WO2019194246A1 - Fibre composite et son procédé de production - Google Patents

Fibre composite et son procédé de production Download PDF

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Publication number
WO2019194246A1
WO2019194246A1 PCT/JP2019/014871 JP2019014871W WO2019194246A1 WO 2019194246 A1 WO2019194246 A1 WO 2019194246A1 JP 2019014871 W JP2019014871 W JP 2019014871W WO 2019194246 A1 WO2019194246 A1 WO 2019194246A1
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amino acid
acid sequence
motif
fibroin
seq
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PCT/JP2019/014871
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English (en)
Japanese (ja)
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弘放 ▲遅▼
オリバ－ セイエッド シャファ－ト
▲郁▼▲群▼ ▲荘▼
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Ｓｐｉｂｅｒ株式会社
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Priority to JP2020512300A priority Critical patent/JP7483263B2/ja
Publication of WO2019194246A1 publication Critical patent/WO2019194246A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
    • D01F8/02—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from cellulose, cellulose derivatives, or proteins

Definitions

  • the present invention relates to a composite fiber and a method for producing the same.
  • Fibroin is a kind of fibrous protein having a ⁇ -pleated sheet structure, and contains a total of 90% of glycine residue, alanine residue and serine residue (Non-patent Document 1).
  • proteins silk protein, hornet silk protein, spider silk protein
  • various amino acid sequence modifications have been carried out.
  • recombinant spider silk proteins that improve toughness and elongation and are suitable for industrial production have been reported (Patent Literature). 1).
  • animal-derived fibers such as wool
  • have the property of shrinking upon contact with moisture for example, immersion in water or hot water, or exposure to a high humidity environment. This property can create a variety of problems during and during the manufacturing process of animal-derived fibers and products using them.
  • the shrinkage prevention method include a method in which a silk fabric using a strong twisted yarn that has been subjected to scouring is immersed in water or another solvent in a tension state and heated for a predetermined time (for example, Patent Document 2).
  • Patent Document 3 A method of fixing the shape by bringing high pressure saturated water vapor into contact with the animal fiber product (for example, Patent Document 3) has been reported.
  • an object of the present invention is to provide an animal-derived fiber with reduced shrinkage due to contact with moisture and a method for producing the same.
  • the present invention relates to the following inventions, for example.
  • a composite fiber comprising a core and an outermost layer covering the core, wherein the core includes a modified fibroin, and the outermost layer includes a structural protein.
  • the structural protein is at least one selected from the group consisting of silk fibroin, spider silk fibroin, collagen, resilin, elastin, and keratin.
  • the modified fibroin is a modified fibroin (third modified fibroin) comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m ,
  • the domain sequence has an amino acid sequence with a reduced content of n motif (A) corresponding to deletion of at least one or more (A) n motifs compared to naturally occurring fibroin,
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin is a modified fibroin (fourth modified fibroin) comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m , Compared with naturally-occurring fibroin, the content of glycine residues corresponding to at least one or more glycine residues in REP was replaced with another amino acid residue in the domain sequence was reduced.
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more. .
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin is a modified fibroin (fifth modified fibroin) comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m ,
  • the domain sequence has one or more amino acid residues in REP substituted with amino acid residues having a higher hydrophobicity index and / or one or more hydrophobicity in REP compared to naturally occurring fibroin.
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif ( A sixth modified fibroin), Glutamine residue content corresponding to the domain sequence having one or more glutamine residues in REP deleted or substituted with other amino acid residues compared to naturally occurring fibroin
  • the composite fiber according to any one of [1] to [4], in which has a reduced amino acid sequence.
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues
  • (A) the number of alanine residues is 80% with respect to the total number of amino acid residues in n motif.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • [4-5] The composite fiber according to any one of [1] to [4], wherein the modified fibroin has a limiting oxygen index (LOI) value of 26.0 or more.
  • LOI limiting oxygen index
  • [4-6] The composite fiber according to any one of [1] to [4], wherein the modified fibroin has a maximum hygroscopic exotherm calculated in accordance with the following formula A of more than 0.025 ° C./g.
  • a method for producing a composite fiber comprising a core part and an outermost layer covering the core part, Preparing a first dope solution comprising modified fibroin and a solvent; Preparing a second dope solution comprising a structural protein and a solvent; And a step of discharging and joining the second dope liquid from the spinneret so as to cover the first dope liquid discharged from the spinneret and forming unstretched composite fibers in the coagulation liquid.
  • the method according to [5] further comprising a step of stretching the unstretched conjugate fiber.
  • the concentration of the modified fibroin in the first dope solution is 5 to 40% by mass based on the total mass of the first dope solution;
  • the above solvents are hexafluoroisopropanol, hexafluoroacetone, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N Selected from the group consisting of an aqueous solution containing methylmorpholine-N-oxide and formic acid and at least one selected from the group consisting of urea, guanidine, sodium dodecyl sulfate, lithium bromide, calcium chloride and lithium thiocyanate
  • the method according to any one of [5] to [7], comprising at least one kind.
  • the modified fibroin is a modified fibroin (third modified fibroin) comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m ,
  • the domain sequence has an amino acid sequence with a reduced content of n motif (A) corresponding to deletion of at least one or more (A) n motifs compared to naturally occurring fibroin, [5] The method according to any one of [12].
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin is a modified fibroin (fourth modified fibroin) comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m , Compared with naturally-occurring fibroin, the content of glycine residues corresponding to at least one or more glycine residues in REP was replaced with another amino acid residue in the domain sequence was reduced.
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more. .
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin is a modified fibroin (fifth modified fibroin) comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m ,
  • the domain sequence has one or more amino acid residues in REP substituted with amino acid residues having a higher hydrophobicity index and / or one or more hydrophobicity in REP compared to naturally occurring fibroin.
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif ( A sixth modified fibroin), Glutamine residue content corresponding to the domain sequence having one or more glutamine residues in REP deleted or substituted with other amino acid residues compared to naturally occurring fibroin
  • the method according to any one of [5] to [12], wherein has a reduced amino acid sequence.
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues
  • (A) the number of alanine residues is 80% with respect to the total number of amino acid residues in n motif.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • [12-5] The method according to any one of [5] to [12], wherein the modified fibroin has a limiting oxygen index (LOI) value of 26.0 or more.
  • LOI limiting oxygen index
  • [12-6] The method according to any one of [5] to [12], wherein the modified fibroin has a maximum hygroscopic exotherm determined in accordance with the following formula A of more than 0.025 ° C./g.
  • a composite fiber comprising a core part and an outermost layer covering the core part, wherein one of the core part and the outermost layer is a modified fibroin.
  • the present invention it is possible to provide a composite fiber in which the contraction rate of the modified fibroin is reduced. Moreover, according to this invention, a high stress is shown by containing modified fibroin in a composite fiber.
  • the composite fiber according to the first embodiment of the present invention includes a core and an outermost layer covering the core, the core includes modified fibroin, and the outermost layer is composed of a structural protein.
  • the composite fiber according to the present embodiment has a two-layer structure including a core portion and an outermost layer, and is also called a core-sheath structure.
  • the core part and the outermost layer may be arranged concentrically, and the center line of the core part may not overlap the center line of the composite fiber.
  • the core portion is completely covered with the intermediate layer or the outermost layer except for the end portion of the fiber (including the cut surface when the fiber is cut), and is not exposed on the surface.
  • the hydrophobicity of the modified fibroin and the hydrophobicity of the structural protein are different from each other.
  • the core of the composite fiber according to the present embodiment includes modified fibroin.
  • the modified fibroin contained in the core portion has a property of contracting when brought into contact with an aqueous medium to be described later (hereinafter also referred to as “water shrinkability”).
  • the modified fibroin is a protein comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further 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 to these, but are typically regions having no amino acid motif repeat characteristic of fibroin and consisting of about 100 amino acids.
  • the modified fibroin is a modified spider silk fibroin, the heat retention, moisture absorption heat generation and / or flame retardancy are more excellent.
  • modified fibroin means an 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 having the same amino acid sequence as that of naturally occurring fibroin.
  • Natural fibroin as used herein is also a protein comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the amino acid sequence of naturally-occurring fibroin may be used as it is, depending on the amino acid sequence of naturally-occurring fibroin.
  • the amino acid sequence may be modified (for example, the amino acid sequence may be modified by modifying the gene sequence of the cloned natural fibroin), and may be artificially designed and used independently of the natural fibroin. It may be synthesized (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).
  • the modified fibroin is a modified spider silk fibroin
  • the composite fiber is superior in heat retention, moisture absorption heat generation and / or flame retardancy.
  • domain sequence refers to a fibroin-specific crystal region (typically corresponding to the (A) n motif in the amino acid sequence) and an amorphous region (typically in the REP of the amino acid sequence). Is equivalent to the amino acid sequence represented by Formula 1: [(A) n motif-REP] m .
  • (A) n motif represents an amino acid sequence mainly composed of alanine residues, and n is 2 to 20, preferably 4 to 20, more preferably 8 to 20, still more preferably 10 to 20, and even more. Preferably, it may be an integer of 4 to 16, still more preferably 8 to 16, particularly preferably 10 to 16.
  • the ratio of the number of alanine residues to the total number of amino acid residues in the n motif may be 40% or more, preferably 60% or more, more preferably 70% or more, and 80 % Or more, more preferably 90% or more, and may be 100% (meaning that it is composed of only alanine residues).
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues, 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 acids. It may be an amino acid sequence composed of residues.
  • m represents an integer of 2 to 300, 8 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, It may be an integer from 20 to 140 or 20 to 120.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • a protein containing the amino acid sequence (PRT410) represented by SEQ ID NO: 13 can be mentioned.
  • the modified fibroin is, for example, a modification of the amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned natural fibroin gene sequence. Can be obtained at Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.
  • Naturally-derived fibroin is a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m , and specific examples include fibroin produced by insects or spiders.
  • fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandarina), Tenaea (Antheraea palaniii), and ⁇ ⁇ (Eriothyraminey). ), Silkworms (Samia cythia), chestnut worms (Caligura japonica), Chussa moth (Anthereaea mylitta), silkworms produced by silkworms such as Antheraea assamata, and vespasima worms Examples include silk proteins.
  • fibroin produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).
  • Fibroin produced by spiders includes, for example, spiders belonging to the genus spider (Araneus spp.) Such as the spider spider, the spider spider, the red spider spider, and the bean spider, the genus spiders of the genus Araneus, the spider spider spider, the spider spider genus e Spiders, spiders such as spiders, spiders belonging to the genus Spider, spiders belonging to the genus Pronos, spiders belonging to the genus Trinofunda, such as Torinofundamas (genus Cyrtarachne) Spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Spider (Ordgarius genus), such as the spiders, the spiders, and the spiders belonging to the genus Ordgarius Spiders belonging to the genus Argiope, such as the genus Argiope, spiders belonging to the genus Arachnura, such as the white-tailed spider, spiders belonging to the
  • Spiders belonging to the genus Azumigumi (Menosira)
  • spiders belonging to the genus Dyschiriognatha (genus Dyschiriognatha) such as the common spider spider, the black spider spider, the genus Spider genus belonging to the genus Spider belonging to the genus and the genus Spider belonging to the genus Spider belonging to the genus
  • Produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops
  • Examples include spider silk protein.
