sample). The test piece was set on a hot plate set at a constant temperature (30°C), and under the condition of a wind speed of 30 cm / second in the wind tunnel, the amount of heat dissipated (a) through the test piece was determined. With the test piece not set, the amount of heat dissipated (b) under the same conditions as above was determined, and the heat retention rate (%) was calculated according to the following formula B. Formula B: Heat retention rate (%) = (1 - a / b) × 100

[0370] From the measurement results, the heat retention property index was determined according to the following formula C. Formula C: Heat retention property index = Heat retention rate (%) / Areal density of the sample (g / m 2 )

[0371] The calculation results of the heat retention property index are shown in Table 14. The higher the heat retention property index, the better the material can be evaluated in terms of heat retention property.

[0372]

Table 14

[0373] As shown in Table 14, it can be seen that the modified fibroin (PRT966 and PRT799) has a higher heat retention property index and is superior in heat retention property compared with the existing materials.

[0374] As shown in Reference Examples 1 to 3, when the modified fibroin is modified spider silk fibroin, the heat retention property, the hygroscopic heat generation property and / or the flame retardancy can be made more excellent. By using the modified spider silk fibroin as a multifilament, a multifilament having more excellent heat retention property, hygroscopic heat generation property and / or flame retardancy and extremely excellent quality stability can be obtained.

Explanation of Signs

[0375] 1... Extrusion device, 2... Undrawn yarn manufacturing device, 3... Wet heat drawing device, 4... Drying device, 6... Dope solution, 10... Spinning device, 20... Coagulation bath, 21... Drawing bath, 25... Spinning device, 36... Multifilament, 38... Modified fibroin multifilament, 40... Manufacturing device, 42... Feed roller, 44... Winders, 46... Water bath, 48... Dryer, 54... Heater, 56... Tension roller, 58... Hot roller, 60... Processing device, 62... Drying device, 64... Dry heat plate, 140... Relaxation and shrinkage means (heating means), 141... Feeding means, 142... Winding means, 146... Speed adjustment means, 147... Temperature adjustment means.

Claims

1. A multifilament comprising modified fibroin, having 1,000 or more and 60,000 or less constituent single filaments, with a coefficient of variation of modulus of elasticity of 0.5% or more and 15% or less, a coefficient of variation of strength of 0.5% or more and 15% or less, and a coefficient of variation of elongation of 1% or more and less than 33%.

2. The multifilament according to claim 1, wherein the coefficient of variation of elongation of the multifilament is 1% or more and 20% or less.

3. The multifilament according to claim 1 or 2, wherein the coefficient of variation of elongation of the multifilament is 1% or more and 10% or less.

4. The multifilament according to any one of claims 1 to 3, wherein the coefficient of variation of fineness of the multifilament is 0.01% or more and 20% or less.

5. The multifilament according to any one of claims 1 to 4, wherein the coefficient of variation of strength of the multifilament is 0.5% or more and 10% or less, and the coefficient of variation of modulus of elasticity of the multifilament is 0.5% or more and 10% or less.

6. The multifilament according to any one of claims 1 to 5, wherein the average hydrophobicity index of the modified fibroin is more than -0.

8.

7. The multifilament according to any one of claims 1 to 6, wherein the modified fibroin is modified spider silk fibroin.

8. The multifilament according to any one of claims 1 to 7, having a shrinkage history that is irreversibly shrunk after spinning.

9. The multifilament according to claim 8, wherein the shrinkage history is a shrinkage history that is irreversibly shrunk by bringing the multifilament into contact with water or a shrinkage history that is irreversibly shrunk by heating and relaxing the multifilament.

10. The multifilament according to claim 8 or 9, wherein the multifilament having a shrinkage history that is irreversibly shrunk after spinning has a shrinkage rate defined by the following formula of 5% or less. Shrinkage rate [%] = (1 - (length of the multifilament when dried from the wet state / length of the multifilament when made wet)) × 100

11. The multifilament according to any one of claims 1 to 10, wherein the coefficient of variation of fineness of the multifilament is 0.01% to 6.5%.

12. The multifilament according to any one of claims 1 to 11, wherein the coefficient of variation of strength of the multifilament is 0.5% to 3.8%.

13. A multifilament comprising a recombinant structural protein, having 1,000 or more and 60,000 or less constituent single filaments, and having a coefficient of variation of elongation of 1% or more and less than 33%.

14. A method for producing a multifilament, comprising the step of discharging a spinning dope containing a modified fibroin and a solvent from a spinneret having 1,000 or more and 60,000 or less holes and bringing it into contact with a coagulating liquid to coagulate the modified fibroin.

15. A method for producing a multifilament, comprising the step of discharging a spinning dope containing a recombinant structural protein and a solvent from a spinneret having 1,000 or more and 60,000 or less holes and bringing it into contact with a coagulating liquid to coagulate the recombinant structural protein.

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