WO2019151424A1 - Fibroin composition, fibroin solution, and method for manufacturing fibroin fiber - Google Patents

Fibroin composition, fibroin solution, and method for manufacturing fibroin fiber Download PDF

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WO2019151424A1
WO2019151424A1 PCT/JP2019/003456 JP2019003456W WO2019151424A1 WO 2019151424 A1 WO2019151424 A1 WO 2019151424A1 JP 2019003456 W JP2019003456 W JP 2019003456W WO 2019151424 A1 WO2019151424 A1 WO 2019151424A1
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fibroin
amino acid
sequence
motif
rep
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PCT/JP2019/003456
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French (fr)
Japanese (ja)
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佑之介 安部
翔太 冨樫
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Ｓｐｉｂｅｒ株式会社
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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

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  • the present invention relates to a fibroin composition, a fibroin solution, and a method for producing a fibroin fiber. More specifically, the present invention relates to a mixed fibroin composition capable of producing a product (fibroin fiber or the like) having excellent physical properties such as water resistance.
  • Fibroin is a kind of fibrous protein and contains up to 90% glycine, alanine and serine residues that lead to the formation of ⁇ -pleated sheets (Non-patent Document 1).
  • proteins salk protein, hornet silk protein, spider silk protein
  • the like that constitute a thread produced by insects and spiders are known.
  • Non-patent document 2 Recombinant spider silk protein and recombinant silk protein have been reported to be produced in protein production systems such as, for example, transgenic goats, transgenic silkworms, recombinant plants or recombinant mammalian cells.
  • Non-patent document 3 Furthermore, as a protein production system capable of mass production, many recombinant fibroin productions using organisms such as yeast, mold, gram-negative bacteria, and gram-positive bacteria as a host have been reported (Patent Document 1).
  • fibroin fiber obtained by spinning fibroin has a property of shrinking by immersion in water or hot water, exposure to a high humidity environment, and the like. This characteristic causes various problems in the manufacturing process and commercialization, and affects products made of fibroin fibers.
  • an anti-shrinking method for preventing the shrinkage of the product for example, a silk fabric using a strong twisted yarn that has been scoured is immersed in water, other solvent, or a mixed system thereof in a tensioned state and heated for a predetermined time.
  • a method for preventing shrinkage of a silk fabric characterized by the above Patent Document 4
  • a method for fixing the shape of animal fiber products (patent document 5), which is characterized by fixing the shape during steam treatment, has been reported.
  • the shrinkage-preventing methods as disclosed in Patent Documents 4 and 5 are industrially disadvantageous because the operation is complicated and the number of steps increases. It is extremely industrially useful if the shrinkage of the fibroin fiber itself can be suppressed or reduced regardless of such a shrinkage prevention method.
  • the present inventors reported (A) fibroin in which the content of n motif was reduced and fibroin having an amino acid sequence in which the content of glutamine residues was reduced as reported in Patent Document 2 (International Publication No. 2017/188434). It has been found that fibroin fibers formed from mixed fibroin mixed with fibroin with increased hydrophobicity such as the above are suppressed or reduced in shrinkage and excellent in stress and elongation.
  • the present invention is based on this novel finding.
  • a fibroin composition comprising: a first fibroin; and at least one fibroin selected from the group consisting of a second fibroin and a third fibroin,
  • the first fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m1 , 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 The maximum sum of the total number of amino acid residues of the two adjacent [(A) n motif-REP] units, which is 11.3, is x, and the total number of amino acid residues of the domain sequence is y.
  • a fibroin having an amino acid sequence in which x / y is 50% or more The second fibroin is represented by a domain sequence represented by Formula 2: [(A) n motif-REP] m2 or represented by Formula 3: [(A) n motif-REP] m3- (A) n motif.
  • the third fibroin contains a domain sequence represented by Formula 4: [(A) n motif-REP] m4 and is located on the most C-terminal side (A) Sequence from the n motif to the C terminus of the domain sequence Z is the total number of amino acid residues contained in a region in which the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs included in the sequence excluding from the domain sequence.
  • a fibroin composition which is a fibroin having an amino acid sequence of [In Formula 1, Formula 2, Formula 3 and Formula 4, (A) n motif independently represents an amino acid sequence composed of 4 to 27 amino acid residues, and (A) all amino acids in n motif
  • the number of alanine residues with respect to the number of residues is 80% or more
  • REP independently represents an amino acid sequence composed of 10 to 200 amino acid residues
  • m1, m2, m3 and m4 are each independently An integer from 8 to 300 is indicated.
  • 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 other amino acid residues in the second fibroin are isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) alanine (A), glycine ( [1] or [1] which is an amino acid residue selected from the group consisting of G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). 2].
  • the second fibroin contains a GPGXX (where X represents an amino acid residue other than a glycine residue) motif in the REP, and the content of the GPGXX motif is 10% or more.
  • the first fibroin comprises an amino acid sequence represented by any one of SEQ ID NOs: 1 to 10, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 10,
  • the second fibroin includes an amino acid sequence represented by any of SEQ ID NOs: 11 to 24, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 11 to 24.
  • the above solvents are hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide
  • HFIP hexafluoroisopropanol
  • HFA hexafluoroacetone
  • DMSO dimethyl sulfoxide
  • DMF N, N-dimethylformamide
  • SDS sodium dodecyl sulfate
  • the fibroin solution according to [9] which is a solvent selected from the group consisting of calcium chloride and lithium thiocyanate, and a mixed solvent of two or more of these.
  • the content ratio of the first fibroin to at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 9.9: 0.1 to 5.0: on
  • the content ratio of the first fibroin to at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 5.0: 5.0 to 0.1:
  • the content ratio of the first fibroin and at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 5.0: 5.0 on a weight basis, [9 ] Or the fibroin solution according to [10].
  • a fibroin composition capable of producing a product excellent in water resistance (fibroin fiber or the like).
  • the fibroin composition according to the present invention comprises at least two types of fibroin. That is, the fibroin composition according to the present invention includes a first fibroin and further includes at least one fibroin selected from the group consisting of a second fibroin and a third fibroin.
  • the first fibroin is (A) a fibroin having a domain sequence in which the content of the n motif is reduced.
  • the second and third fibroins are fibroin with increased hydrophobicity.
  • the second fibroin is a fibroin having a domain sequence with a reduced content of glutamine residues
  • Fibroin is a fibroin having a domain sequence including a region having a large hydrophobic index locally.
  • the fibroin according to the present invention is a protein containing a domain sequence represented by formula A: [(A) n motif-REP] m or formula B: [(A) n motif-REP] m- (A) n motif. is there.
  • 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 “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). Corresponding amino acid sequence).
  • the n motif represents an amino acid sequence composed of 4 to 20 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in the n motif is 80% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 8 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 n motif may be (A) the number of alanine residues relative to the total number of amino acid residues in the n motif is 80% or more, preferably 83% or more, and 86% or more. More preferably, it is more preferably 90% or more, still more preferably 95% or more, and particularly preferably 100% (meaning that it is composed only of alanine residues). It is preferable that at least seven of the (A) n motifs present in the domain sequence are composed of only alanine residues.
  • (A) n motif is (A) n (A represents an alanine residue, n represents an integer of 4 to 20, preferably an integer of 4 to 16). It has the amino acid sequence represented by these.
  • the first fibroin according to the present invention includes a domain sequence represented by Formula 1: [(A) n motif-REP] m1 , and two adjacent [[A] from the N-terminal side toward the C-terminal side.
  • the ratio of the number of amino acid residues in the other REP is 1
  • the maximum sum of the total number of amino acid residues of the two adjacent [(A) n motif-REP] units, which is .8 to 11.3, is x
  • the total number of amino acid residues of the domain sequence Is a fibroin having an amino acid sequence where x / y is 50% or more, except for fibroin corresponding to the second fibroin or the third fibroin.
  • (A) n motif represents an amino acid sequence composed of 4 to 27 amino acid residues
  • (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 80%.
  • REP represents an amino acid sequence composed of 10 to 200 amino acid residues
  • m1 represents an integer of 8 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.
  • X / y in the first fibroin is preferably 60% or more, more preferably 65% or more, still more preferably 70% or more, and even more preferably 75% or more, It is still more preferable that it is 80% or more, and it is especially preferable that it is 85% or more.
  • x / y is in this range, it becomes a fibroin from which a fibroin fiber excellent in stress and elongation can be obtained.
  • X / y may be calculated according to the method described in Patent Document 2 (International Publication No. 2017/188434).
  • the first fibroin may be a modified fibroin described in Patent Document 2 (International Publication No. 2017/188434). That is, the first fibroin is obtained, for example, by deleting one or more of the sequences encoding the (A) n motif from the gene sequence of naturally occurring fibroin so that x / y is 50% or more. be able to. In addition, for example, an amino acid sequence corresponding to the deletion of one or more (A) n motifs so that x / y is 50% or more from the amino acid sequence of naturally derived fibroin is designed and designed. It can also be obtained by chemically synthesizing a nucleic acid encoding the 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.
  • Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.
  • Naturally occurring fibroin include fibroin produced by insects or spiders.
  • fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandaraina), Tengea (Antheraea yamanai), ⁇ ⁇ (Antereaperanii), ⁇ ⁇ (Eriothyraminey) ), Silkworms (Samia cythia), chestnut worms (Caligula japonica), Chussa moth (Anthereaea mylitta), silkworms produced by silkworms such as Antheraea assamata, vespasam 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 (L) and the genus Spider belonging to the genus Usd 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 spidolin 1 [derived from Nephila clavipes (GenBank accession number AAC4, amino acid sequence A04) U37520 (base sequence)), major angul11ate spidroin 1 [La rodectus hesperus origin] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Nephila clavata origin] (GenBank accession number AAL32447, amino acid sequence 45, amino acid sequence) major
  • 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 first 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 first fibroin.
  • Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 25 (amino acid sequence containing a His tag).
  • the first fibroin (i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 10 (see Table 1), or (ii) an amino acid represented by any one of SEQ ID NOs: 1 to 10 Mention may be made of fibroin comprising an amino acid sequence having 90% or more sequence identity to the sequence.
  • the fibroin may consist of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 10.
  • the fibroin of (ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 10.
  • Fibroin of (ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m1 .
  • the sequence identity is preferably 95% or more.
  • x / y is preferably within the above-described range.
  • the second fibroin according to the present invention is a domain sequence represented by Formula 2: [(A) n motif-REP] m2 or Formula 3: [(A) n motif-REP] m3- (A) n motif. It is a fibroin having an amino acid sequence that includes the represented domain sequence and has a glutamine residue content of 9% or less.