  • the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.
  • fibroin produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin- 4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidrin 1 [derived from Nephila clavies (GenBank accession number AAC4, amino acid sequence A04) U37520 (base sequence)), major sample spidroin 1 [La rodectus hesperus derived] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Nephila clavata derived] (GenBank accession number AAL32447, amino acid sequence 45, amino acid sequence) major ampulate s
  • Naturally derived fibroin include fibroin whose sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidin, sample, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION from sequences including INV as DIVISION among the sequence information registered in NCBI GenBank. It can be confirmed by extracting a character string of a specific product from the sequence, CDS, and a sequence in which the specific character string is described from SOURCE to TISSUE TYPE.
  • the modified fibroin may be modified silk fibroin (modified from the amino acid sequence of silk protein produced by silkworm) or modified spider silk fibroin (modified from the amino acid sequence of spider silk protein produced by spiders). There may be.
  • modified fibroin a modified spider silk fibroin is preferable.
  • modified fibroin derived from a large sphincter bookmarker protein produced in a spider large bottle-like line
  • first modified fibroin modified fibroin with reduced glycine residue content
  • second modified fibroin modified fibroin with reduced content of n motif
  • third modified fibroin modified fibroin with reduced content of n motif
  • content of glycine residue and (A) content of n motif was reduced
  • a modified fibroin (4th modified fibroin) is mentioned.
  • modified fibroin is excellent in flame retardancy, moisture absorption heat generation, and heat retention, fireproof clothing (for example, for firefighting clothing, rescue), fireproof gloves (for example, laboratory, industrial, cooking), gloves It is also suitable for use in winter clothes such as mufflers, sweaters, outerwear and jackets, padding for winter clothes, innerwear, sportswear, shirts, bedding, and padding for bedding.
  • Examples of the first modified fibroin include a protein comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • (A) the number of amino acid residues of the 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, and an integer of 10 to 20 Is more preferable, an integer of 4 to 16 is still more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable.
  • the number of amino acid residues constituting REP is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues.
  • the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by the formula 1: [(A) n motif-REP] m is an amino acid residue.
  • the total number is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more.
  • the first modified fibroin comprises an amino acid sequence unit represented by Formula 1: [(A) n motif-REP] m , and the C-terminal sequence is represented by any one of SEQ ID NOs: 1 to 3 or It may be a polypeptide that is an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.
  • the amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is the sequence
  • the amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminal, and the amino acid sequence shown in SEQ ID NO: 3 has 29 residues removed from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
  • modified fibroin As a more specific example of the first modified fibroin, (1-i) an amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) an amino acid sequence represented by SEQ ID NO: 4 and 90 Mention may be made of modified fibroin comprising an amino acid sequence having a sequence identity of at least%. The sequence identity is preferably 95% or more.
  • the amino acid sequence represented by SEQ ID NO: 4 is an amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminus.
  • the 13th repeat region was increased to approximately double, and the translation was mutated to terminate at the 1154th amino acid residue.
  • the C-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4 is identical to the amino acid sequence shown in SEQ ID NO: 3.
  • the modified fibroin (1-i) may be composed of the amino acid sequence represented by SEQ ID NO: 4.
  • the second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues compared to naturally occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP substituted with another amino acid residue as compared with naturally occurring fibroin. .
  • the second modified fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine) as compared to naturally occurring fibroin.
  • G is a glycine residue
  • P is a proline residue
  • X is an amino acid residue other than glycine
  • at least one glycine residue in at least one or more of the motif sequences is substituted with another amino acid residue. May be.
  • the ratio of the motif sequence in which the above glycine residue is replaced with another amino acid residue may be 10% or more with respect to the entire motif sequence.
  • the second modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and is located on the most C-terminal side from the domain sequence (A) from the n motif to the domain sequence.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. More preferably, it is 100% (meaning that it is composed only of alanine residues).
  • the second modified fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue.
  • 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, % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.
  • a fibroin modified fibroin or naturally-occurring fibroin containing a domain sequence represented by Formula 1: [(A) n motif-REP] m , (A) n located closest to the C-terminal side from the domain sequence
  • An amino acid sequence consisting of XGX is extracted from all REPs included in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence.
  • z / w (%) can be calculated by dividing z by w.
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70% or more. It is still more preferable that it is 80% or more. Although there is no restriction
  • the second modified fibroin is obtained by, for example, modifying a cloned natural fibroin gene sequence so as to encode another amino acid residue by substituting at least a part of a base sequence encoding a glycine residue.
  • a glycine residue in GGX motif and GPGXX motif may be selected as a glycine residue to be modified, or substitution may be performed so that z / w is 50.9% or more.
  • an amino acid sequence satisfying the above-described aspect can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues are further substituted or deleted.
  • the amino acid sequence corresponding to the insertion and / or addition may be modified.
  • the other amino acid residue is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( M) hydrophobic amino acid residues such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues are preferred, and valine (V) residues, phenylalanine (F) residues, leucine (L) residues, isoleucine ( I) residue and glutamine (Q) residue are more preferred, and glutamine (Q) residue is more preferred.
  • the second modified fibroin examples include (2-i) 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 shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.
  • the modified fibroin (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin.
  • the amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence represented by SEQ ID NO: 6, wherein every two (A) n motifs are deleted from the N-terminal side to the C-terminal side, and further before the C-terminal sequence.
  • One [(A) n motif-REP] is inserted into the.
  • the amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid on the C-terminal side is deleted.
  • the amino acid sequence represented by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). The Hinge and His tags are added to the C-terminus of the sequence repeated 4 times.
  • the value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%.
  • the z / w values of the amino acid sequence shown by SEQ ID NO: 6, the amino acid sequence shown by SEQ ID NO: 7, the amino acid sequence shown by SEQ ID NO: 8, and the amino acid sequence shown by SEQ ID NO: 9 are 58.7%, 70.1%, 66.1% and 70.0%.
  • the value of x / y at the ratio of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 (described later) 1: 1.8 to 11.3 is: 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.
  • the modified fibroin (2-i) may be composed of 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 (2-ii) includes 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 .
  • the sequence identity is preferably 95% or more.
  • 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 is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • the second modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
  • tag sequences include affinity tags that use specific affinity (binding property, affinity) with other molecules.
  • affinity tag include a histidine tag (His tag).
  • His tag is a short peptide with about 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel. Therefore, the isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography) Can be used.
  • Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • an “epitope tag” using an antigen-antibody reaction can also be used.
  • a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound.
  • HA peptide sequence of hemagglutinin of influenza virus
  • myc tag peptide sequence of hemagglutinin of influenza virus
  • FLAG tag peptide sequence of hemagglutinin of influenza virus
  • a tag sequence that can be separated with a specific protease can also be used.
  • the modified fibroin from which the tag sequence has been separated can also be recovered.
  • modified fibroin containing a tag sequence (2-iii) the amino acid represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799)
  • Examples include (2-iv) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in (2-iv) SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .
  • amino acid sequences represented by SEQ ID NO: 16 are SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • amino acid sequence shown in SEQ ID NO: 11 is added to the N-terminus of the amino acid sequence shown.
  • the modified fibroin may be composed of 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 (2-iv) includes 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 Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (2-iv) has an XGX (which 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 is contained in REP ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • 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 host.
  • the third modified fibroin has an amino acid sequence in which the domain sequence has a reduced content of (A) n motif compared to naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or more (A) n motifs, as compared to naturally occurring fibroin.
  • the third modified fibroin may have an amino acid sequence corresponding to 10% to 40% deletion of the (A) n motif from naturally occurring fibroin.
  • the third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal side toward the C-terminal one to three (A) n motif every one (A) n motif May have an amino acid sequence corresponding to deletion of.
  • the third modified fibroin has a domain sequence that is at least two consecutive from the N-terminal side to the C-terminal side compared to the naturally occurring fibroin (A) deletion of the n motif, and one (A ) It may have an amino acid sequence corresponding to the deletion of the n motif repeated in this order.
  • the third modified fibroin may have an amino acid sequence whose domain sequence corresponds to that at least every two (A) n motifs are deleted from the N-terminal side to the C-terminal side. .
  • the third modified fibroin includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) n motifs from the N-terminal side toward the C-terminal side. -REP]
  • the ratio of the number of amino acid residues in the other REP is 1.8 to
  • x the maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units that becomes 11.3
  • x the total number of amino acid residues in 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.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. More preferably, it is 100% (meaning that it is composed only of alanine residues).
  • FIG. 1 shows a 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) It has a sequence called n motif.
  • FIG. 1 includes 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 4th REP and 5th REP), pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison), pattern 4 (first REP and Comparison of the second REP).
  • pattern 1 compare 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 4th REP and 5th REP
  • pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison
  • pattern 4 first REP and Comparison of the second REP
  • the number of amino acid residues of each REP in the two adjacent [(A) n motif-REP] units selected is compared.
  • each pattern the number of all amino acid residues of two adjacent [(A) n motif-REP] units indicated by solid lines is added (not only REP but also (A) the number of amino acid residues of the n motif. is there.). Then, the total value added is compared, and the total value (maximum value of the total value) of the pattern having the maximum total value is set as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
  • x / y (%) can be calculated by dividing x by the total number of amino acid residues y of the domain sequence.
  • x / y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, and even more preferably 70% or more. Preferably, it is still more preferably 75% or more, and particularly preferably 80% or more. There is no restriction
  • x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x / y / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.
  • a plurality of third modified fibroins are present in the domain sequence (A)
  • x / y is 46.4% or more It is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, and more preferably 80% or more. It is particularly preferred.
  • x / y in naturally derived fibroin will be described.
  • 663 types of fibroin (of which 415 types were derived from spiders) were extracted.
  • x / y is calculated from the amino acid sequence of naturally derived fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above-described calculation method.
  • FIG. 3 shows the results when the jagged ratio is 1: 1.9 to 4.1.
  • the horizontal axis indicates x / y (%), and the vertical axis indicates frequency.
  • x / y in naturally derived fibroin is less than 64.2% (the highest, 64.14%).
  • one or a plurality of sequences encoding the n motif is deleted so that x / y is 64.2% or more from the cloned gene sequence of naturally occurring fibroin.
  • an amino acid sequence corresponding to the deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and / or added.
  • the amino acid sequence corresponding to this may be modified.
  • third modified fibroin examples include (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met -PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.
  • the modified fibroin (3-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 17 is derived from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin from the N-terminal side to the C-terminal side every two (A) n
  • the motif is deleted, and one [(A) n motif-REP] is inserted before the C-terminal sequence.
  • the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified fibroin.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) at a jagged ratio of 1: 1.8 to 11.3 is 15.0%.
  • the value of x / y in the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by 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 z / w values in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1% and 66. respectively. 1% and 70.0%.
  • the modified fibroin (3-i) 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.
  • the modified fibroin (3-ii) 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 a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (3-ii) 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
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other
  • x / y is 64.2% or more, where x is the maximum total value of the total number of bases and y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may contain the tag sequence described above at one or both of the N-terminal and C-terminal.
  • modified fibroin containing a tag sequence 3-iii) amino acids represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799)
  • a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the sequence or (3-iv) the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .
  • amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are SEQ ID NO: 11 at 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. Are added to the amino acid sequence (including His tag sequence and hinge sequence).
  • the modified fibroin may be composed of 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 (3-iv) comprises 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) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (3-iv) 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.
  • the other X is the maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3.
  • x / y is preferably 64.2% or more.
  • 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 host.
  • the fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced glycine residue content in addition to (A) a reduced content of the n motif compared to naturally occurring fibroin. It is what you have.
  • the domain sequence of the fourth modified fibroin has at least one or more (A) n motifs deleted as compared to naturally occurring fibroin, and at least one or more glycine residues in the REP. It can be said to have an amino acid sequence corresponding to the substitution with another amino acid residue. That is, it is a modified fibroin having the characteristics of the second modified fibroin described above and the third modified fibroin. Specific embodiments and the like are as described in the second modified fibroin and the third modified fibroin.
  • 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) 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
  • a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by Specific embodiments of the modified fibroin comprising 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.
  • the structural protein constituting the outermost layer may be a synthetic fiber or a modified fibroin.
  • Synthetic fibers are generally difficult to get wet with water and have poor water absorption and hygroscopic properties.
  • Examples of synthetic fibers include polyesters such as polyethylene terephthalate, polycaproamide (nylon 6), polyamides such as nylon 66, polyacryl, and polyvinyl formal (vinylon).
  • Synthetic fibers generally have the property of not easily shrinking even when brought into contact with an aqueous medium.
  • the component constituting the outermost layer may be a chemical fiber.
  • chemical fibers include natural polymers (natural fibers), semi-synthetic polymers (semi-synthetic fibers), and synthetic polymers (synthetic fibers).
  • natural polymers natural fibers
  • natural fibers include cellulose regenerated fibers such as rayon, cupra, polynosic, and lyocell.
  • semi-synthetic fiber examples include acetate fibers such as acetate (diacetate) and triacetate, and promix.
  • polyesters such as the above-mentioned polyethylene terephthalate, polycaproamide (nylon 6), polyamides such as nylon 66, polyacryl, polyvinyl formal (vinylon), and polyurethane (spandex). Can be mentioned.
  • cellulose regenerated fiber, acetate fiber, promix, polyacryl, polyvinyl formal, or polyurethane is used as the component constituting the outermost layer, it is dissolved in a known solvent (solution) to form a spinning solution (dope solution), and spinning
  • a spinning solution dip solution
  • a core-sheath type composite fiber can be obtained by discharging from the base and solidifying it in a coagulation liquid using the first component (modified fibroin) as the core and the second component (chemical fiber) as the sheath.
  • polyester or polyamide When polyester or polyamide is used as the component constituting the outermost layer, these raw materials are melted to form a liquid, discharged from the spinneret, the first component (modified fibroin) as the core, and the second component (chemical fiber) Can be made into a core-sheath type composite fiber.
  • the component constituting the outermost layer may be modified fibroin.
  • modified fibroin constitutes the outermost layer, a core-sheath type composite fiber excellent in flame retardancy, moisture absorption exothermic property, and heat retention can be obtained.
  • hydrophobic modified fibroin means a modified fibroin fiber having poor water absorption or hygroscopicity, for example, a hydrophobicity index of each amino acid (hydropathic index, hereinafter also referred to as “HI”). You may judge using.
  • the high hydrophobicity index increases the hydrophobicity of the fiber itself, and the shrinkage rate can be reduced even when contacted with an aqueous medium.
  • the structural protein is not particularly limited, and may be a protein produced by a microorganism or the like by a gene recombination technique, or a protein produced by synthesis. Alternatively, the structural protein may be a product obtained by purifying a naturally derived structural protein.
  • the structural protein may be, for example, a structural protein and an artificial structural protein derived from the structural protein.
  • the structural protein means a structural protein that forms or maintains a structure and a form in a living body. That is, the structural protein may be a naturally derived structural protein, and a modified protein obtained by modifying a part of the amino acid sequence (for example, 10% or less of the amino acid sequence) based on the amino acid sequence of the naturally derived structural protein. It may be.
  • the structural protein include fibroin, keratin, collagen, elastin, and resilin.
  • the fibroin may be one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin, for example.
  • the structural protein may be silk fibroin, spider silk fibroin or a combination thereof.
  • the ratio of silk fibroin may be, for example, 40 parts by mass or less, 30 parts by mass or less, or 10 parts by mass or less with respect to 100 parts by mass of spider silk fibroin.
  • the silk thread is a fiber (a silk thread) obtained from a silkworm made by a silkworm, Bombyx mori larva.
  • one silk thread is composed of two silk fibroins and glue quality (sericin) covering them from the outside.
  • Silk fibroin is composed of many fibrils.
  • Silk fibroin is covered with four layers of sericin. Practically, silk filaments obtained by dissolving and removing outer sericin by scouring are used for clothing.
  • a general silk thread has a specific gravity of 1.33, an average fineness of 3.3 decitex, and a fiber length of about 1300 to 1500 m.
  • Silk fibroin can be obtained from natural or domestic silkworms, or used or discarded silk fabrics.
  • the silk fibroin may be sericin-removed silk fibroin, sericin-unremoved silk fibroin, or a combination thereof.
  • Sericin-removed silk fibroin is purified by removing sericin covering silk fibroin and other fats.
  • the silk fibroin thus purified is preferably used as a lyophilized powder.
  • the sericin unremoved silk fibroin is an unpurified silk fibroin from which sericin and the like have not been removed.
  • the spider silk fibroin may contain a spider silk polypeptide selected from the group consisting of a natural spider silk structure protein and a polypeptide derived from the natural spider silk structure protein (artificial spider silk structure protein).
  • Examples of the natural spider silk structure protein include a large sphincter bookmark structure protein, a weft protein, and a small bottle-like gland structure protein. Since the large spout bookmarker has a repeating region composed of a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and stretchability.
  • the weft of spider silk has a feature that it does not have a crystalline region but has a repeating region consisting of an amorphous region. The weft thread is less stressed than the large spout bookmarker thread, but has high stretchability.
  • Large splint bookmarker structure protein is produced in spider large bottle-like glands and has a characteristic of excellent toughness.
  • Examples of the large sphincter dragline structure protein include large bottle-shaped gland spiders MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from two spider spiders (Araneus diadematus).
  • ADF3 is one of the two main dragline proteins of the elder spider.
  • the polypeptide derived from the natural spider silk structure protein may be a polypeptide derived from these bookmarker thread structure proteins.
  • a polypeptide derived from ADF3 is relatively easy to synthesize and has excellent properties in terms of strength and toughness.
  • the weft structure protein is produced in the flagellate gland of the spider.
  • Examples of the weft structure protein include flagellum silk protein derived from the American spider (Nephila clavies).
  • the polypeptide derived from the natural spider silk structure protein may be a recombinant spider silk structure protein.
  • recombinant spider silk structure proteins include mutants, analogs or derivatives of natural spider silk structure proteins.
  • a preferred example of such a polypeptide is a recombinant spider silk structure protein (also referred to as “polypeptide derived from a large sphincter bookmark thread protein”).
  • Examples of the structural protein derived from the large sphincter bookmark thread that is a fibroin-like structural protein and the structural protein derived from silkworm silk include, for example, a protein comprising a domain sequence represented by Formula 1: [(A) n motif-REP1] m Is mentioned.
  • (A) n of the motif A represents an alanine residue, and n is preferably an integer of 2 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16 and an integer of 10 to 16, and (A) the number of alanine residues relative to the total number of amino acid residues in the n motif may be 40% or more, 60% or more, 70% or more, 80% or more 90% or more and 100% (meaning that it is composed of only alanine residues).
  • REP1 represents an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REP1s may have the same amino acid sequence or different amino acid sequences.
  • (A) n protein in formula 1 may be deleted, and the structural protein whose industrial productivity is improved while maintaining the strength and elongation may be used.
  • the frequency of deletion for example, from the N-terminal side to the C-terminal side, the number of amino acid residues of REP of two adjacent [(A) n motif-REP1] units is sequentially compared, and amino acid residues When the number of amino acid residues of a small number of REP is 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3.
  • the two adjacent [(A) n motif-REP1] units include structural proteins in which x / y is 50% or more, where x is the maximum sum of the total number of amino acid residues and y is the total number of amino acid residues in the domain sequence.
  • An example of such a structural protein is a structural protein in which the ratio of a motif sequence in which a glycine residue is replaced with another amino acid residue is 10% or more based on the total motif sequence.
  • structural protein derived from the large sputum bookmarker thread include structural proteins including the amino acid sequences represented by SEQ ID NO: 13 and SEQ ID NO: 15.
  • a structural protein derived from the weft structural protein for example, a structural protein containing a domain sequence represented by Formula 2: [REP2] o (where REP2 is derived from Gly-Pro-Gly-Gly-X in Formula 2). And X represents one amino acid selected from the group consisting of alanine (Ala), serine (Ser), tyrosine (Tyr), and valine (Val), and o represents an integer of 8 to 300. .).
  • a structural protein containing the amino acid sequence represented by SEQ ID NO: 13 can be exemplified.
  • the amino acid sequence represented by SEQ ID NO: 41 corresponds to a repeat portion and a motif of a partial sequence (NCBI accession number: AAF36090, GI: 7106224) of a partial sequence of flagellar silk structure protein of American spider spider obtained from the NCBI database.
  • PR1 sequence An amino acid sequence from the 1220th residue to the 1659th residue from the N-terminal (referred to as PR1 sequence), and a partial sequence of the flagellar silk structure protein of American spider spider obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649), the C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminal is linked, and the amino acid sequence (tag sequence and hinge sequence) represented by SEQ ID NO: 11 is at the N-terminal of the combined sequence With added ones That.
  • a structural protein derived from collagen for example, a structural protein containing a domain sequence represented by Formula 3: [REP3] p (wherein, in Formula 3, p represents an integer of 5 to 300.
  • REP3 represents Gly-X -Y represents an amino acid sequence composed of Y, and X and Y represent any amino acid residue other than Gly, and a plurality of REP3 may be the same amino acid sequence or different amino acid sequences.