  • (A) n motif independently represents an amino acid sequence composed of 4 to 27 amino acid residues, and (A) alanine residues relative to the total number of amino acid residues in n motif The number is 80% or more, REP independently represents an amino acid sequence composed of 10 to 200 amino acid residues, and m2 and m3 each independently represents an integer of 8 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 second fibroin may have a glutamine residue content of 9% or less, preferably 7% or less, more preferably 4% or less, and particularly preferably 0%.
  • glutamine residue content is in this range, the fibroin can be obtained to obtain a fibroin fiber in which shrinkage is suppressed or reduced.
  • FIG. 1 is a schematic diagram showing the domain sequence of fibroin.
  • the method for calculating the glutamine residue content will be specifically described with reference to FIG.
  • Formula A [(A) n motif-REP] m or
  • Formula B [(A) n motif-REP] m-
  • A) In fibroin containing a domain sequence represented by n motif, the most C-terminal side Located in (A) all REPs contained in a sequence (sequence corresponding to “region A” in FIG.
  • the glutamine residue content is calculated as a / b.
  • sequence that is 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 may include a sequence that is not highly correlated with the sequence characteristic of fibroin, where m is If it is small (that is, if the domain sequence is short), it affects the calculation result of the glutamine residue content, so this effect is eliminated.
  • the second fibroin is, for example, deleting one or more glutamine residues in REP from the gene sequence of naturally occurring fibroin so that the glutamine residue content is 9% or less, and / or It can be obtained by replacing one or more glutamine residues in REP with other amino acid residues.
  • one or more glutamine residues in REP are deleted from the amino acid sequence of naturally occurring fibroin so that the glutamine residue content is 9% or less, and / or 1 in REP.
  • it can be obtained by designing an amino acid sequence corresponding to substitution of a plurality of glutamine residues with other amino acid residues, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • 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.
  • the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further performed.
  • the method for substitution, deletion, insertion and / or addition of amino acid residues, and naturally-occurring fibroin are as described for the first fibroin.
  • 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.
  • 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).
  • the hydrophobicity index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 2 below.
  • 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 second fibroin is at least one selected from the GGX motif and the GPGXX motif (G represents a glycine residue, P represents a phenylalanine residue, and X represents an amino acid residue other than a glycine residue) in the amino acid sequence of REP.
  • G represents a glycine residue
  • P represents a phenylalanine residue
  • X represents an amino acid residue other than a glycine residue
  • one motif is included.
  • the content ratio of the GPGXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more. Thereby, the elongation of the second fibroin can be further improved.
  • the “GPGXX motif content” is a value calculated by the following method.
  • Formula A [(A) n motif-REP] m or Formula B: [(A) n motif-REP] m-
  • FIG. 1 is a schematic diagram showing the domain sequence of fibroin.
  • the calculation method of the content ratio of GPGXX motif will be specifically described with reference to FIG.
  • all REPs are “most C-terminally located ( A) GPGXX for calculating c because it is included in the “sequence excluding the sequence from the n motif to the C-terminus of the domain sequence from the domain sequence” (the sequence indicated by “region A” in FIG. 1).
  • 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. Even more preferably, it is particularly preferably 0.4 or more.
  • the hydrophobicity of REP It may be 1.0 or less and may be 0.7 or less.
  • the “hydrophobicity of REP” is a value calculated by the following method.
  • Formula A [(A) n motif-REP] m or Formula B: [(A) n motif-REP] m- (A) In fibroin containing a domain sequence represented by n motif, the most C-terminal side Positioned (A) In all REPs included in the sequence (sequence corresponding to “region A” in FIG.
  • the second 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 second fibroin.
  • Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 25 (amino acid sequence containing a His tag).
  • the amino acid sequence represented by any one of SEQ ID NOs: 11 to 24 (see Table 3) or (iv) the amino acid represented by any one of SEQ ID NOs: 11 to 24 Mention may be made of fibroin comprising an amino acid sequence having 90% or more sequence identity to the sequence.
  • the fibroin may comprise an amino acid sequence represented by any of SEQ ID NOs: 11 to 24.
  • the fibroin (iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 11 to 24.
  • Fibroin is also a protein comprising a domain sequence represented by Formula 2: [(A) n motif-REP] m2 or Formula 3: [(A) n motif-REP] m3- (A) n motif It is.
  • the sequence identity is preferably 95% or more.
  • the fibroin (iv) preferably has a glutamine residue content within the above-described range. Moreover, it is preferable that the fibroin of (iv) has the content ratio of GPGXX motif in the above-mentioned range.
  • the third fibroin according to the present invention includes a domain sequence represented by Formula 4: [(A) n motif-REP] m4 , and is located on the most C-terminal side (A) from the n motif to the C terminal of the domain sequence.
  • Z is the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs included in the sequence excluding the sequence up to the domain sequence.
  • (A) When the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence is represented by w (6) z / w is 6. Fibroin having an amino acid sequence of 2% or more (excluding fibroin corresponding to the second fibroin).
  • (A) n motif represents an amino acid sequence composed of 4 to 27 amino acid residues
  • (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 80%.
  • REP represents an amino acid sequence composed of 10 to 200 amino acid residues
  • m4 represents an integer of 8 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 third fibroin may have a z / w of 6.2% or more, preferably 7% or more, more preferably 10% or more, still more preferably 20% or more, and 30 It is still more preferable that it is% or more.
  • the upper limit of z / w is not particularly limited, but may be 45% or less, for example.
  • z / w is a value calculated by the following method.
  • sequence A a sequence obtained by removing the sequence from the domain sequence to the C-terminal side of the domain sequence (A) n motif to the C-terminus of the domain sequence.
  • sequence 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 z.
  • the total number of amino acid residues contained in sequence A is w.
  • the average value of the hydrophobicity index of four consecutive amino acid residues is 2
  • 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, it is as shown in Table 2.
  • the third fibroin is, for example, one or more hydrophilic amino acid residues in the REP (for example, the hydrophobicity index is negative so that the amino acid sequence of naturally derived fibroin satisfies the above z / w condition).
  • a hydrophobic amino acid residue eg, an amino acid residue having a positive hydrophobicity index
  • It can be obtained by locally modifying the amino acid sequence including a region having a large hydrophobicity index.
  • an amino acid sequence satisfying the above z / w condition 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 method for substitution, deletion, insertion and / or addition of amino acid residues, and naturally-occurring fibroin are as described for the first fibroin.
  • 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 third 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 third fibroin.
  • Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 25 (amino acid sequence containing a His tag).
  • the amino acid sequence represented by any one of SEQ ID NOs: 27 to 32 (see Table 4) or (vi) the amino acid represented by any one of SEQ ID NOs: 27 to 32 Mention may be made of fibroin comprising an amino acid sequence having 90% or more sequence identity to the sequence.
  • the fibroin may consist of an amino acid sequence represented by any of SEQ ID NOs: 27 to 32.
  • the fibroin (vi) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 27 to 32.
  • the fibroin in (vi) is also a protein containing a domain sequence represented by Formula 4: [(A) n motif-REP] m4 .
  • the sequence identity is preferably 95% or more.
  • the fibroin (vi) preferably has z / w within the above-mentioned range.
  • the first fibroin, the second fibroin, and the third fibroin can be obtained by the method described in Patent Document 2 (International Publication No. 2017/188434). That is, a host is transformed with an expression vector having one or more regulatory sequences operably linked to the nucleic acid sequence encoding the fibroin according to the present embodiment, and the nucleic acid according to the present invention is transformed in the host. Can be produced to produce the fibroin according to the present embodiment. Further, purified fibroin can be obtained by the method described in Patent Document 2 (International Publication No. 2017/188434).
  • the content ratio of at least one selected from the first fibroin and the second fibroin and the third fibroin is not particularly limited, and is suitable for use of the fibroin composition, etc. Accordingly, it can be set as appropriate.
  • the first fibroin has excellent properties with respect to stress and elongation
  • the second and third fibroin have excellent properties with respect to water resistance. Therefore, the content ratio of at least one selected from the first fibroin and the second fibroin and the third fibroin in the fibroin composition depends on whether stress or elongation or water resistance is enhanced.
  • the ratio of the first fibroin may be increased, and at least one selected from the first fibroin, the second fibroin and the third fibroin
  • the content ratio is preferably in the range of 9.9: 0.1 to 5.0: 5.0 (weight ratio).
  • the ratio of at least one selected from the second fibroin and the third fibroin may be increased, and the first fibroin, the second fibroin and the third fibroin are selected.
  • the content ratio of at least one selected from the above is preferably in the range of 5.0: 5.0 to 0.1: 9.9.
  • the content ratio of the first fibroin and at least one selected from the second fibroin and the third fibroin is 5.0: 5. It may be 0.
  • the fibroin composition according to the present invention may be in any form of powder, paste, or liquid (for example, a suspension or a fibroin solution described later).
  • the fibroin composition according to the present invention is in the form of a molded body containing the fibroin composition or made of the fibroin composition (for example, a product such as fibroin fiber described later). May be.
  • the fibroin composition according to the present invention may contain a component other than the first fibroin and at least one selected from the second fibroin and the third fibroin depending on the form and the like.
  • the fibroin composition according to the present invention may be a product containing or consisting of the fibroin composition.
  • examples of products include fibers, yarns, films, foams, spheres, nanofibrils, gels (hydrogels, etc.) and resins. These can be produced according to the methods described in JP-A-2009-505668, JP-A-5678283, JP-A-4638735 and the like.
  • the fibroin fiber formed from the fibroin composition according to the present invention is a fiber (long fiber, short fiber, multifilament, monofilament, etc.) or yarn (spun yarn, twisted yarn, false twisted yarn, processed yarn, blended yarn, blended yarn). Etc.) and can be applied to woven fabrics, knitted fabrics, braided fabrics, non-woven fabrics and the like. It can also be applied to high-strength applications such as ropes, surgical sutures, flexible stops for electrical components, and bioactive materials for transplantation (eg, artificial ligaments and aortic bands).
  • the fibroin solution according to the present invention is obtained by dissolving the fibroin composition according to the present invention in a solvent.
  • the fibroin solution can be used, for example, as a dope solution for forming fibroin fibers, fibroin films and the like.
  • solvent examples include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and urea, guanidine, sodium dodecyl sulfate (SDS), Examples include an aqueous solution containing lithium bromide, calcium chloride, lithium thiocyanate, and the like. These solvents may be used alone or in combination of two or more.
  • an inorganic salt may be added to the fibroin solution according to the present embodiment as necessary.
  • the inorganic salt which consists of the following Lewis acid and Lewis base is mentioned, for example.