  • a structural protein including the amino acid sequence represented by SEQ ID NO: 42 can be exemplified.
  • amino acid sequence shown in SEQ ID NO: 42 corresponds to the repeat portion and motif of the partial sequence of human collagen type 4 (NCBI GenBank accession number: CAA56335.1, GI: 3702452) obtained from the NCBI database.
  • An amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence from the 301st residue to the 540th residue.
  • a structural protein derived from resilin for example, a structural protein containing a domain sequence represented by Formula 4: [REP4] q (wherein, q represents an integer of 4 to 300 in Formula 4.
  • REP4 represents Ser-J- An amino acid sequence composed of J-Tyr-Gly-U-Pro, wherein J represents an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr. Represents any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser.
  • Plural REP4s may have the same or different amino acid sequences. Good).
  • a structural protein including the amino acid sequence represented by SEQ ID NO: 43 can be exemplified.
  • the amino acid sequence represented by SEQ ID NO: 43 is the amino acid sequence of resilin (NCBI GenBank accession number NP 611157, Gl: 24654243), wherein Thr at the 87th residue is replaced with Ser, and the Asn at the 95th residue.
  • the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced with Asp.
  • structural proteins derived from elastin include structural proteins having amino acid sequences such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine).
  • a structural protein including the amino acid sequence represented by SEQ ID NO: 44 can be exemplified.
  • the amino acid sequence represented by SEQ ID NO: 44 is the amino acid sequence represented by SEQ ID NO: 11 at the N-terminus of the amino acid sequence of residues 121 to 390 of the amino acid sequence of NCBI GenBank accession number AAC98395 (tag sequence). And a hinge arrangement).
  • Examples of the keratin-derived structural protein include type I keratin of Capra hircus.
  • a structural protein containing the amino acid sequence represented by SEQ ID NO: 45 (PRT798) (the amino acid sequence of NCBI GenBank accession number ACY30466) can be given.
  • the amino acid sequence represented by SEQ ID NO: 45 is obtained by adding the amino acid sequence represented by SEQ ID NO: 11 (tag sequence and hinge sequence) to the N-terminal of the amino acid sequence of NCBI GenBank accession number ACY30466.
  • Structural proteins or structural proteins derived therefrom can be used singly or in combination of two or more. By combining two or more structural proteins, the overall hydrophobicity may be adjusted to a desired value.
  • Synthetic fibers are generally known to be hydrophobic fibers because they are difficult to wet with water and have poor water absorption and hygroscopic properties.
  • the chemical fiber include polyester such as polyethylene terephthalate, polycaproamide (nylon 6), polyamide such as nylon 66, polyacryl, and polyvinyl formal (vinylon).
  • Nylon 6 official moisture content: 4.5
  • polyester official moisture content: 0.4
  • the hydrophobic fiber is, for example, a fiber having an official moisture content of 7% or less.
  • 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 having the same amino acid sequence as that of naturally-occurring fibroin.
  • the modified fibroin used for the outermost layer may be, for example, an artificially modified domain sequence so as to impart hydrophobicity to naturally derived fibroin.
  • modified fibroin As a specific example of the modified fibroin used in the outermost layer, the content of a modified fibroin having a domain sequence including a region having a large hydrophobic index locally (fifth modified fibroin) or glutamine residue is reduced. And modified fibroin having a domain sequence (sixth modified fibroin).
  • the hydrophobicity index of each amino acid will be described later.
  • the fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a higher hydrophobicity index and / or REP compared to naturally occurring fibroin. It may have an amino acid sequence including a region having a large hydrophobicity index corresponding to the insertion of one or more amino acid residues having a large hydrophobicity index.
  • the region where the hydrophobic index is locally large is preferably composed of 2 to 4 amino acid residues.
  • the above-mentioned “region with a large hydrophobicity index” means that the total or average of the hydrophobicity indices of 2 to 4 amino acid residues in succession is the hydrophobicity index of the amino acid residue at the same position in the corresponding naturally occurring fibroin. The area is larger than the total or average value.
  • amino acid residue having a large hydrophobicity index is an amino acid residue having a larger hydrophobicity index than the amino acid residue at the same position in the corresponding naturally occurring fibroin.
  • the amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). More preferably, it is a residue.
  • the fifth modified fibroin has one or more amino acid residues in REP substituted with amino acid residues having a higher hydrophobicity index and / or one or more in REP compared to naturally occurring fibroin.
  • substitution, deletion, insertion and / or addition of one or more amino acid residues as compared with naturally occurring fibroin There may be amino acid sequence modifications corresponding to the above.
  • the fifth modified fibroin is obtained by removing one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned natural fibroin gene sequence. It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in REP.
  • hydrophilic amino acid residues for example, amino acid residues having a negative hydrophobicity index
  • a group for example, an amino acid residue having a positive hydrophobicity index
  • one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to insertion of, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin and / or one or more hydrophobic amino acids in REP
  • the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.
  • the fifth modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and is located on the most C-terminal side (A) from the n motif to the C terminus of the domain sequence.
  • p is the total number of amino acid residues included in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more
  • (A) When the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence, which is located at the most C-terminal side, from the domain sequence is q, p / q is 6 It may have an amino acid sequence that is 2% or more.
  • hydrophobicity index of amino acid residues As for 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 bio.p. 7”. 105-132). Specifically, the hydrophobicity index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
  • a sequence obtained by removing the sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m to the most C-terminal side from the domain (A) n motif to the C terminus of the domain sequence. (Hereinafter referred to as “array A”).
  • array A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated.
  • the average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in the four consecutive amino acid residues by 4 (number of amino acid residues).
  • the average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of “four consecutive amino acid residues whose average value of hydrophobicity index is 2.6 or more”, it should be included as one amino acid residue in the region. become.
  • the total number of amino acid residues contained in the region is p.
  • the total number of amino acid residues contained in sequence A is q.
  • the average value of the hydrophobicity index of four consecutive amino acid residues is 2
  • p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and preferably 20% or more. Even more preferably, it is still more preferably 30% or more.
  • the upper limit of p / q is not particularly limited, but may be 45% or less, for example.
  • the fifth modified fibroin is, for example, one or a plurality of hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP so that the amino acid sequence of the naturally-derived fibroin thus cloned satisfies the above p / q condition. Is replaced with a hydrophobic amino acid residue (for example, an amino acid residue with a positive hydrophobicity index) and / or one or more hydrophobic amino acid residues are inserted in the REP By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index.
  • hydrophilic amino acid residues for example, a hydrophobicity index
  • an amino acid sequence satisfying the above p / q conditions can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index and / or one or more amino acid residues in REP.
  • modifications corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. .
  • the amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
  • the fifth modified fibroin (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 comprising 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.
  • the modified fibroin (5-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 19 is an amino acid sequence consisting of 3 amino acid residues for every other REP with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) except for the terminal on the C-terminal side ( VLI) is inserted at two positions, a part of glutamine residue (G) is substituted with a serine residue (S), and a part of amino acids on the C-terminal side is deleted.
  • the amino acid sequence represented by SEQ ID NO: 20 is an amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP. is there.
  • the amino acid sequence shown in SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues into the amino acid sequence shown in SEQ ID NO: 8 every other REP.
  • the modified fibroin (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin (5-ii) comprises 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.
  • the modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (5-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the fifth modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal.
  • modified fibroin containing the tag sequence examples include (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv And a modified fibroin comprising 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.
  • amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences represented by SEQ ID NO: 11 (His tag) at the N-terminus of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Including a sequence and a hinge sequence).
  • the modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
  • the modified fibroin (5-iv) comprises 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.
  • the modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (5-iv) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located on the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • 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 host.
  • the sixth modified fibroin has an amino acid sequence in which the content of glutamine residues is reduced compared to naturally occurring fibroin.
  • the sixth modified fibroin preferably contains at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence of REP.
  • the content ratio of the GPGXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more.
  • the upper limit of GPGXX motif content rate 50% or less may be sufficient and 30% or less may be sufficient.
  • GPGXX motif content is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m- (A) fibroin (modified fibroin or naturally derived) containing a domain sequence represented by the n motif In fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the domain sequence (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
  • the number obtained by multiplying the total number by three is s, and is located at the most C-terminal side.
  • the sequence from the n motif to the C-terminal of the domain sequence is determined from the domain sequence.
  • the content ratio of the GPGXX motif is calculated as s / t, where t is the total number of amino acid residues of all REPs excluding the n motif. It is.
  • “A sequence located at the most C-terminal side (A) excluding the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence” (A)
  • the sequence from the n motif to the C terminus of the domain sequence ”(sequence corresponding to REP) may include a sequence that is not highly correlated with the sequence characteristic of fibroin, and m is small In this case (that is, when the domain sequence is short), the calculation result of the content ratio of the GPGXX motif is affected, so this influence is excluded.
  • the “GPGXX motif” is located at the C-terminus of REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.
  • FIG. 5 is a schematic diagram showing the domain sequence of the modified fibroin.
  • all REPs are “a sequence located at the most C-terminal side (A)
  • the sequence from the n motif to the C-terminal of the domain sequence is excluded from the domain sequence” (the sequence indicated by “region A” in FIG. ))
  • 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%. .
  • the “glutamine residue content” is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m-
  • A) the sequence from the n- motif to the C-terminal of the domain sequence located on the most C-terminal side is included.
  • the total number of glutamine residues contained in the region is u
  • the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence
  • (A) n The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif.
  • the reason why "A sequence located at the most C-terminal side (A) excluding the sequence from the n motif to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.
  • the sixth modified fibroin corresponds to its domain sequence having one or more glutamine residues in REP deleted or replaced with other amino acid residues compared to naturally occurring fibroin. It may have an amino acid sequence.
  • the “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. Table 1 shows the hydrophobicity index of amino acid residues.
  • amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can.
  • an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. More preferably, it is an amino acid residue selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
  • the hydrophobicity of REP is preferably ⁇ 0.8 or more, more preferably ⁇ 0.7 or more, still more preferably 0 or more, and 0.3 or more. It is still more preferable that it is and it is especially preferable that it is 0.4 or more.
  • the “hydrophobicity of REP” is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m-
  • A) the sequence from the n- motif to the C-terminal of the domain sequence located on the most C-terminal side is included.
  • the sum of the hydrophobicity index of each amino acid residue in the region is represented by v, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence, and ( A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding the n motif.
  • t is the total number of amino acid residues of all REPs excluding the n motif.
  • the sixth modified fibroin has its domain sequence deleted one or more glutamine residues in REP and / or one or more glutamine residues in REP compared to naturally occurring fibroin.
  • modifications corresponding to substitution of other amino acid residues there may also be amino acid sequence modifications corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues. .
  • the sixth modified fibroin is, for example, deleting one or more glutamine residues in REP from the cloned gene sequence of naturally occurring fibroin and / or other one or more glutamine residues in REP. It can obtain by substituting to the amino acid residue.
  • one or more glutamine residues in REP are deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP are replaced with other amino acid residues.
  • it can also be obtained by designing a corresponding amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • sixth modified fibroin examples include (6-i) 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-PRT1009) or SEQ ID NO: 46 (Met-PRT966)
  • (6-ii) 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 or SEQ ID NO: 46 An amino acid sequence having 90% or more sequence identity with the amino acid sequence shown Mention may be made of a non-modified fibroin.
  • the (6-i) modified fibroin will be described.
  • the amino acid sequence represented by SEQ ID NO: 25 is obtained by substituting VL for QQ in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410).
  • the amino acid sequence represented by SEQ ID NO: 26 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with TS and replacing the remaining Q with A.
  • the amino acid sequence represented by SEQ ID NO: 27 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VL and replacing the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO: 28 is obtained by replacing all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VI and replacing the remaining Q with L.
  • the amino acid sequence represented by SEQ ID NO: 29 is one in which all QQs in the amino acid sequence represented by SEQ ID NO: 7 are substituted with VF, and the remaining Q is substituted with I.
  • the amino acid sequence represented by SEQ ID NO: 30 is obtained by substituting VL for all QQs in the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525).
  • the amino acid sequence shown in SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO: 8 with VL and replacing the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO: 32 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) (provided that several amino acid residues on the C-terminal side of the region are substituted) QQ in the sequence which is repeated twice) is replaced with VF, and the remaining Q is replaced with I.
  • 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 and SEQ ID NO: 32 all have a glutamine residue content of 9% or less. Yes (Table 2).
  • the modified fibroin of (6-i) is an 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 or SEQ ID NO: 46 It may consist of.
  • the modified fibroin of (6-ii) is an 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 or SEQ ID NO: 46 And an amino acid sequence having a sequence identity of 90% or more.
  • the modified fibroin of (6-ii) is also represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less.
  • the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
  • modified fibroin containing a tag sequence (6-iii) 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 (PRT1009), or a modified fibroin comprising the amino acid sequence shown by SEQ ID NO: 47 (PRT966), or (6-iv) SEQ ID NO: 33 An amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 or SEQ ID NO: 47
  • the modified fibroin containing can be mentioned.
  • 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 and SEQ ID NO: 47 are SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
  • SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 46 the amino acid sequence represented by SEQ ID NO: 11 (His tag sequence and (Including hinge sequence).
  • the amino acid sequences represented by SEQ ID NO: 40 and SEQ ID NO: 47 all have a glutamine residue content of 9% or less (Table 3).
  • the modified fibroin of (6-iii) is 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 or SEQ ID NO: 47 It may consist of.
  • the modified fibroin of (6-iv) is 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 or SEQ ID NO: 47. And an amino acid sequence having a sequence identity of 90% or more.
  • the modified fibroin of (6-iv) is also a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less.
  • the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.
  • 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 host.
  • the modified (artificial) fibroin fiber has a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more, or 26 or more. It may be 28 or more, 29 or more, or 30 or more.
  • LOI limiting oxygen index
  • the above LOI value is measured in accordance with “Test method for powdered or low melting point synthetic resin” described in “Head of Fire and Disaster Management Agency, Hazardous Materials Regulation Section, Fire Hazard No. 50 (May 31, 1995)”. Value.
  • the maximum moisture absorption exotherm required in accordance with the following formula A of the modified (artificial) fibroin fiber may be greater than 0.025 ° C./g, may be 0.026 ° C./g or more, and may be 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 may be sufficient and 0.040 ° C / g or more may be sufficient.
  • the upper limit of the maximum moisture absorption exotherm is not particularly limited, but is usually 0.060 ° C./g or less.
  • the heat retention index of the modified fibroin fiber may be 0.22 or more, may be 0.24 or more, may be 0.26 or more, may be 0.28 or more, and may be 0.30 or more. It may be 0.32 or more. Although there is no restriction
  • the modified fibroin fiber preferably has excellent heat retention, and the heat retention index determined according to the following formula C may be 0.20 or more.
  • Thermal insulation index Heat retention rate (%) / Sample weight (g / m 2 )
  • the composite fiber according to the present embodiment may include a plurality of core portions.
  • a composite fiber is generally called a sea-island structure, and the island part corresponds to the core part described above, and the sea part has the same characteristics as the outermost layer described above.
  • the second embodiment of the present invention includes a core, an outermost layer covering the core, and one or a plurality of intermediate layers between the core and the outermost layer. At least one contains modified fibroin and the outermost layer is composed of structural proteins.
  • the conjugate fiber according to the present embodiment at least one of the core and the intermediate layer has water shrinkability. Moreover, both the core part and the intermediate layer may have water shrinkability. When only one of the core part and the intermediate layer has water shrinkage, the material constituting the other is not limited.
  • the conjugate fiber according to the present embodiment includes, for example, a conjugate fiber in which the core portion and the outermost layer are individually composed of structural proteins and the intermediate layer has water-shrinkability.
  • FIG. 7 shows an example of a composite fiber according to each embodiment of the present invention.
  • Fig.7 (a) is a schematic diagram which shows the composite fiber 50 by which the core part 51 and the outermost layer 52 (sheath part) are arrange
  • the center line of the core part 51 is arrange
  • FIG. 7B is a schematic diagram showing the composite fiber 50 in which the core portion 51 and the outermost layer 52 (sheath portion) are arranged non-concentrically.
  • the center line of the core 51 does not overlap with the center line of the composite fiber, and the thickness of the outermost layer 52 covering the core 51 is not constant.
  • FIG.7 (c) is a schematic diagram which shows the composite fiber 50 provided with the some core part 51 and the outermost layer 52 (sheath part).
  • FIG. 7D is a schematic diagram showing a composite fiber 50 having a three-layer structure including an intermediate layer 53 between the core 51 and the outermost layer 52.
  • the hydrophobicity of the modified fibroin contained in the core and the hydrophobicity of the structural protein contained in the outermost layer are different from each other.
  • the hydrophobicity of the modified fibroin or structural protein the total of each HI of each amino acid residue (excluding the amino acid residue corresponding to the tag sequence and the hinge sequence) constituting each is calculated, and the amino acid residue is calculated. The value divided by the radix.
  • the hydrophobicity of the modified fibroin contained in the core is, for example, preferably ⁇ 0.8 or less, and more preferably ⁇ 0.55 or less.
  • the hydrophobicity of the structural protein contained in the outermost layer (or the intermediate layer) is, for example, more than ⁇ 0.8 and more preferably more than ⁇ 0.55.
  • the hydrophobicity of the core is calculated as the average value according to the proportion of each component by calculating the hydrophobicity of each modified fibroin contained in the core. Also good. For example, the value obtained by multiplying the hydrophobicity of each modified fibroin by the content of the modified fibroin in the core and totaling it and dividing by the number of modified fibroin may be used.
  • the hydrophobicity of the outermost layer (or intermediate layer) is calculated by calculating the hydrophobicity of each structural protein contained in the outermost layer (or intermediate layer). Then, it may be calculated as an average value corresponding to the ratio of each component. For example, the value obtained by multiplying the hydrophobicity of each structural protein by the content rate in the core of the structural protein may be added and divided by the number of structural proteins.
  • the content rate is small (for example, the content rate is 10%).
  • the modified fibroin or structural protein has a sufficiently small contribution to the hydrophobicity of the whole component, the hydrophobicity of each component is calculated without considering the hydrophobicity of the modified fibroin or structural protein. May be.
  • the silk thread is composed of about 75% silk fibroin and about 25% sericin (UniProt database, Entry No. P07856).
  • Silk fibroin is composed of fibroin H chain (UniProt database, Entry No.
  • fibroin H chain occupies the majority.
  • hydrophobicity of the silk fibroin is calculated by calculating the sum of each HI of each amino acid residue of the fibroin H chain, which is the main component. It is good also as hydrophobicity 0.216 which is the value which remove
  • the hydrophobicity of the amino acid sequence is as shown in Table 4. In calculating the degree of hydrophobicity of each amino acid sequence, it was calculated by excluding a sequence unrelated to the modified fibroin (that is, a sequence corresponding to the amino acid sequence represented by SEQ ID NO: 11).
  • Table 5 shows the degree of hydrophobicity of the amino acid sequences represented by SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45.
  • the degree of hydrophobicity of each amino acid sequence it was calculated by excluding a sequence unrelated to the structural protein (that is, a sequence corresponding to the amino acid sequence represented by SEQ ID NO: 11).
  • the modified fibroin expresses the nucleic acid, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced.
  • the method for producing the nucleic acid encoding the modified fibroin is not particularly limited.
  • the nucleic acid is produced by a method such as amplification by polymerase chain reaction (PCR), cloning, modification by genetic engineering techniques, or chemical synthesis. can do.
  • the method for chemically synthesizing nucleic acids is not particularly limited. For example, based on the amino acid sequence information of fibroin obtained from the NCBI web database, etc., AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) A gene can be chemically synthesized by a method of linking oligonucleotides that are synthesized automatically by PCR or the like.
  • a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N terminus of the above amino acid sequence is synthesized. May be.
  • Regulatory sequences are sequences that control the expression of modified fibroin in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.), and can be appropriately selected depending on the type of host.
  • an inducible promoter that functions in the host cell and can induce expression of the modified fibroin may be used.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure or pH value.
  • the type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like.
  • a vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed is preferably used.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • microorganisms belonging to the genus Escherichia include Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like.
  • microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis.
  • microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • vectors for introducing a nucleic acid encoding a modified fibroin include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238696) and the like can be mentioned.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.).
  • yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like.
  • Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma and the like.
  • examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEp13 (ATCC37115) and YEp24 (ATCC37051).
  • a method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • electroporation method electroporation method
  • spheroplast method protoplast method
  • lithium acetate method competent method, and the like.
  • a method for expressing a nucleic acid by a host transformed with an expression vector in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd edition, etc. .
  • the modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting from the culture medium.
  • the method for culturing a host in a culture medium can be performed according to a method usually used for culturing a host.
  • the culture medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.
  • Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism.
  • Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid, etc.
  • Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source examples include 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 cells and digested products thereof can be used.
  • inorganic salts for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and agitation culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter
  • indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter.
  • An acid or the like may be added to the medium.
  • Isolation and purification of the expressed modified fibroin can be performed by a commonly used method.
  • the host cells are recovered by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a manton.
  • the host cells are disrupted with a Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract.
  • a method usually used for isolation and purification of modified fibroin that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method by DE, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), or a resin such as S-Sepharose FF (manufactured by Pharmacia) Electrophoresis such as cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing Use methods such as law alone or in combination It is possible to obtain a purified product.
  • a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA
  • the modified fibroin when expressed by forming an insoluble substance in the cell, the host cell is similarly collected, crushed, and centrifuged to collect the modified fibroin insoluble substance as a precipitate fraction.