  • the Lewis base include oxo acid ions (nitrate ions, perchlorate ions, etc.), metal oxo acid ions (permanganate ions, etc.), halide ions, thiocyanate ions, cyanate ions, and the like.
  • the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, complex ions, and the like.
  • inorganic salts composed of a Lewis acid and a Lewis base include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate, calcium chloride, calcium bromide.
  • Calcium salts such as calcium iodide, calcium nitrate, calcium perchlorate and calcium thiocyanate
  • iron salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate and iron thiocyanate
  • Aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate
  • Sodium salts such as sodium uride, sodium nitrate, sodium perchlorate and sodium thiocyanate
  • zinc salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, zinc perchlorate and zinc thiocyanate
  • chloride Magnesium salts such as magnesium, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium thiocyanate, barium chloride, barium bromide, barium iodide, barium nitrate, barium perchlorate, and barium thiocyanate
  • strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate.
  • the first fibroin and at least one selected from the second fibroin and the third fibroin are mixed (hereinafter also referred to as “mixed fibroin”). It can be obtained by preparing a dissolved solution. The mixing may be performed by mixing a solution obtained by dissolving the first fibroin in a solvent and a solution obtained by dissolving at least one selected from the second fibroin and the third fibroin in the solvent. Fibroin and at least one selected from the second fibroin and the third fibroin may be mixed and dissolved in the same solvent.
  • the fibroin solution In the preparation of the fibroin solution according to this embodiment, it may be heated to 30 to 90 ° C. What is necessary is just to set the temperature which can be melt
  • the viscosity of the fibroin solution according to this embodiment may be appropriately set according to the use of the fibroin solution.
  • the viscosity may be appropriately set according to the spinning method, for example, 100 to 15,000 cP (centipoise) at 35 ° C., What is necessary is just to set to 100-30,000 cP (centipoise) etc. in 40 degreeC.
  • the viscosity of the spinning solution can be measured using, for example, a trade name “EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.
  • the content ratio of at least one selected from the first fibroin and the second fibroin and the third fibroin is not particularly limited and depends on the use of the fibroin solution and the like. And can be set as appropriate. Specifically, for example, when stress and elongation are emphasized, the content ratio of the first fibroin and at least one selected from the second fibroin and the third fibroin is 9.9: 0.1. It is preferably in the range of up to 5.0: 5.0 (weight ratio). When water resistance is important, the content ratio of the first fibroin and at least one selected from the second fibroin and the third fibroin is 5.0: 5.0 to 0.1: 9. Is preferably in the range of .9. Furthermore, in the case where stress, elongation, and water resistance are exhibited in a balanced manner, the content ratio of the first fibroin and at least one selected from the second fibroin and the third fibroin is 5.0: 5. It may be 0.
  • the fibroin fiber according to the present invention can be obtained by using the fibroin solution according to the present invention as a spinning solution (dope solution) and spinning it by a method usually used for spinning fibroin.
  • the method for producing the fibroin fiber according to the present invention may include a step of using the fibroin solution according to the present invention as a dope liquid and extruding the dope liquid from a die into a coagulating liquid to obtain an undrawn yarn.
  • Examples of the solvent for the spinning solution include the same solvents as those exemplified in the description of the fibroin solution.
  • An inorganic salt may be added to the spinning solution as necessary.
  • an inorganic salt the same kind as the inorganic salt exemplified in the description of the fibroin solution can be exemplified.
  • the content ratio of at least one selected from the first fibroin and the second fibroin and the third fibroin in the spinning solution is as described in the fibroin solution.
  • fibroin solution By appropriately blending the first fibroin and at least one selected from the second fibroin and the third fibroin, fibroin superior in strength and elongation to the fibroin fiber spun only with the first fibroin. Fiber (mixed fibroin fiber) can be obtained. Further, it is possible to obtain a fibroin fiber (mixed fibroin fiber) superior in water resistance to a fibroin fiber spun with at least one selected from the second fibroin and the third fibroin. In any case, by spinning the mixed fibroin, it is possible to obtain mixed fibroin fibers that are superior in stress and elongation or water resistance compared to the case of spinning fibroin alone.
  • the first fibroin: second fibroin The mixed fibroin fiber spun using a spinning solution (dope solution) mixed and dissolved at a ratio of 7: 3 is stronger than the fibroin fiber spun using a spinning solution containing only PRT799 (SEQ ID NO: 9) protein. It becomes a mixed fibroin fiber excellent in elongation.
  • the mixed fibroin fiber has better water resistance than the spun fibroin fiber.
  • the spinning method is not particularly limited as long as it is a method capable of spinning the mixed fibroin according to the present invention, and examples thereof include dry spinning, melt spinning, and wet spinning.
  • a preferred spinning method includes wet spinning.
  • a solvent in which mixed fibroin is dissolved is extruded from a spinneret (nozzle) into a coagulating liquid (coagulating liquid tank), and the mixed fibroin is solidified in the coagulating liquid, whereby the shape of the yarn is not yet formed.
  • a drawn yarn can be obtained.
  • the coagulation liquid 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. Water may be appropriately added to the coagulation liquid.
  • the temperature of the coagulation liquid is preferably 0 to 30 ° C.
  • the extrusion speed may be 0.001 to 0.50 mL / min per hole. It is preferably from 01 to 0.50 mL / min, more preferably from 0.01 to 0.40 mL / min, further preferably from 0.01 to 0.35 mL / min, and from 0.02 to 0 Particularly preferred is .35 mL / min.
  • the length of the coagulating liquid tank is not limited as long as the solvent can be efficiently removed, and is, for example, 200 to 500 mm.
  • the undrawn yarn take-up speed may be, for example, 0.001 to 100 m / min, 0.01 to 100 m / min, 0.1 to 80 m / min, 0.1 to It may be 60 m / min, 0.1 to 40 m / min, 0.1 to 30 m / min, 0.1 to 25 m / min, 0.1 to 20 m / min Minutes, 0.1 to 15 m / min, 1 to 15 m / min, 1 to 13 m / min, 1 to 10 m / min, 1 to It may be 5 m / min and may be 1 to 3 m / min.
  • the take-up speed is less than 0.001 m / min, sufficient productivity cannot be obtained.
  • the residence time may be any time as long as the dope solvent is removed from the undrawn yarn.
  • the residence time may be 0.01 to 3 minutes, preferably 0.01 to 1.5 minutes. More preferably, the time is 01 to 0.2 minutes, more preferably 0.03 to 0.2 minutes, and particularly preferably 0.05 to 0.15 minutes.
  • stretching pre-stretching
  • the coagulation liquid may be kept at a low temperature and taken up in an undrawn yarn state.
  • the coagulating liquid tank may be provided in multiple stages, and the stretching may be performed at each stage or a specific stage as required.
  • the undrawn yarn (or predrawn yarn) obtained by the above method can be made into a drawn yarn (mixed fibroin fiber) through a drawing process.
  • Examples of the stretching method include wet heat stretching and dry heat stretching.
  • Wet and hot stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, and in steam heating.
  • the temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C.
  • undrawn yarn or predrawn yarn
  • Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, or the like.
  • the temperature may be, for example, 140 ° C. to 270 ° C., and preferably 160 ° C. to 230 ° C.
  • an undrawn yarn or predrawn yarn
  • an undrawn yarn can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
  • Wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and the third stage stretching is performed by dry heat stretching.
  • wet heat stretching and dry heat stretching can be appropriately combined.
  • the final draw ratio in the drawing step is, for example, 5 to 20 times, preferably 6 to 11 times that of the undrawn yarn (or predrawn yarn).
  • the mixed fibroin according to the present invention may be drawn into a mixed fibroin fiber and then chemically cross-linked between polypeptide molecules in the mixed fibroin fiber.
  • 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 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 a C 1-6 alkyl group or a cycloalkyl group). 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 cross-linking treatment is preferably performed by adding a cross-linking agent to the mixed fibroin fiber and performing cross-linking by vacuum heating and drying.
  • a cross-linking agent a pure product may be imparted to the mixed fibroin fiber, or a diluted product of 0.005 to 10% by mass with a lower alcohol having 1 to 5 carbon atoms or a buffer solution may be imparted to the mixed fibroin fiber. May be.
  • the crosslinking treatment is preferably performed at a temperature of 20 to 45 ° C. for 3 to 42 hours. By the crosslinking treatment, a higher stress (strength) can be applied to the mixed fibroin fiber.
  • the physical properties of the mixed fibroin fiber can be measured and evaluated as follows.
  • a mixed fibroin fiber sample may be attached to a mold made of cardboard, and the distance between the grippers may be 20 mm, and the pulling speed may be 10 mm / min.
  • the load cell capacity is 10N, and the gripping jig may be a clip type.
  • Fibroin fibers have the property of shrinking (primary shrinkage) by contact (wetting) with water below the boiling point. After the primary shrinkage, when it is dried, it further shrinks (secondary shrinkage). After the secondary shrinkage, when it is again brought into contact with water below the boiling point, it expands to the length before the secondary shrinkage, and after that, when drying and wetting are repeated, the width is about the same as the secondary shrinkage (“stretch rate” in FIG. 3). ), The contraction and the expansion are repeated (FIG. 3). In the fibroin fiber, the smaller the shrinkage, the better. However, in the product such as a woven fabric made of fibroin fiber, the secondary shrinkage is preferably small.
  • Secondary shrinkage can be evaluated using the secondary shrinkage rate obtained by the following method as an index.
  • ⁇ Secondary shrinkage> A plurality of fibroin fibers having a length of about 30 cm are bundled to form a fiber bundle having a fineness of 150 denier.
  • a 0.8 g lead weight is attached to the fiber bundle, and in this state, the fiber bundle is immersed in water at 40 ° C. for 10 minutes to undergo primary shrinkage, and the length of the fiber bundle is measured in water.
  • the primary-shrinked 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.
  • a fibroin fiber spun from naturally derived fibroin usually has a secondary shrinkage of 11 to 20%, but a fibroin fiber obtained by spinning the second fibroin according to the present invention alone (for example, Reference Example 11 described later). Is a fiber whose secondary shrinkage is reduced to 8% or less.
  • the mixed fibroin fiber which concerns on this embodiment shows the secondary shrinkage rate equivalent or more reduced than the fibroin fiber which spun only the 2nd fibroin.
  • the fibroin film according to the present invention can be obtained by using the mixed fibroin solution according to the present invention as a dope solution, casting the dope solution on a substrate surface, drying and / or removing the solvent.
  • the solvent for the dope solution examples include the same solvents as those exemplified in the description of the fibroin solution.
  • the solvent is preferably a polar solvent such as formic acid, hexafluoro-2-propanol (HFIP), or dimethyl sulfoxide.