  • the recovered insoluble form of modified fibroin can be solubilized with a protein denaturant.
  • a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.
  • the modified fibroin when secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture with a technique 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.
  • the first dope solution includes a modified fibroin and a solvent.
  • the second dope solution contains a structural protein and a solvent.
  • the concentration of the modified fibroin in the first dope solution is not particularly limited, depending on factors such as the crimpability and fiber diameter of the desired composite fiber, the combination with the structural protein contained in the outermost layer (or intermediate layer), etc. It can be set appropriately.
  • the concentration of the modified fibroin is preferably 5 to 40% by mass, based on the total mass of the first dope solution (when the total mass of the first dope solution is 100% by mass), preferably 7 to 40% by mass More preferably, it is 10 to 40% by mass, more preferably 7 to 35% by mass, more preferably 10 to 35% by mass, and 12 to 35% by mass.
  • the concentration of the modified fibroin is 5% by mass or more, the productivity of the composite fiber tends to be further improved.
  • concentration of the modified fibroin is 40% by mass or less, the dope solution can be discharged more stably from the spinneret, and the productivity tends to be further improved.
  • the concentration of the structural protein in the second dope solution is not particularly limited, and may be appropriately set according to factors such as the crimpability and fiber diameter of the desired composite fiber, the combination with the structural protein contained in the core, and the like. it can.
  • the concentration of the modified fibroin is preferably 5 to 40% by mass based on the total mass of the second dope solution (when the total mass of the second dope solution is 100% by mass), preferably 7 to 40% by mass More preferably, it is 10 to 40% by mass, more preferably 7 to 35% by mass, more preferably 10 to 35% by mass, and 12 to 35% by mass.
  • the concentration of the modified fibroin is 5% by mass or more, the productivity of the composite fiber tends to be further improved.
  • concentration of the modified fibroin is 40% by mass or less, the dope solution can be discharged more stably from the spinneret, and the productivity tends to be further improved.
  • the solvent of the first dope liquid may be any solvent that can dissolve the modified fibroin.
  • the solvent of 2nd dope liquid should just be a thing which can melt
  • solvents include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), 1,3 -Dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine-N-oxide (NMO), formic acid and the like.
  • HFIP hexafluoroisopropanol
  • HFA hexafluoroacetone
  • DMSO dimethyl sulfoxide
  • DMF N-dimethylformamide
  • DMA N-dimethylacetamide
  • DMI 1,3 -Dimethyl
  • the solvent may be an aqueous solution, specifically, an aqueous solution containing at least one selected from the group consisting of urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide, calcium chloride, and lithium thiocyanate. Can be mentioned. These solvent may be used individually by 1 type, and may be used in combination of 2 or more type.
  • the method for preparing the dope solution may be a method well known to those skilled in the art, and the modified fibroin or structural protein and a solvent may be mixed in any order.
  • An inorganic salt may be added to the first dope liquid and the second dope liquid as necessary.
  • the inorganic salt can function as a dissolution promoter for the modified fibroin.
  • examples of inorganic salts include alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, and the like.
  • Specific examples of inorganic salts 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.
  • the viscosity of the dope solution can be appropriately set according to the use of the composite fiber and the spinning method.
  • the viscosity of the dope solution may be, for example, 5000 to 40000 mPa ⁇ sec at 20 ° C., 7000 to 40000 mPa ⁇ sec, 10000 to 40000 mPa ⁇ sec, 7000 to 35000 mPa ⁇ sec, 10000 to 35000 mPa ⁇ sec, and 10000 to 30000 mPa ⁇ sec. sec, or 10,000 to 25000 mPa ⁇ sec.
  • the viscosity of the dope solution can be measured, for example, using a trade name “EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.
  • the dope solution may be stirred or shaken for a certain period of time in order to promote dissolution.
  • the dope solution may be heated to 30 ° C. or higher, 40 ° C. or higher, 50 ° C. or higher, 60 ° C. or higher, 70 ° C. or higher, 80 ° C. or higher, or 90 ° C. or higher.
  • the upper limit of the heating temperature is, for example, not more than the boiling point of the solvent.
  • the composite fiber can be produced by a known spinning method.
  • the composite fiber can be obtained by spinning using a first dope solution and a second dope solution by a known spinning method such as dry spinning, melt spinning, wet spinning, and dry wet spinning.
  • Preferred spinning methods include wet spinning or dry temperature spinning.
  • the first dope liquid and the second dope liquid are ejected from a spinneret (nozzle), joined, and the modified fibroin is solidified in the coagulation liquid, thereby forming the composite fiber in an undrawn yarn state.
  • FIG. 6 is an explanatory view schematically showing an example of a spinning device for producing a composite fiber.
  • a spinning device 10 illustrated in FIG. 6 is an example of a spinning device for dry and wet spinning, and includes an extrusion device 1, an undrawn yarn production device 2, a wet heat drawing device 3, and a drying device 4.
  • the dope solution 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8.
  • the dope solution may be filled into a cylinder and extruded from a nozzle using a syringe pump.
  • the extruded dope liquid 6 is supplied through the air gap 19 into the coagulation liquid 11 in the coagulation liquid tank 20, the solvent is removed, the protein is coagulated, and a fibrous coagulum is formed.
  • the fibrous solidified body is supplied into the hot water 12 in the drawing bath 21 and drawn. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14.
  • the stretched fibrous solidified body is supplied to the drying device 4 and dried in the yarn path 22, and the composite fiber 36 is obtained as the wound body 5.
  • Reference numerals 18a to 18g denote thread guides.
  • a spinneret (nozzle) for producing a composite fiber for example, a nozzle for producing a composite fiber having a core-sheath structure, a nozzle for producing a composite fiber having a sea-island structure, and the like are known.
  • a first base for extruding a first dope liquid (corresponding to the core part of the composite fiber) is disposed at the center of the nozzle, and a second dope liquid ( A second base for extruding) corresponding to the outermost layer is arranged.
  • the second base may be inclined toward the first base such that the second dope liquid covers the first dope liquid extruded from the first base.
  • the second base may be circular so as to surround the first base. Moreover, the 1st nozzle
  • die may be united and the nozzle pack may be formed. Each die is preferably designed so that the discharge amount is constant by size, temperature control and the like.
  • the second dope liquid extruded from the second base is integrated so as to cover the first dope liquid extruded from the first base, and the unstretched yarn having a core-sheath structure is obtained by contacting with the coagulation liquid. Let it form.
  • the first dope liquid (corresponding to the core portion of the composite fiber) is coated with the second dope liquid (corresponding to the intermediate layer of the composite fiber), and then the third die
  • the third dope solution (corresponding to the outermost layer) is extruded so as to further cover the second dope solution, thereby forming an undrawn yarn having a three-layer structure.
  • by adding a nozzle it is possible to form an undrawn yarn having a four-layer structure or a five-layer structure.
  • the nozzle for the composite fiber having the sea-island structure is the above-described nozzle for manufacturing the composite fiber having the core-sheath structure, except that a plurality of first caps for extruding the first dope solution are arranged at the center of the nozzle. It is the same.
  • the position of each die can be appropriately adjusted according to the spinning conditions such as the type of fiber raw material to be used, the viscosity of each dope solution, the extrusion speed, and the temperature.
  • the coagulation liquid 11 may be any solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone.
  • the coagulation liquid 11 may appropriately contain water.
  • the temperature of the coagulation liquid 11 is preferably 0 to 30 ° C.
  • the distance that the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) may be a length that allows efficient desolvation, for example, 200 to 500 mm. It is.
  • the residence time in the coagulation liquid 11 may be a time for removing the dope solvent from the undrawn yarn.
  • stretching may be performed in the coagulating liquid 11.
  • the coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as necessary.
  • the coagulation liquid may be kept at a low temperature and taken up in an undrawn yarn state. Further, the undrawn yarn may be drawn in the coagulation liquid (pre-drawing).
  • the undrawn yarn (or predrawn yarn) obtained by the above method can be in the state of drawn yarn (composite fiber) by the drawing process.
  • Examples of the stretching method include wet heat stretching and dry heat stretching.
  • Wet heat stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in 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 drawing may be, for example, 1 to 30 times, 2 to 25 times, or 2 to 20 times the undrawn yarn (or the predrawn yarn). It may be ⁇ 15 times, may be 2 to 10 times, may be 2 to 8 times, may be 2 to 6 times, and may be 2 to 4 times. However, the draw ratio is not limited as long as desired properties such as fiber thickness and mechanical properties are obtained.
  • the dry heat drawing can be performed using an apparatus such as a contact-type hot plate and a non-contact type furnace, but is not particularly limited, and the fiber is heated to a predetermined temperature, Any device capable of stretching at a magnification may be used.
  • 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., 160 ° C. to 180 ° C. It may be in ° C.
  • the draw ratio in the dry heat drawing step may be, for example, 1 to 30 times, 2 to 30 times, or 2 to 20 times the undrawn yarn (or predrawn yarn). It may be 3 to 15 times, preferably 3 to 10 times, more preferably 3 to 8 times, and still more preferably 4 to 8 times. However, the draw ratio is not limited as long as desired properties such as fiber thickness and mechanical properties are obtained.
  • wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, as the stretching process, wet heat stretching is performed as the first stage stretching, dry heat stretching is performed as the second stage stretching, or wet heat stretching is performed as the first stage stretching, wet heat stretching is performed as the second stage stretching, and further the third stage stretching is performed. May be performed by dry heat stretching, and may be performed by appropriately combining wet heat stretching and dry heat stretching.
  • the lower limit value of the final draw ratio of the composite fiber that has undergone the drawing step is preferably 1 time, 2 times, 3 times, 4 times, 5 times, 6 times with respect to the undrawn yarn (or the predrawn yarn). , 7 times, 8 times, or 9 times.
  • the upper limit of the final draw ratio of the composite fiber that has undergone the drawing step may preferably be 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. . Also, for example, it may be 3 to 40 times, 3 to 30 times, 5 to 30 times, 5 to 20 times, 5 to 15 times, 5 It may be up to 13 times.
  • the shape of the spinneret, the hole shape, the number of holes and the like are not particularly determined, and can be appropriately selected according to the desired fiber diameter and the number of single yarns.
  • an oil agent may be applied to the undrawn yarn (or predrawn yarn) or the drawn yarn for the purpose of imparting charge suppression, convergence, lubricity, and the like.
  • the kind of oil agent to be applied, the amount to be applied, and the like are not particularly limited, and can be appropriately adjusted in consideration of the use of the composite fiber, the handleability of the bonded fiber, and the like.
  • a hole diameter of 0.1 mm to 0.6 mm can be exemplified.
  • the hole diameter is 0.1 mm or more, pressure loss can be reduced, and equipment costs can be reduced.
  • the hole diameter is 0.6 mm or less, the stretching operation for reducing the fiber diameter can be omitted, and the possibility of tearing (stretching break) between ejection and take-up can be further reduced.