  • An inorganic salt may be added to the dope solution as necessary.
  • an inorganic salt the same kind as the inorganic salt exemplified in the description of the fibroin solution can be exemplified.
  • the viscosity of the dope solution when forming the fibroin film is preferably 15 to 80 cP (centipoise), more preferably 20 to 70 cP.
  • the concentration of the mixed fibroin according to the present invention is preferably 3 to 50% by mass, more preferably 3.5 to 35% by mass, and 4.2. More preferably, it is ⁇ 15.8% by mass.
  • the base material may be a resin substrate, a glass substrate, a metal substrate, or the like.
  • the base material is preferably a resin substrate from the viewpoint that the film after cast molding can be easily peeled off.
  • the resin substrate may be, for example, a polyethylene terephthalate (PET) film, a fluororesin film such as polytetrafluoroethylene, a polypropylene (PP) film, or a release film in which a silicone compound is immobilized on the surface of these films.
  • PET polyethylene terephthalate
  • PP polypropylene
  • the base material is stable to HFIP, DMSO solvent, etc., the dope solution can be stably cast-molded, and the silicone compound is immobilized on the PET film or PET film surface from the viewpoint that the film after molding can be easily peeled off. It is more preferable that it is a peeled film.
  • a dope solution is cast on the surface of a substrate, and a predetermined thickness (for example, drying and / or removing) using a film thickness control means such as an applicator, a knife coater, or a bar coater.
  • a film thickness control means such as an applicator, a knife coater, or a bar coater.
  • a wet film having a thickness of 1 to 1000 ⁇ m after the solvent is prepared.
  • Drying and / or desolvation can be performed dry or wet.
  • Examples of the dry method include vacuum drying, hot air drying, and air drying.
  • Examples of the wet method include a method in which a cast film is immersed in a solvent removal liquid (also referred to as a coagulation liquid) to remove the solvent.
  • a solvent removal liquid also referred to as a coagulation liquid
  • the solvent removal liquid include water, alcohol liquids such as lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and mixed liquids of water and alcohol.
  • the temperature of the solvent removal liquid (coagulation liquid) is preferably 0 to 90 ° C.
  • the unstretched film after drying and / or desolvation can be uniaxially stretched or biaxially stretched in water.
  • Biaxial stretching may be sequential stretching or simultaneous biaxial stretching.
  • Multi-stage stretching of two or more stages may be performed.
  • the stretching ratio is preferably 1.01 to 6 times, more preferably 1.05 to 4 times in both length and width. Within this range, it is easy to balance stress-strain.
  • the stretching in water is preferably performed at a water temperature of 20 to 90 ° C.
  • the stretched film is preferably heat-set for 5 to 600 seconds with a dry heat of 50 to 200 ° C. This heat setting provides dimensional stability at room temperature.
  • a uniaxially stretched film becomes a uniaxially oriented film
  • a biaxially stretched film becomes a biaxially oriented film.
  • the water resistance of the film can be evaluated by measuring the degree of moisture absorption under high humidity using a saturated salt method using a saturated aqueous solution of salts.
  • salts include potassium sulfate, potassium chloride, sodium chloride, sodium bromide, potassium carbonate, magnesium chloride and the like.
  • the water resistance of the film is set, for example, in a sealed container such as a falcon tube containing a saturated aqueous solution of potassium sulfate so that the film cut to an appropriate size is not immersed in the aqueous solution. It can be evaluated by allowing the film to stand for 20 to 48 hours in air at high humidity and in an equilibrium state, measuring the weight and moisture content of the film, and determining the moisture content from the moisture content per weight. .
  • Fibroin having the amino acid sequence shown by SEQ ID NO: 1 to 10 was designed as the first fibroin, and fibroin having the amino acid sequence shown by SEQ ID NO: 11 to 24 was designed as the second fibroin.
  • These fibroins are designed based on the base sequence and amino acid sequence of Nephila clavies (GenBank accession number: P46804.1, GI: 1174415), which is a fibroin derived from nature.
  • amino acid sequences shown in SEQ ID NOs: 6 to 10 are obtained by adding the amino acid sequence shown in SEQ ID NO: 25 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NOs: 1 to 5, respectively.
  • the amino acid sequences represented by SEQ ID NOs: 18 to 24 are obtained by adding the amino acid sequence represented by SEQ ID NO: 25 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequences represented by SEQ ID NOs: 11 to 17, respectively.
  • the fibroin having the amino acid sequence represented by SEQ ID NOs: 1 to 10 corresponds to the first fibroin according to the present invention (see Table 1).
  • Fibroin having the amino acid sequence shown by SEQ ID NO: 11 to 24 corresponds to the second fibroin according to the present invention (see Table 3).
  • the amino acid sequence represented by SEQ ID NO: 1 is such that the number of consecutive alanine residues is five in the amino acid sequence in which the alanine residues in the (A) n motif of the naturally derived fibroin are continuous.
  • (A) n motif ((A) 5 ) is deleted from the deleted amino acid sequence every two from the N-terminal side to the C-terminal side, and [(A) n is inserted before the C-terminal sequence.
  • a motif-REP] is inserted.
  • the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) is obtained by replacing all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 1 (Met-PRT399) with GQX.
  • the amino acid sequence represented by SEQ ID NO: 3 (Met-PRT587) is obtained by performing substitution, insertion and deletion of amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of the natural fibroin. .
  • the amino acid sequence represented by SEQ ID NO: 4 (Met-PRT799) is obtained by performing substitution, insertion and deletion of amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of the natural fibroin. .
  • the amino acid sequence represented by SEQ ID NO: 5 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: 2 (Met-PRT410), and Some glutamine (Q) residues are substituted with serine (S) residues, and some amino acids on the N-terminal side are deleted so as to be approximately the same as the molecular weight of SEQ ID NO: 2.
  • the amino acid sequence represented by SEQ ID NO: 11 is obtained by replacing all QQs in REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with VL.
  • the amino acid sequence represented by SEQ ID NO: 12 is obtained by substituting all QQs in REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with TS and substituting the remaining Q with A. .
  • the amino acid sequence represented by SEQ ID NO: 13 is obtained by replacing all QQs in the REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with VL and replacing the remaining Q with I. .
  • the amino acid sequence represented by SEQ ID NO: 14 is obtained by substituting all QQs in REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with VI and replacing the remaining Q with L. .
  • the amino acid sequence represented by SEQ ID NO: 15 is obtained by replacing all QQs in the REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with VF and replacing the remaining Q with I. .
  • the amino acid sequence represented by SEQ ID NO: 16 has two alanine residues on the C-terminal side of each (A) n motif (A 5 ) with respect to the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410).
  • the two C-terminal domain sequences were deleted so that the molecular weight of the amino acid sequence shown in SEQ ID NO: 2 (Met-PRT410) was approximately the same, and 13 glutamine residues (Q) remained as serine residues.
  • the amino acid sequence represented by SEQ ID NO: 17 (M_PRT698) is obtained by replacing all QQs in the REP of the amino acid sequence represented by SEQ ID NO: 26 (M_PRT525) with VL and replacing the remaining Q with I.
  • the nucleic acids encoding the proteins having the amino acid sequences shown by SEQ ID NOs: 6 to 10 and SEQ ID NOs: 18 to 24 were synthesized.
  • the nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon.
  • These five types of nucleic acids were cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequence shown in SEQ ID NO: 6-10 and SEQ ID NO: 18-24.
  • the transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium (Table 5) containing ampicillin so that the OD 600 was 0.005.
  • the culture temperature was kept at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours) to obtain a seed culture solution.
  • the seed culture was added to a jar fermenter to which 500 mL of production medium (Table 6) was added so that the OD 600 was 0.05, and transformed E. coli was inoculated.
  • 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 target protein.
  • the culture solution was centrifuged, and the cells were collected. SDS-PAGE was performed using cells prepared from the culture solution before and after the addition of IPTG, and the expression of the target protein was confirmed by the appearance of a band of the desired protein size depending on the addition of IPTG.
  • 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 (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, the water was removed with a freeze dryer, and the lyophilized powder was recovered.
  • 8M guanidine buffer 8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • the degree of purification of the target protein in the obtained lyophilized powder was confirmed by image analysis of the result of polyacrylamide gel electrophoresis of the powder using Totallab (nonlinear dynamics ltd.).
  • Fibroin fibers were produced using the first fibroin prepared above or the second fibroin alone, and the contractility was evaluated.
  • the spinning solution was filled in a reserve tank and discharged from a monohole nozzle having a diameter of 0.1 or 0.2 mm into a 100 mass% methanol coagulation bath using a gear pump. The discharge rate was adjusted to 0.01 to 0.08 mL / min. After solidification, washing and stretching were performed in a 100 mass% methanol washing bath. After washing and drawing, it was dried using a dry heat plate, and the obtained raw yarn (fibroin fiber) was wound up.
  • the fiber spun with PRT410 (Reference Example 1) protein showed a secondary contraction rate of 12%, but it contained a glutamine residue by replacing the glutamine residue (Q) in the domain with another amino acid residue.
  • PRT888 SEQ ID NO: 18
  • a reduced rate 6.3%)
  • This effect of reducing the secondary contraction rate was recognized without further increasing the hydrophobicity of REP as in PRT888 by further reducing (0%) the glutamine residue content (Reference Example 3).
  • the content of glutamine residues was further reduced (0%), and the effect of reducing the secondary contraction rate was more remarkably observed by substituting amino acid residues with higher hydrophobicity (Reference Example 4). To 6).
  • a fibroin film was produced using the first fibroin prepared above or the second fibroin alone, and the water resistance was evaluated.
  • the molded wet film was allowed to stand for 12 hours in a constant temperature bath (manufactured by espec) at 55 ° C. and dried. Thereafter, the dried film was peeled off from the substrate and immersed in methanol for 12 hours. After immersion, it was allowed to stand again in a constant temperature bath (manufactured by espec) for 60 hours and dried. The obtained film was cut into 30 mm squares and subjected to the following water resistance evaluation.
  • Fibroin fibers were prepared by mixing the first fibroin prepared above and the second fibroin alone or mixed, and the physical properties were evaluated.
  • Table 9 shows the secondary shrinkage rates of the fibroin fibers of the examples and reference examples.
  • the secondary shrinkage shown in Table 9 is a relative value when the secondary shrinkage of the fibroin fiber of Reference Example 10 (PRT799 alone) is 100.
  • (6-4) Evaluation by physical property measurement of fibroin fiber The physical property of the raw yarn obtained in (6-2) was measured by the following method.