  • the temperature at the time of passing through the spinneret and the temperature of the spinneret are not particularly limited, and may be appropriately adjusted depending on the concentration and viscosity of the dope liquid to be used, the type of solvent, and the like.
  • the temperature of the spinneret is preferably 30 ° C. to 100 ° C. from the viewpoint of preventing deterioration of the modified fibroin and the structural protein.
  • the upper limit of the temperature may be lower than the boiling point of the solvent to be used from the viewpoint of further reducing the possibility of pressure increase due to volatilization of the solvent and clogging in the piping due to solidification of the dope solution. preferable. Thereby, process stability improves.
  • the method according to the present embodiment may further include a step of filtering the dope solution before discharging the dope solution (filtering step) and / or a step of defoaming the dope solution before discharging (defoaming step).
  • the manufacturing method of the composite fiber according to the present embodiment may further include a crimping process.
  • the crimping step is a step of crimping the composite fiber by bringing it into contact with an aqueous medium (hereinafter sometimes referred to as “water crimping”).
  • water crimping an aqueous medium
  • the eccentric core-sheath type composite fiber as shown in FIG. 7B has a latent crimping ability, and can show more excellent crimpability when brought into contact with an aqueous medium.
  • the aqueous medium is a liquid or gas (steam) medium containing water (including water vapor).
  • the aqueous medium may be water or a mixed solution of water and a hydrophilic solvent.
  • the hydrophilic solvent include volatile solvents such as ethanol and methanol, and vapors thereof.
  • the aqueous medium may be a mixed liquid of water and a volatile solvent such as ethanol or methanol, and is preferably water or a mixed liquid of water and ethanol.
  • the ratio of water to the volatile solvent or its vapor is not particularly limited, and for example, the water: volatile solvent may have a mass ratio of 10:90 to 90:10.
  • the water content is preferably 30% by mass or more based on the total mass of the aqueous medium, and may be 40% by mass or 50% by mass or more.
  • the aqueous medium is a liquid, it is preferable to disperse an oil agent in the aqueous medium. In this case, water crimping and oil adhesion can be performed simultaneously.
  • the oil agent may be, for example, a known oil agent used for general purposes such as process passability and functionality imparting such as antistatic, friction reducing, flexibility imparting, or water repellency imparting. Any of them can be used.
  • the amount of the oil agent is not particularly limited, and may be, for example, 1 to 10% by mass or 2 to 5% by mass with respect to the total mass of the oil agent and the aqueous medium.
  • the aqueous medium is preferably a liquid or gas at 10 to 230 ° C. containing water (including water vapor).
  • the temperature of the aqueous medium may be 10 ° C or higher, 25 ° C or higher, 40 ° C or higher, 60 ° C or higher, or 100 ° C or higher, and 230 ° C or lower, 120 ° C or lower, or 100 ° C or lower. More specifically, when the aqueous medium is a gas (steam), the temperature of the aqueous medium is preferably from 100 to 230 ° C, more preferably from 100 to 120 ° C. When the steam of the aqueous medium is 230 ° C. or lower, thermal denaturation of the composite fiber can be prevented.
  • the temperature of the aqueous medium is preferably 10 ° C. or higher, 25 ° C. or higher, or 40 ° C. or higher from the viewpoint of efficiently imparting crimp, and from the viewpoint of keeping the strength of the composite fiber high. C. or lower is preferable.
  • the time of contact with the aqueous medium is not particularly limited, but may be 30 seconds or more, may be 1 minute or more, or 2 minutes or more, and is preferably 10 minutes or less from the viewpoint of productivity. In the case of steam, it is considered that a large shrinkage rate can be obtained in a shorter time than liquid.
  • the contact with the aqueous medium may be performed under normal pressure or under reduced pressure (for example, vacuum).
  • Examples of the method of contacting the aqueous medium include a method of immersing the composite fiber in the aqueous medium, a method of spraying the aqueous medium with steam on the composite fiber, and a method of exposing the composite fiber to an environment filled with the aqueous medium of steam.
  • the aqueous medium is steam
  • the contact of the aqueous medium with the composite fiber can be performed using a general steam setting apparatus.
  • Specific examples of the steam setting device include devices such as product name: FMSA type steam setter (manufactured by Fukushin Kogyo Co., Ltd.) and product name: EPS-400 (manufactured by Sakurai Dyeing Machinery Co., Ltd.).
  • the composite fiber is accommodated in the predetermined storage chamber, while the aqueous medium steam is introduced into the storage chamber, and the temperature of the storage chamber is set to the predetermined temperature.
  • steam is brought into contact with the composite fiber while adjusting to (for example, 100 ° C. to 230 ° C.).
  • the crimping step of the composite fiber by contact with the aqueous medium is preferably performed in a state where no tensile force is applied to the composite fiber (no tension is applied in the fiber axis direction) or a predetermined amount (fiber). (Strained in the axial direction by a predetermined amount). In this case, the degree of crimp can be controlled by adjusting the tensile force applied to the composite fiber.
  • a method for adjusting the tensile force applied to the composite fiber for example, a method of adjusting the load applied to these fibers by suspending weights of various weights on the composite fiber, or the state where the fiber is loosened
  • the method of changing the amount of looseness, winding the fiber around a wound body such as a paper tube or bobbin, and the winding force (clamping force on the paper tube or bobbin) at that time The method etc. which change suitably are mentioned.
  • the composite fiber may be further dried after contacting with the aqueous medium.
  • the drying method is not particularly limited, and the drying may be natural drying or may be performed by a thermal storm or a hot roller.
  • the drying temperature is not particularly limited, and may be, for example, 20 to 150 ° C., preferably 40 to 120 ° C., and more preferably 60 to 100 ° C.
  • the core portion containing the modified fibroin and the outermost layer containing the structural protein have different hydrophobicities.
  • the difference between the hydrophobicity of the core and the outermost layer (difference between the hydrophobicity of the modified fibroin and the hydrophobicity of the structural protein) in the composite fiber can be appropriately selected according to the desired crimping property.
  • 0.1 or more is preferable, 0.2 or more is more preferable, 0.3 or more is more preferable, 0.4 or more is more preferable, 0.5 or more is more preferable, 0.6 or more is more preferable, 0.7 or more Is more preferably 0.8 or more, more preferably 0.9 or more, more preferably 1.0 or more, more preferably 1.1 or more, still more preferably 1.2 or more, and particularly preferably 1.3 or more. preferable.
  • the greater the difference in the degree of hydrophobicity the more stable the potential crimpability can be stably imparted.
  • the core portion and the outermost layer (if present, the intermediate layer) of the composite fiber have different shrinkage rates due to water shrinkage.
  • the modified fibroin used for the core part contracts upon contact with an aqueous medium.
  • the structural protein used in the outermost layer (or intermediate layer) does not shrink even when contacted with an aqueous medium, or has a smaller shrinkage rate than the core.
  • Table 6 shows the shrinkage ratio of the modified fibroin fiber obtained by spinning the modified fibroin under the same conditions to the aqueous medium. The shrinkage rate was calculated by the following method. ⁇ Shrinkage rate> A plurality of modified fibroin fibers having a length of about 30 cm are bundled into a fiber bundle having a fineness of 150 denier.
  • a 0.8 g lead weight is attached to the fiber bundle, and in that state, the fiber bundle is immersed in water at 40 ° C. for 10 minutes to be shrunk to remove shrinkage due to residual stress derived from the manufacturing process.
  • the fiber bundle is taken out of the water and dried at room temperature for 2 hours with a 0.8 g lead weight attached. After drying, the length of the fiber bundle is measured. Again, it is immersed in water at 40 ° C. for 10 minutes to shrink, and the length of the fiber bundle is measured in water. These wetting and drying are repeated at least three times, and an average length when wet (Lwet) and an average length when drying (Ldry) are determined.
  • the composite ratio of the core part and the outermost layer in the composite fiber is not particularly limited, and can be appropriately set according to the combination of the core part and the outermost layer, the desired crimpability, the composite form, and the like.
  • the composite ratio of the core portion and the outermost layer may be, for example, in the range of 90:10 to 10:90 on a weight basis, in the range of 80:20 to 20:80, and 75:25 to 25:75. May be in the range of 75:25 to 35:65, may be in the range of 70:30 to 30:70, may be in the range of 65:35 to 35:65, 65 : 35 to 45:55, or 60:40 to 40:60.
  • the cross-sectional shape of the composite fiber is not particularly limited, and may be any of a round cross section, a triangular cross section, a multi-lopal cross section, a daruma-shaped cross section, a flat cross section, and other known cross-sectional shapes.
  • the conjugate fiber is an eccentric core-sheath type conjugate fiber as shown in FIG. 7B (the sectional shape of the conjugate fiber is not a concentric double circle), the balance between crimp development and texture is more excellent.
  • the fiber diameter of the composite fiber is not particularly limited and can be set as appropriate according to the application, for example, 10 to 125 ⁇ m, 10 to 100 ⁇ m, or 10 to 80 ⁇ m. It may be 10 to 60 ⁇ m, may be 10 to 40 ⁇ m, may be 10 to 35 ⁇ m, and may be 10 to 30 ⁇ m. When the fiber diameter is 125 ⁇ m or less, the solvent removal rate in the spinning process is difficult to increase. When the fiber diameter is 10 ⁇ m or more, it becomes easy to obtain a composite fiber stably.
  • the number of crimps of the composite fiber can be appropriately set depending on the application, for example, the number of crimps may be 5/25 mm or more, the number of crimps may be 10/25 mm or more, and the number of crimps may be The number of crimps may be 15 pieces / 25 mm or more, the number of crimps may be 20 pieces / 25 mm or more, and the number of crimps may be 25 pieces / 25 mm or more.
  • the composite fiber may be further chemically cross-linked between the polypeptide molecules in the composite fiber depending on the application.
  • functional groups that can be crosslinked include amino groups, carboxyl groups, thiol groups, and hydroxy groups.
  • the amino group of the lysine side chain contained in the polypeptide can be crosslinked with an amide bond by dehydration condensation with the carboxyl group of the glutamic acid or aspartic acid side chain.
  • Crosslinking may be performed by performing a dehydration condensation reaction under vacuum heating, or may be crosslinked by a dehydration condensation agent such as carbodiimide.
  • 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.
  • the carbodiimide is represented by the general formula R 1 N ⁇ C ⁇ NR 2 (wherein R 1 and R 2 each independently represents an organic group containing an alkyl group having 1 to 6 carbon atoms or a cycloalkyl group). It is a compound.
  • carbodiimide examples include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N, N′-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide. And diisopropylcarbodiimide (DIC).
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • DCC N, N′-dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC and DIC are preferable because they have a high ability to form an amide bond between polypeptide molecules and easily undergo a crosslinking reaction.
  • the crosslinking treatment is preferably performed by applying a crosslinking agent to the composite fiber and crosslinking by vacuum heating and drying.