  • B) The stress, initial elastic modulus, and elongation (displacement at break) of the fibroin fiber were measured using a tensile tester (INSTRON 3342) under the conditions of a temperature of 20 ° C. and a relative humidity of 65%. In the tensile test, measurements were made at 10 msec intervals. Each sample was attached to a mold made of cardboard, the distance between the gripping jigs was 20 mm, and the pulling speed was 10 mm / min. The load cell capacity was 10N, and the gripping jig was a clip type. The measured value was an average value of the number of samples n 5.
  • the results of measuring the stress and elongation of each raw yarn are shown in Table 9.
  • the stress and elongation shown in Table 9 are relative values when the value of stress and elongation of the fibroin fiber of Reference Example 10 (PRT799 alone) is 100.
  • the mixed fibroin fiber (Example 1) in which PRT918 was mixed with PRT799 at a ratio equal to or less than that of PRT799 has an effect that the stress is further improved as compared with the fibroin fiber of PRT799 alone (Reference Example 10).
  • the result was an unexpected and excellent result.
  • the fibroin fiber of PRT918 alone (Reference Example 11) was seen as a reference, it was found that the mixed fibroin fiber mixed with PRT799 can improve the stress as the proportion of PRT799 increases (implementation). Examples 1 to 3).
  • the mixed fibroin fiber (Example 1) obtained by mixing PRT918 at a ratio equal to or less than that of PRT799 has an effect that the elongation is further improved as compared with the fibroin fiber of PRT799 alone (Reference Example 10).
  • the fibrin fiber of PRT918 alone (Reference Example 11) was seen as a reference, it was confirmed that the mixed fibroin fiber mixed with PRT799 can improve the elongation as the proportion of PRT799 increases ( Examples 1 to 3).
  • Table 10 shows the measurement results of the secondary shrinkage rate, stress and elongation.
  • the secondary shrinkage rate, stress, and elongation shown in Table 10 are relative values when the value of the fibroin fiber (Reference Example 12) of PRT918 alone is 100.

Abstract

The present invention relates to a fibroin composition that contains a first fibroin, and at least one fibroin selected from the group consisting of a second fibroin and a third fibroin, wherein the first fibroin has a domain sequence in which the (A)n motif content is lowered, the second fibroin has a domain sequence in which the glutamine residue content is lowered, and the third fibroin has a domain sequence that contains a localized region having a high hydrophobicity index.

Description

フィブロイン組成物、フィブロイン溶液、及びフィブロイン繊維の製造方法Fibroin composition, fibroin solution, and method for producing fibroin fiber
　本発明は、フィブロイン組成物、フィブロイン溶液、及びフィブロイン繊維の製造方法に関する。より具体的には、本発明は、耐水性等の物性に優れた製品（フィブロイン繊維等）を製造可能な混合フィブロイン組成物に関する。 The present invention relates to a fibroin composition, a fibroin solution, and a method for producing a fibroin fiber. More specifically, the present invention relates to a mixed fibroin composition capable of producing a product (fibroin fiber or the like) having excellent physical properties such as water resistance.
　フィブロインは、繊維状のタンパク質の一種であり、βプリーツシートの形成につながるグリシン残基、アラニン残基及びセリン残基を最大９０％含有する（非特許文献１）。フィブロインとして、昆虫及びクモ類が産生する糸を構成するタンパク質（絹タンパク質、ホーネットシルクタンパク質、スパイダーシルクタンパク質）等が知られている。 Fibroin is a kind of fibrous protein and contains up to 90% glycine, alanine and serine residues that lead to the formation of β-pleated sheets (Non-patent Document 1). As fibroin, proteins (silk protein, hornet silk protein, spider silk protein) and the like that constitute a thread produced by insects and spiders are known.
　組換えスパイダーシルクタンパク質、及び組換え絹タンパク質は、例えば、トランスジェニック・ヤギ、トランスジェニック・カイコ、組換え植物又は組換え哺乳類細胞等のタンパク質生産系での産生が報告されている。（非特許文献２）。更に、大量生産が可能なタンパク質生産系として、酵母、カビ、グラム陰性細菌及びグラム陽性細菌等の生物を宿主とした組換えフィブロイン生産も多数報告されている（特許文献１）。 Recombinant spider silk protein and recombinant silk protein have been reported to be produced in protein production systems such as, for example, transgenic goats, transgenic silkworms, recombinant plants or recombinant mammalian cells. (Non-patent document 2). Furthermore, as a protein production system capable of mass production, many recombinant fibroin productions using organisms such as yeast, mold, gram-negative bacteria, and gram-positive bacteria as a host have been reported (Patent Document 1).
　より改良されたフィブロインを得るために、様々なアミノ酸配列の改変が行われており、例えば、タフネス及び伸度を向上させ、工業生産に適した組換えスパイダーシルクタンパク質が報告されている（特許文献２）。 In order to obtain a more improved fibroin, various amino acid sequence modifications have been carried out. For example, recombinant spider silk proteins that improve toughness and elongation and are suitable for industrial production have been reported (Patent Literature). 2).
　また、スパイダーシルクタンパク質とウール等といった異なる種類のフィブロイン繊維を混合して紡糸することで物性の改変されたフィブロイン繊維が報告されている（特許文献３）。 Also, fibroin fibers whose properties have been modified by mixing and spinning different kinds of fibroin fibers such as spider silk protein and wool have been reported (Patent Document 3).
　しかしながら、例えば、フィブロインを紡糸して得られるフィブロイン繊維は、水又は湯への浸漬、高湿度環境への暴露等により収縮する特性を有する。この特性は、製造工程及び製品化において様々な問題を発生させ、フィブロイン繊維よりなる製品にも影響が及ぶ。 However, for example, fibroin fiber obtained by spinning fibroin has a property of shrinking by immersion in water or hot water, exposure to a high humidity environment, and the like. This characteristic causes various problems in the manufacturing process and commercialization, and affects products made of fibroin fibers.
　当該製品の収縮を防止するための防縮方法として、例えば、精練を完了した強撚糸使用の絹織物を、緊張した状態で水、その他の溶媒、又はその混合系に浸漬して所定時間加温することを特徴とする絹織物の防縮加工法（特許文献４）、所要形状に成形された状態にある動物繊維製品に、１２０～２００℃の高圧飽和水蒸気を接触させる処理を施して、当該繊維製品に水蒸気処理時の形状を固定することを特徴とする動物繊維製品の形状固定化方法（特許文献５）等が報告されている。 As an anti-shrinking method for preventing the shrinkage of the product, for example, a silk fabric using a strong twisted yarn that has been scoured is immersed in water, other solvent, or a mixed system thereof in a tensioned state and heated for a predetermined time. A method for preventing shrinkage of a silk fabric characterized by the above (Patent Document 4), subjecting an animal fiber product that has been formed into a required shape to contact with high-pressure saturated steam at 120 to 200 ° C. A method for fixing the shape of animal fiber products (patent document 5), which is characterized by fixing the shape during steam treatment, has been reported.
国際公開第２０１５／０４２１６４号International Publication No. 2015/042164 国際公開第２０１７／１８８４３４号International Publication No. 2017/188434 国際公開第２０１６／２０１３６９号International Publication No. 2016/201369 特公平２－６８６９号公報Japanese Patent Publication No.2-6869 特開平６－２９４０６８号公報JP-A-6-294068
　特許文献４及び５に開示されるような防縮方法は、操作が煩雑であり、また工程が増えるため、工業的に不利である。このような防縮方法によらず、フィブロイン繊維自体の収縮を抑制又は低減させることができれば、極めて工業的に有用である。 The shrinkage-preventing methods as disclosed in Patent Documents 4 and 5 are industrially disadvantageous because the operation is complicated and the number of steps increases. It is extremely industrially useful if the shrinkage of the fibroin fiber itself can be suppressed or reduced regardless of such a shrinkage prevention method.
　本発明は、耐水性に優れた製品（フィブロイン繊維等）を製造可能なフィブロイン組成物の提供を目的とする。本発明はまた、収縮が抑制又は低減されたフィブロイン繊維、及びその製造方法の提供も目的とする。 The object of the present invention is to provide a fibroin composition capable of producing a product excellent in water resistance (such as fibroin fiber). Another object of the present invention is to provide a fibroin fiber with reduced or reduced shrinkage and a method for producing the same.
　本発明者らは、特許文献２（国際公開第２０１７／１８８４３４号）で報告した（Ａ）ｎモチーフの含有量が低減されたフィブロインと、グルタミン残基含有率が低減されたアミノ酸配列を有するフィブロイン等の疎水性を高めたフィブロインとを混合した混合フィブロインから形成したフィブロイン繊維は、収縮が抑制又は低減されると共に、応力及び伸度にも優れることを見い出した。本発明はこの新規な知見に基づく。 The present inventors reported (A) fibroin in which the content of n motif was reduced and fibroin having an amino acid sequence in which the content of glutamine residues was reduced as reported in Patent Document 2 (International Publication No. 2017/188434). It has been found that fibroin fibers formed from mixed fibroin mixed with fibroin with increased hydrophobicity such as the above are suppressed or reduced in shrinkage and excellent in stress and elongation. The present invention is based on this novel finding.
　すなわち、本発明は、例えば、以下の各発明に関する。
［１］
　第１のフィブロインと、第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインと、を含むフィブロイン組成物であって、
　上記第１のフィブロインが、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ１で表されるドメイン配列を含み、Ｎ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる上記隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、上記ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが５０％以上であるアミノ酸配列を有するフィブロインであり、
　上記第２のフィブロインが、式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ２で表されるドメイン配列又は式３：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ３－（Ａ）ｎモチーフで表されるドメイン配列を含み、グルタミン残基含有率が９％以下であるアミノ酸配列を有するフィブロインであり、
　上記第３のフィブロインが、式４：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ４で表されるドメイン配列を含み、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｚとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｗとしたときに、ｚ／ｗが６．２％以上であるアミノ酸配列を有するフィブロインである、フィブロイン組成物。
［式１、式２、式３及び式４中、（Ａ）ｎモチーフは、それぞれ独立に、４～２７アミノ酸残基から構成されるアミノ酸配列を示し、かつ（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が８０％以上であり、ＲＥＰは、それぞれ独立に、１０～２００アミノ酸残基から構成されるアミノ酸配列を示し、ｍ１、ｍ２、ｍ３及びｍ４は、それぞれ独立に、８～３００の整数を示す。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。］
［２］
　上記第２のフィブロインのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１若しくは複数のグルタミン残基を欠失したこと、又は他のアミノ酸残基に置換したことに相当する、グルタミン残基の含有量が低減されたアミノ酸配列である、［１］に記載のフィブロイン組成物。
［３］
　上記第２のフィブロインにおける上記他のアミノ酸残基が、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）アラニン（Ａ）、グリシン（Ｇ）、スレオニン（Ｔ）、セリン（Ｓ）、トリプトファン（Ｗ）、チロシン（Ｙ）、プロリン（Ｐ）及びヒスチジン（Ｈ）からなる群より選択されるアミノ酸残基である、［１］又は［２］に記載のフィブロイン組成物。
［４］
　上記第２のフィブロインは、ＲＥＰ中にＧＰＧＸＸ（但し、Ｘはグリシン残基以外のアミノ酸残基を示す。）モチーフを含み、ＧＰＧＸＸモチーフ含有率が１０％以上である、［１］～［３］のいずれかに記載のフィブロイン組成物。
［５］
　上記第２のフィブロインは、ＲＥＰの疎水性度が－０．８以上である、［１］～［４］のいずれかに記載のフィブロイン組成物。
［６］
　上記第１のフィブロインは、配列番号１～１０のいずれかで示されるアミノ酸配列、又は配列番号１～１０のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、［１］～［５］のいずれかに記載のフィブロイン組成物。
［７］
　上記第２のフィブロインは、配列番号１１～２４のいずれかで示されるアミノ酸配列、又は配列番号１１～２４のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、［１］～［６］のいずれかに記載のフィブロイン組成物。
［８］
　［１］～［７］のいずれかに記載のフィブロイン組成物を含み、
　繊維、糸、フィルム、発泡体、球体、ナノフィブリル、ゲル及び樹脂からなる群から選択される製品。
［９］
　［１］～［７］のいずれかに記載のフィブロイン組成物が、溶媒に溶解してなる、フィブロイン溶液。
［１０］
　上記溶媒が、ヘキサフルオロイソプロパノール（ＨＦＩＰ）、ヘキサフルオロアセトン（ＨＦＡ）、ジメチルスルホキシド（ＤＭＳＯ）、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）、ギ酸、尿素、グアニジン、ドデシル硫酸ナトリウム（ＳＤＳ）、臭化リチウム、塩化カルシウム及びチオシアン酸リチウム、並びにこれら２種以上の混合溶媒からなる群より選ばれる溶媒である、［９］に記載のフィブロイン溶液。
［１１］
　上記第１のフィブロインと、上記第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインとの含有比率が、重量基準で９．９：０．１～５．０：５．０である、［９］又は［１０］に記載のフィブロイン溶液。
［１２］
　上記第１のフィブロインと、上記第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインとの含有比率が、重量基準で５．０：５．０～０．１：９．９である、［９］又は［１０］に記載のフィブロイン溶液。
［１３］
　上記第１のフィブロインと、上記第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインとの含有比率が、重量基準で５．０：５．０である、［９］又は［１０］に記載のフィブロイン溶液。
［１４］
　［９］～［１３］のいずれかに記載のフィブロイン溶液を使用したフィブロイン繊維の製造方法であって、
　上記フィブロイン溶液をドープ液とし、
　上記ドープ液を口金から凝固液に押し出し、未延伸糸を得る工程を含む、フィブロイン繊維の製造方法。
［１５］
　上記未延伸糸を延伸する工程を更に含む、［１４］に記載のフィブロイン繊維の製造方法。
［１６］
　上記凝固液が、メタノール、エタノール及び２－プロパノールからなる群より選ばれる少なくとも１種である、［１４］又は［１５］に記載のフィブロイン繊維の製造方法。 That is, the present invention relates to the following inventions, for example.
[1]
A fibroin composition comprising: a first fibroin; and at least one fibroin selected from the group consisting of a second fibroin and a third fibroin,
The first fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m1 , and two adjacent [(A) n motifs from the N-terminal side toward the C-terminal side -REP] When the number of amino acid residues in the REP of the unit is sequentially compared, and the number of amino acid residues in the REP with a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to The maximum sum of the total number of amino acid residues of the two adjacent [(A) n motif-REP] units, which is 11.3, is x, and the total number of amino acid residues of the domain sequence is y. A fibroin having an amino acid sequence in which x / y is 50% or more,
The second fibroin is represented by a domain sequence represented by Formula 2: [(A) n motif-REP] m2 or represented by Formula 3: [(A) n motif-REP] m3- (A) n motif. A fibroin having an amino acid sequence comprising a domain sequence and having a glutamine residue content of 9% or less;
The third fibroin contains a domain sequence represented by Formula 4: [(A) n motif-REP] m4 and is located on the most C-terminal side (A) Sequence from the n motif to the C terminus of the domain sequence Z is the total number of amino acid residues contained in a region in which the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs included in the sequence excluding from the domain sequence. (A) z / w is 6.2% or more when the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence is w A fibroin composition, which is a fibroin having an amino acid sequence of
[In Formula 1, Formula 2, Formula 3 and Formula 4, (A) n motif independently represents an amino acid sequence composed of 4 to 27 amino acid residues, and (A) all amino acids in n motif The number of alanine residues with respect to the number of residues is 80% or more, REP independently represents an amino acid sequence composed of 10 to 200 amino acid residues, m1, m2, m3 and m4 are each independently An integer from 8 to 300 is indicated. 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. ]
[2]
Glutamine, wherein the domain sequence of the second fibroin corresponds to deletion of one or more glutamine residues in REP or substitution with other amino acid residues as compared to naturally occurring fibroin The fibroin composition according to [1], which is an amino acid sequence having a reduced residue content.
[3]
The other amino acid residues in the second fibroin are isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) alanine (A), glycine ( [1] or [1] which is an amino acid residue selected from the group consisting of G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). 2].
[4]
The second fibroin contains a GPGXX (where X represents an amino acid residue other than a glycine residue) motif in the REP, and the content of the GPGXX motif is 10% or more. [1] to [3] The fibroin composition according to any one of the above.
[5]
The fibroin composition according to any one of [1] to [4], wherein the second fibroin has a hydrophobicity of REP of −0.8 or more.
[6]
The first fibroin comprises an amino acid sequence represented by any one of SEQ ID NOs: 1 to 10, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 10, [1] The fibroin composition according to any one of [5].
[7]
The second fibroin includes an amino acid sequence represented by any of SEQ ID NOs: 11 to 24, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 11 to 24. [1] The fibroin composition according to any one of [6].
[8]
Including the fibroin composition according to any one of [1] to [7],
A product selected from the group consisting of fibers, yarns, films, foams, spheres, nanofibrils, gels and resins.
[9]
A fibroin solution obtained by dissolving the fibroin composition according to any one of [1] to [7] in a solvent.
[10]
The above solvents are hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide The fibroin solution according to [9], which is a solvent selected from the group consisting of calcium chloride and lithium thiocyanate, and a mixed solvent of two or more of these.
[11]
The content ratio of the first fibroin to at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 9.9: 0.1 to 5.0: on a weight basis. The fibroin solution according to [9] or [10], which is 5.0.
[12]
The content ratio of the first fibroin to at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 5.0: 5.0 to 0.1: The fibroin solution according to [9] or [10], which is 9.9.
[13]
The content ratio of the first fibroin and at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 5.0: 5.0 on a weight basis, [9 ] Or the fibroin solution according to [10].
[14]
A method for producing fibroin fiber using the fibroin solution according to any one of [9] to [13],
The fibroin solution as a dope solution,
A method for producing fibroin fiber, comprising a step of extruding the dope solution from a die into a coagulation solution to obtain an undrawn yarn.
[15]
The method for producing fibroin fiber according to [14], further comprising a step of drawing the undrawn yarn.
[16]
The method for producing a fibroin fiber according to [14] or [15], wherein the coagulating liquid is at least one selected from the group consisting of methanol, ethanol and 2-propanol.
　本発明によれば、耐水性に優れた製品（フィブロイン繊維等）を製造可能なフィブロイン組成物の提供が可能となる。 According to the present invention, it is possible to provide a fibroin composition capable of producing a product excellent in water resistance (fibroin fiber or the like).
一実施形態に係るフィブロインのドメイン配列を示す模式図である。It is a schematic diagram which shows the domain arrangement | sequence of the fibroin which concerns on one Embodiment. 一実施形態に係るフィブロインのドメイン配列を示す模式図である。It is a schematic diagram which shows the domain arrangement | sequence of the fibroin which concerns on one Embodiment. 水等との接触によるフィブロイン繊維の長さ変化の例を示す図である。It is a figure which shows the example of the length change of the fibroin fiber by contact with water etc.
　以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
〔フィブロイン組成物〕
　本発明に係るフィブロイン組成物は、少なくとも２種のフィブロインを含む。すなわち、本発明に係るフィブロイン組成物は、第１のフィブロインを含み、更に第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインを含む。ここで、第１のフィブロインは、（Ａ）ｎモチーフの含有量が低減されたドメイン配列を有するフィブロインである。第２及び第３のフィブロインは、疎水性を高めたフィブロインであり、具体的には、第２のフィブロインは、グルタミン残基の含有量が低減されたドメイン配列を有するフィブロインであり、第３のフィブロインは、局所的に疎水性指標の大きい領域を含むドメイン配列を有するフィブロインである。 [Fibroin composition]
The fibroin composition according to the present invention comprises at least two types of fibroin. That is, the fibroin composition according to the present invention includes a first fibroin and further includes at least one fibroin selected from the group consisting of a second fibroin and a third fibroin. Here, the first fibroin is (A) a fibroin having a domain sequence in which the content of the n motif is reduced. The second and third fibroins are fibroin with increased hydrophobicity. Specifically, the second fibroin is a fibroin having a domain sequence with a reduced content of glutamine residues, Fibroin is a fibroin having a domain sequence including a region having a large hydrophobic index locally.
　本発明に係るフィブロインは、式Ａ：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ又は式Ｂ：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。本発明に係るフィブロインは、ドメイン配列のＮ末端側及びＣ末端側のいずれか一方又は両方に更にアミノ酸配列（Ｎ末端配列及びＣ末端配列）が付加されていてもよい。Ｎ末端配列及びＣ末端配列は、これに限定されるものではないが、典型的には、フィブロインに特徴的なアミノ酸モチーフの反復を有さない領域であり、１００残基程度のアミノ酸からなる。 The fibroin according to the present invention is a protein containing a domain sequence represented by formula A: [(A) n motif-REP] m or formula B: [(A) n motif-REP] m- (A) n motif. is there. In the fibroin according to the present invention, 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.