  • a crosslinking agent a pure product may be imparted to the composite fiber, or a product diluted with a lower alcohol having 1 to 5 carbon atoms and a buffer solution to a concentration of 0.005 to 10% by mass may be imparted to the composite fiber.
  • the crosslinking treatment is preferably performed at a temperature of 20 to 45 ° C. for 3 to 42 hours. By the crosslinking treatment, higher stress (strength) can be applied to the composite fiber.
  • the crimpability of the composite fiber can be evaluated by, for example, confirming the number of crimps in a certain length of the composite fiber that has developed latent crimps through a crimping process.
  • the conjugate fiber according to this embodiment can be applied to various products.
  • examples of such products include fibers, yarns, fabrics, knitted fabrics, braids, nonwoven fabrics, paper, and cotton.
  • fibers include long fibers, short fibers, monofilaments, and multifilaments.
  • yarns include spun yarns, twisted yarns, false twisted yarns, processed yarns, mixed yarns, and mixed yarns.
  • fabrics such as woven fabrics, knitted fabrics, braided fabrics, non-woven fabrics, paper, cotton, etc. can be produced from these fibers and yarns. These products can be produced by known methods.
  • modified fibroin (1) Preparation of expression vector Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession numbers: P46804.1, GI: 1174415) derived from Nephila clavipes, SEQ ID NO: 15, SEQ ID NO: Modified fibroin having the amino acid sequence shown by 37 and SEQ ID NO: 47 (hereinafter also referred to as “PRT799”, “PRT918” and “PRT966”, respectively) were designed.
  • the amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence in which substitution, insertion and deletion of amino acid residues are performed for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes.
  • amino acid sequence represented by SEQ ID NO: 11 is added to the N-terminus.
  • the amino acid sequence represented by SEQ ID NO: 47 (PRT966) is an amino acid sequence represented by SEQ ID NO: 7 (amino acid sequence before the amino acid sequence represented by SEQ ID NO: 11 is added to the N-terminal) for the purpose of improving hydrophobicity.
  • QQ in the sequence repeated twice was replaced with VF, the remaining Q was replaced with I, and the amino acid sequence represented by SEQ ID NO: 11 at the N-terminus ( Tag sequence and hinge sequence).
  • nucleic acids encoding PRT799, PRT918 and PRT966 were synthesized.
  • the nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon.
  • the nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b (+) to obtain expression vectors.
  • the seed culture was added to a jar fermenter to which 500 mL of production medium (Table 8) was added so that the OD 600 was 0.05.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • 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 the culture was performed at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and cultured for 20 hours.
  • 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the modified fibroin.
  • the culture solution was centrifuged, and the cells were collected. Perform SDS-PAGE using cells prepared from the culture before and after adding IPTG, and confirm the expression of the desired modified fibroin by the appearance of the desired modified fibroin size band depending on the addition of IPTG. did.
  • the washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 60 ° C. And stirred for 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (manufactured by Sanko Junyaku Co., Ltd., cellulose tube 36/32). The white aggregated protein obtained after dialysis was collected by centrifugation, water was removed with a freeze dryer, and lyophilized powder was collected to obtain modified fibroin (PRT799, PRT918 and PRT966).
  • 8M guanidine buffer 8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • This aqueous solution was placed in a cellulose dialysis membrane (Seamless Cellulose Tubing, 36/32 manufactured by VISKASESELESCOAP), and dialyzed for 3 to 4 days using distilled water.
  • the recovered solution after dialysis was centrifuged at 20 ° C. and 15000 rpm for 1 hour to remove undissolved residues and impurities. Furthermore, it diluted with deionized water so that a density
  • the silk fibroin aqueous solution was frozen at ⁇ 80 ° C. and lyophilized overnight. After confirming that water was sufficiently removed, silk fibroin powder was obtained. SDS-PAGE was performed using the obtained silk fibroin, and the appearance of a band confirmed that the molecular weight of silk fibroin was 70 kDa.
  • Example 2 Preparation of dope solution
  • the first dope solution was prepared in the same manner as in Example 1.
  • As the second dope solution 10% by mass of the modified fibroin (PRT966, hydrophobicity: ⁇ 0.80) obtained in the modified fibroin production process and 90% by mass of formic acid were mixed, and the heating temperature was 70 ° C. Otherwise, the second dope solution was prepared in the same manner as the first dope solution.
  • (2) Wet spinning The prepared first dope solution and second dope solution were filled in reserve tanks, respectively.
  • Table 7 shows the physical property evaluation results of the raw yarn.
  • the stress values in Table 7 are relative values when the stress value of the modified fibroin fiber of Comparative Example 1 (PRT799 alone) is 100.
  • Table 9 shows the shrinkage of the composite fibers of Examples 1 and 2.
  • the shrinkage rate in Table 9 is a relative value when the value of the shrinkage rate of the modified fibroin fiber of Comparative Example 1 (PRT799 alone) is 100.
  • the composite fibers of Examples 1 and 2 can obtain an excellent shrinkage reduction effect as compared with the fiber of Comparative Example 1 (PRT799 alone), and the greater the degree of hydrophobicity (the higher the degree of hydrophobicity), the more It was recognized that the shrinkage reduction effect can be imparted. Furthermore, in the composite fiber of Example 1, an improvement in stress value was recognized.
  • Reference Example 1 Flammability test of modified fibroin Addition of lyophilized powder of modified fibroin (PRT799) to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0 mass%) to a concentration of 24 mass%, and shaker Used and mixed for 3 hours to dissolve. Thereafter, insoluble matters and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
  • the obtained spinning dope is heated to 90 ° C., filtered through a metal filter having an opening of 5 ⁇ m, and then left to stand in a 30 mL stainless syringe and defoamed, and then 100 mass from a solid nozzle having a needle diameter of 0.2 mm.
  • the solution was discharged into a% 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 fiber (raw fiber).
  • a knitted fabric (thickness: 180 denier, number of gauges: 18) was manufactured by circular knitting using a circular knitting machine using twisted yarn obtained by twisting raw material fibers. 20 g of the obtained knitted fabric was cut out and used as a test piece.
  • the flammability test complied with “Testing method for powdered or low melting point synthetic resin” described in “Head of Fire and Disaster Management, Hazardous Materials Regulation Section, Fire Safety No. 50 (May 31, 1995)”. The test was performed under conditions of a temperature of 22 ° C., a relative humidity of 45%, and an atmospheric pressure of 1021 hPa. Table 10 shows the measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)).
  • the critical oxygen index (LOI) value of the knitted fabric knitted with the modified fibroin (PRT799) fiber was 27.2.
  • the LOI value is 26 or more, it is known to be flame retardant. It can be seen that the modified fibroin is excellent in flame retardancy.
  • Reference Example 2 Hygroscopic exothermic evaluation of modified fibroin Add lyophilized powder of modified fibroin to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0% by mass) to a concentration of 24% by mass and use a shaker. And then mixed for 3 hours to dissolve. Thereafter, insoluble matters and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
  • the obtained spinning dope is heated to 60 ° C., filtered through a metal filter having an opening of 5 ⁇ m, and then left to stand in a 30 mL stainless syringe and defoamed, and then 100 mass from a solid nozzle having a needle diameter of 0.2 mm.
  • the solution was discharged into a% 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 fiber (raw fiber).
  • Table 10 shows the thickness of the knitted fabric using PRT918 fiber or PRT799 fiber and the number of gauges.
  • the knitted fabric using other raw material fibers was adjusted in thickness and gauge number so as to have almost the same cover factor as that of the modified fibroin fiber knitted fabric. Specifically, it is as follows.
  • test piece 2 pieces of knitted fabric cut to 10 cm ⁇ 10 cm were put together, and four sides were sewn together to obtain 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 is transferred to a high humidity environment (temperature 20 ⁇ 2 ° C., relative humidity 90 ⁇ 5%). The temperature was measured at intervals of 1 minute by a temperature sensor attached to the center of the interior for 30 minutes.
  • FIG. 8 is a graph showing an example of the results of the hygroscopic exothermic test.
  • the horizontal axis of the graph represents the time (minutes) left in the high humidity environment, with the time when the sample was transferred from the low humidity environment to the high humidity environment as 0.
  • the vertical axis of the graph indicates the temperature (sample temperature) measured by the temperature sensor.
  • the point indicated by M corresponds to the maximum value of the sample temperature.
  • Table 12 shows the calculation results of the maximum moisture absorption exotherm of each knitted fabric.
  • the modified fibroin (PRT918 and PRT799) has a high maximum moisture absorption exotherm and excellent moisture absorption exothermicity as compared with existing materials.
  • Reference Example 3 Evaluation of heat retention of modified fibroin Add lyophilized powder of modified fibroin to a dimethyl sulfoxide solution of lithium chloride (concentration: 4.0% by mass) to a concentration of 24% by mass, and use a shaker. It was dissolved by mixing for 3 hours. Thereafter, insoluble matters and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
  • the obtained spinning dope is heated to 60 ° C., filtered through a metal filter having an opening of 5 ⁇ m, and then left to stand in a 30 mL stainless syringe and defoamed, and then 100 mass from a solid nozzle having a needle diameter of 0.2 mm.
  • the solution was discharged into a% 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 fiber (raw fiber).
  • Each raw material fiber was used to produce knitted fabrics by flat knitting using a flat knitting machine.
  • Table 13 shows the count, the number of twists, the number of gauges, and the basis weight of the knitted fabric using the PRT966 fiber or the PRT799 fiber.
  • the knitted fabric using the other raw material fibers was adjusted so as to have almost the same cover factor as that of the modified fibroin fiber knitted fabric. Specifically, it is as follows.
  • the heat retention was evaluated by using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd., using a dry contact method (a method assuming that the skin and clothes are directly touched in a dry state).
  • a test piece One knitted fabric cut into a 20 cm ⁇ 20 cm rectangle was used as a test piece (sample).
  • the test piece was set on a hot plate set at a constant temperature (30 ° C.), and the amount of heat (a) dissipated through the test piece was determined under the condition of the wind speed in the wind tunnel of 30 cm / second. With the test piece not set, the amount of heat (b) dissipated under the same conditions as described above was determined, and the heat retention rate (%) was calculated according to the following formula B.
  • Table 14 shows the calculation results of the heat retention index. It can be evaluated that the higher the heat retention index, the better the heat retention material.
  • the modified fibroin (PRT966 and PRT799) has a higher heat retention index than the existing materials and is superior in heat retention.

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Abstract

La présente invention concerne une fibre composite ayant une partie coeur et une couche la plus externe qui recouvre la partie coeur, la partie coeur contenant une fibroïne modifiée et la couche la plus externe contenant une protéine structurale.
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WO2021241546A1 (fr) * 2020-05-28 2021-12-02 Ｓｐｉｂｅｒ株式会社 Procédé de production de protéine cible

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