　本明細書において「ドメイン配列」とは、フィブロイン特有の結晶領域（典型的には、アミノ酸配列の（Ａ）ｎモチーフに相当する。）と非晶領域（典型的には、アミノ酸配列のＲＥＰに相当する。）を生じるアミノ酸配列である。（Ａ）ｎモチーフは４～２０アミノ酸残基から構成されるアミノ酸配列を示し、かつ（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が８０％以上である。ＲＥＰは１０～２００アミノ酸残基から構成されるアミノ酸配列を示す。ｍは８～３００の整数を示す。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。 In the present specification, the “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). Corresponding amino acid sequence). (A) The n motif represents an amino acid sequence composed of 4 to 20 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in the n motif is 80% or more. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 8 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.
　（Ａ）ｎモチーフは、（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が８０％以上であればよいが、８３％以上であることが好ましく、８６％以上であることがより好ましく、９０％以上であることが更に好ましく、９５％以上であることが更により好ましく、１００％であること（アラニン残基のみで構成されることを意味する）が特に好ましい。ドメイン配列中に複数存在する（Ａ）ｎモチーフは、少なくとも７つがアラニン残基のみで構成されることが好ましい。アラニン残基のみで構成されるとは、（Ａ）ｎモチーフが、（Ａ）ｎ（Ａはアラニン残基を示し、ｎは４～２０の整数、好ましくは４～１６の整数を示す。）で表されるアミノ酸配列を有することを意味する。 (A) The n motif may be (A) the number of alanine residues relative to the total number of amino acid residues in the n motif is 80% or more, preferably 83% or more, and 86% or more. More preferably, it is more preferably 90% or more, still more preferably 95% or more, and particularly preferably 100% (meaning that it is composed only of alanine residues). It is preferable that at least seven of the (A) n motifs present in the domain sequence are composed of only alanine residues. (A) n motif is (A) n (A represents an alanine residue, n represents an integer of 4 to 20, preferably an integer of 4 to 16). It has the amino acid sequence represented by these.
（第１のフィブロイン）
　本発明に係る第１のフィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ１で表されるドメイン配列を含み、Ｎ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる上記隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、上記ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが５０％以上であるアミノ酸配列を有するフィブロインである（但し、第２のフィブロイン又は第３のフィブロインに該当するフィブロインを除く。）。 (First fibroin)
The first fibroin according to the present invention includes a domain sequence represented by Formula 1: [(A) n motif-REP] m1 , and two adjacent [[A] from the N-terminal side toward the C-terminal side. ) When the number of amino acid residues in the REP of the n motif-REP] unit is sequentially compared, and the number of amino acid residues in the REP with a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1 The maximum sum of the total number of amino acid residues of the two adjacent [(A) n motif-REP] units, which is .8 to 11.3, is x, and the total number of amino acid residues of the domain sequence Is a fibroin having an amino acid sequence where x / y is 50% or more, except for fibroin corresponding to the second fibroin or the third fibroin.
　ここで、式１中、（Ａ）ｎモチーフは、４～２７アミノ酸残基から構成されるアミノ酸配列を示し、かつ（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が８０％以上であり、ＲＥＰは、１０～２００アミノ酸残基から構成されるアミノ酸配列を示し、ｍ１は、８～３００の整数を示す。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。 Here, in Formula 1, (A) n motif represents an amino acid sequence composed of 4 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 80%. In the above, REP represents an amino acid sequence composed of 10 to 200 amino acid residues, and m1 represents an integer of 8 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.
　第１のフィブロインは、（Ａ）ｎモチーフの含有量が低減されているため、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数の比（以下、「ギザ比率」ともいう。）が、上記ｘ／ｙで示す範囲に入る割合が高くなっている。第１のフィブロインにおけるｘ／ｙは、６０％以上であることが好ましく、６５％以上であることがより好ましく、７０％以上であることが更に好ましく、７５％以上であることが更により好ましく、８０％以上であることが更によりまた好ましく、８５％以上であることが特に好ましい。ｘ／ｙの上限に特に制限はなく、例えば、１００％以下であってよい。ｘ／ｙがこの範囲にあることにより、応力及び伸度に優れたフィブロイン繊維を得ることができるフィブロインとなる。 First fibroin, (A) since the content of n motif is reduced, two adjacent of [(A) n motif -rep] The ratio of the number of amino acid residues units REP (hereinafter, "Giza Ratio ")" Is higher in the range indicated by x / y. X / y in the first fibroin is preferably 60% or more, more preferably 65% or more, still more preferably 70% or more, and even more preferably 75% or more, It is still more preferable that it is 80% or more, and it is especially preferable that it is 85% or more. There is no restriction | limiting in particular in the upper limit of x / y, For example, you may be 100% or less. When x / y is in this range, it becomes a fibroin from which a fibroin fiber excellent in stress and elongation can be obtained.
　ｘ／ｙは、特許文献２（国際公開第２０１７／１８８４３４号）に記載した方法に従って算出すればよい。 X / y may be calculated according to the method described in Patent Document 2 (International Publication No. 2017/188434).
　第１のフィブロインは、特許文献２（国際公開第２０１７／１８８４３４号）に記載した改変フィブロインであってよい。すなわち、第１のフィブロインは、例えば、天然由来のフィブロインの遺伝子配列から、ｘ／ｙが５０％以上になるように（Ａ）ｎモチーフをコードする配列の１又は複数を欠失させることにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から、ｘ／ｙが５０％以上になるように１又は複数の（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列から（Ａ）ｎモチーフが欠失したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 The first fibroin may be a modified fibroin described in Patent Document 2 (International Publication No. 2017/188434). That is, the first fibroin is obtained, for example, by deleting one or more of the sequences encoding the (A) n motif from the gene sequence of naturally occurring fibroin so that x / y is 50% or more. be able to. In addition, for example, an amino acid sequence corresponding to the deletion of one or more (A) n motifs so that x / y is 50% or more from the amino acid sequence of naturally derived fibroin is designed and designed. It can also be obtained by chemically synthesizing a nucleic acid encoding the sequence. In any case, in addition to the modification corresponding to the deletion of the (A) n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. The amino acid sequence corresponding to this may be modified.
　アミノ酸残基の置換、欠失、挿入及び／又は付加は、部分特異的突然変異誘発法等の当業者に周知の方法により行うことができる。具体的には、Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓ．１０，６４８７（１９８２）、Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，１００，４４８（１９８３）等の文献に記載されている方法に準じて行うことができる。 Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.
　天然由来のフィブロインとして、具体的には、例えば、昆虫又はクモ類が産生するフィブロインが挙げられる。 Specific examples of naturally occurring fibroin include fibroin produced by insects or spiders.
　昆虫が産生するフィブロインとしては、例えば、ボンビックス・モリ（Ｂｏｍｂｙｘ　ｍｏｒｉ）、クワコ（Ｂｏｍｂｙｘ　ｍａｎｄａｒｉｎａ）、天蚕（Ａｎｔｈｅｒａｅａ　ｙａｍａｍａｉ）、柞蚕（Ａｎｔｅｒａｅａ　ｐｅｒｎｙｉ）、楓蚕（Ｅｒｉｏｇｙｎａ　ｐｙｒｅｔｏｒｕｍ）、蓖蚕（Ｐｉｌｏｓａｍｉａ　Ｃｙｎｔｈｉａ　ｒｉｃｉｎｉ）、樗蚕（Ｓａｍｉａ　ｃｙｎｔｈｉａ）、栗虫（Ｃａｌｉｇｕｒａ　ｊａｐｏｎｉｃａ）、チュッサー蚕（Ａｎｔｈｅｒａｅａ　ｍｙｌｉｔｔａ）、ムガ蚕（Ａｎｔｈｅｒａｅａ　ａｓｓａｍａ）等のカイコが産生する絹タンパク質、スズメバチ（Ｖｅｓｐａ　ｓｉｍｉｌｌｉｍａ　ｘａｎｔｈｏｐｔｅｒａ）の幼虫が吐出するホーネットシルクタンパク質が挙げられる。 Examples of fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandaraina), Tengea (Antheraea yamanai), 柞 蚕 (Antereaperanii), 楓 蚕 (Eriothyraminey) ), Silkworms (Samia cythia), chestnut worms (Caligula japonica), Chussa moth (Anthereaea mylitta), silkworms produced by silkworms such as Antheraea assamata, vespasam worms Examples include silk proteins.
　昆虫が産生するフィブロインのより具体的な例としては、例えば、カイコ・フィブロインＬ鎖（ＧｅｎＢａｎｋアクセッション番号Ｍ７６４３０（塩基配列）、ＡＡＡ２７８４０．１（アミノ酸配列））が挙げられる。 More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).
　クモ類が産生するフィブロインとしては、例えば、オニグモ、ニワオニグモ、アカオニグモ、アオオニグモ及びマメオニグモ等のオニグモ属（Ａｒａｎｅｕｓ属）に属するクモ、ヤマシロオニグモ、イエオニグモ、ドヨウオニグモ及びサツマノミダマシ等のヒメオニグモ属（Ｎｅｏｓｃｏｎａ属）に属するクモ、コオニグモモドキ等のコオニグモモドキ属（Ｐｒｏｎｕｓ属）に属するクモ、トリノフンダマシ及びオオトリノフンダマシ等のトリノフンダマシ属（Ｃｙｒｔａｒａｃｈｎｅ属）に属するクモ、トゲグモ及びチブサトゲグモ等のトゲグモ属（Ｇａｓｔｅｒａｃａｎｔｈａ属）に属するクモ、マメイタイセキグモ及びムツトゲイセキグモ等のイセキグモ属（Ｏｒｄｇａｒｉｕｓ属）に属するクモ、コガネグモ、コガタコガネグモ及びナガコガネグモ等のコガネグモ属（Ａｒｇｉｏｐｅ属）に属するクモ、キジロオヒキグモ等のオヒキグモ属（Ａｒａｃｈｎｕｒａ属）に属するクモ、ハツリグモ等のハツリグモ属（Ａｃｕｓｉｌａｓ属）に属するクモ、スズミグモ、キヌアミグモ及びハラビロスズミグモ等のスズミグモ属（Ｃｙｔｏｐｈｏｒａ属）に属するクモ、ゲホウグモ等のゲホウグモ属（Ｐｏｌｔｙｓ属）に属するクモ、ゴミグモ、ヨツデゴミグモ、マルゴミグモ及びカラスゴミグモ等のゴミグモ属（Ｃｙｃｌｏｓａ属）に属するクモ、及びヤマトカナエグモ等のカナエグモ属（Ｃｈｏｒｉｚｏｐｅｓ属）に属するクモが産生するスパイダーシルクタンパク質、並びにアシナガグモ、ヤサガタアシナガグモ、ハラビロアシダカグモ及びウロコアシナガグモ等のアシナガグモ属（Ｔｅｔｒａｇｎａｔｈａ属）に属するクモ、オオシロカネグモ、チュウガタシロカネグモ及びコシロカネグモ等のシロカネグモ属（Ｌｅｕｃａｕｇｅ属）に属するクモ、ジョロウグモ及びオオジョロウグモ等のジョロウグモ属（Ｎｅｐｈｉｌａ属）に属するクモ、キンヨウグモ等のアズミグモ属（Ｍｅｎｏｓｉｒａ属）に属するクモ、ヒメアシナガグモ等のヒメアシナガグモ属（Ｄｙｓｃｈｉｒｉｏｇｎａｔｈａ属）に属するクモ、クロゴケグモ、セアカゴケグモ、ハイイロゴケグモ及びジュウサンボシゴケグモ等のゴケグモ属（Ｌａｔｒｏｄｅｃｔｕｓ属）に属するクモ、及びユープロステノプス属（Ｅｕｐｒｏｓｔｈｅｎｏｐｓ属）に属するクモ等のアシナガグモ科（Ｔｅｔｒａｇｎａｔｈｉｄａｅ科）に属するクモが産生するスパイダーシルクタンパク質が挙げられる。スパイダーシルクタンパク質としては、例えば、ＭａＳｐ（ＭａＳｐ１及びＭａＳｐ２）、ＡＤＦ（ＡＤＦ３及びＡＤＦ４）等の牽引糸タンパク質、ＭｉＳｐ（ＭｉＳｐ１及びＭｉＳｐ２）等が挙げられる。 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 genus Acusilas such as the common spider, the spider belonging to the genus Acusilas, and the genus Spider Spiders belonging to (genus Cytophora), spiders belonging to the genus Spider belonging to the genus Spider (genus Poltys) such as spiders, genus Spider, spiders belonging to the genus Spider belonging to the genus Cyclosa (genus Cyclosa), and spiders belonging to the genus Cyclosa (genus Cyclosa) Spider silk proteins produced by spiders belonging to the genus Chorizopes), and Asina, such as Asagaaga spider, Yasagata Asaga spider, Harabiro Ashida spider and Urokoa Asaga spider Spiders belonging to the genus Tetragnata, spiders belonging to the genus Spider genus (Leucage sp.) Such as the white spider spider and the white spider spider, the spider genus belonging to the spider spider genus (Nephila spp. 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 (L) and the genus Spider belonging to the genus Usd Produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops Examples include spider silk protein. Examples of 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.
　クモ類が産生するフィブロインのより具体的な例としては、例えば、ｆｉｂｒｏｉｎ－３（ａｄｆ－３）［Ａｒａｎｅｕｓ　ｄｉａｄｅｍａｔｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ４７０１０（アミノ酸配列）、Ｕ４７８５５（塩基配列））、ｆｉｂｒｏｉｎ－４（ａｄｆ－４）［Ａｒａｎｅｕｓ　ｄｉａｄｅｍａｔｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ４７０１１（アミノ酸配列）、Ｕ４７８５６（塩基配列））、ｄｒａｇｌｉｎｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ｓｐｉｄｒｏｉｎ　１［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ０４５０４（アミノ酸配列）、Ｕ３７５２０（塩基配列））、ｍａｊｏｒ　ａｎｇｕ１１ａｔｅ　ｓｐｉｄｒｏｉｎ　１［Ｌａｔｒｏｄｅｃｔｕｓ　ｈｅｓｐｅｒｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＢＲ６８８５６（アミノ酸配列）、ＥＦ５９５２４６（塩基配列））、ｄｒａｇｌｉｎｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ｓｐｉｄｒｏｉｎ　２［Ｎｅｐｈｉｌａ　ｃｌａｖａｔａ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＬ３２４７２（アミノ酸配列）、ＡＦ４４１２４５（塩基配列））、ｍａｊｏｒ　ａｎｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　１［Ｅｕｐｒｏｓｔｈｅｎｏｐｓ　ａｕｓｔｒａｌｉｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＣＡＪ００４２８（アミノ酸配列）、ＡＪ９７３１５５（塩基配列））、及びｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　２［Ｅｕｐｒｏｓｔｈｅｎｏｐｓ　ａｕｓｔｒａｌｉｓ］（ＧｅｎＢａｎｋアクセッション番号ＣＡＭ３２２４９．１（アミノ酸配列）、ＡＭ４９０１６９（塩基配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　１［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ１４５８９．１（アミノ酸配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　２［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ１４５９１．１（アミノ酸配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ－ｌｉｋｅ　ｐｒｏｔｅｉｎ［Ｎｅｐｈｉｌｅｎｇｙｓ　ｃｒｕｅｎｔａｔａ］（ＧｅｎＢａｎｋアクセッション番号ＡＢＲ３７２７８．１（アミノ酸配列）等が挙げられる。 More specific examples of 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 spidolin 1 [derived from Nephila clavipes (GenBank accession number AAC4, amino acid sequence A04) U37520 (base sequence)), major angul11ate spidroin 1 [La rodectus hesperus origin] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Nephila clavata origin] (GenBank accession number AAL32447, amino acid sequence 45, amino acid sequence) major antiderate spirodin 1 [from Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampulative spirodin 2 (EuprosentropisGastrospus nk accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampilate silk protein 1 [Nephila clavies] (GenBank accession number AAC1458.11 (amino acid sequence)), minor ampulativesilk2 ] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplitude spiroin-like protein [Nephilegenes cruentata] (GenBank accession number ABR37278. 1 (amino acid sequence), and the like.
　天然由来のフィブロインのより具体的な例としては、更に、ＮＣＢＩ　ＧｅｎＢａｎｋに配列情報が登録されているフィブロインを挙げることができる。例えば、ＮＣＢＩ　ＧｅｎＢａｎｋに登録されている配列情報のうちＤＩＶＩＳＩＯＮとしてＩＮＶを含む配列の中から、ＤＥＦＩＮＩＴＩＯＮにｓｐｉｄｒｏｉｎ、ａｍｐｕｌｌａｔｅ、ｆｉｂｒｏｉｎ、「ｓｉｌｋ及びｐｏｌｙｐｅｐｔｉｄｅ」、又は「ｓｉｌｋ及びｐｒｏｔｅｉｎ」がキーワードとして記載されている配列、ＣＤＳから特定のｐｒｏｄｕｃｔの文字列、ＳＯＵＲＣＥからＴＩＳＳＵＥ　ＴＹＰＥに特定の文字列の記載された配列を抽出することにより確認することができる。 More specific examples of naturally derived fibroin include fibroin whose sequence information is registered in NCBI GenBank. For example, 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.
　第１のフィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、第１のフィブロインの単離、固定化、検出及び可視化等が可能となる。タグ配列の具体例として、例えば、配列番号２５で示されるアミノ酸配列（Ｈｉｓタグを含むアミノ酸配列）が挙げられる。 The first 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 first fibroin. Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 25 (amino acid sequence containing a His tag).
　第１のフィブロインのより具体的な例として、（ｉ）配列番号１～１０のいずれかで示されるアミノ酸配列（表１参照）、又は（ｉｉ）配列番号１～１０のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むフィブロインを挙げることができる。 As a more specific example of the first fibroin, (i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 10 (see Table 1), or (ii) an amino acid represented by any one of SEQ ID NOs: 1 to 10 Mention may be made of fibroin comprising an amino acid sequence having 90% or more sequence identity to the sequence.
　（ｉ）のフィブロインは、配列番号１～１０のいずれかで示されるアミノ酸配列からなるものであってもよい。 (I) The fibroin may consist of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 10.
　（ｉｉ）のフィブロインは、配列番号１～１０のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（ｉｉ）のフィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ１で表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。（ｉｉ）のフィブロインは、ｘ／ｙが上述した範囲内にあることが好ましい。 The fibroin of (ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 10. Fibroin of (ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m1 . The sequence identity is preferably 95% or more. In the fibroin (ii), x / y is preferably within the above-described range.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（第２のフィブロイン）
　本発明に係る第２のフィブロインは、式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ２で表されるドメイン配列又は式３：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ３－（Ａ）ｎモチーフで表されるドメイン配列を含み、グルタミン残基含有率が９％以下であるアミノ酸配列を有するフィブロインである。 (Second fibroin)
The second fibroin according to the present invention is a domain sequence represented by Formula 2: [(A) n motif-REP] m2 or Formula 3: [(A) n motif-REP] m3- (A) n motif. It is a fibroin having an amino acid sequence that includes the represented domain sequence and has a glutamine residue content of 9% or less.
　式２及び式３中、（Ａ）ｎモチーフは、それぞれ独立に、４～２７アミノ酸残基から構成されるアミノ酸配列を示し、かつ（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が８０％以上であり、ＲＥＰは、それぞれ独立に、１０～２００アミノ酸残基から構成されるアミノ酸配列を示し、ｍ２及びｍ３は、それぞれ独立に、８～３００の整数を示す。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。 In Formula 2 and Formula 3, (A) n motif independently represents an amino acid sequence composed of 4 to 27 amino acid residues, and (A) alanine residues relative to the total number of amino acid residues in n motif The number is 80% or more, REP independently represents an amino acid sequence composed of 10 to 200 amino acid residues, and m2 and m3 each independently represents an integer of 8 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.
　第２のフィブロインは、グルタミン残基含有率が９％以下であればよく、７％以下であることが好ましく、４％以下であることがより好ましく、０％であることが特に好ましい。グルタミン残基含有率がこの範囲にあることにより、収縮が抑制又は低減されたフィブロイン繊維を得ることができるフィブロインとなる。 The second fibroin may have a glutamine residue content of 9% or less, preferably 7% or less, more preferably 4% or less, and particularly preferably 0%. When the glutamine residue content is in this range, the fibroin can be obtained to obtain a fibroin fiber in which shrinkage is suppressed or reduced.
　本明細書において、「グルタミン残基含有率」は、以下の方法により算出される値である。図１は、フィブロインのドメイン配列を示す模式図である。図１を参照しながらグルタミン残基含有率の算出方法を具体的に説明する。式Ａ：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式Ｂ：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロインにおいて、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列（図１の「領域Ａ」に相当する配列。）に含まれる全てのＲＥＰにおいて、その領域に含まれるグルタミン残基の総数をａとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｂとしたときに、グルタミン残基含有率はａ／ｂとして算出される。 In the present specification, the “glutamine residue content” is a value calculated by the following method. FIG. 1 is a schematic diagram showing the domain sequence of fibroin. The method for calculating the glutamine residue content will be specifically described with reference to FIG. Formula A: [(A) n motif-REP] m or Formula B: [(A) n motif-REP] m- (A) In fibroin containing a domain sequence represented by n motif, the most C-terminal side Located in (A) all REPs contained in a sequence (sequence corresponding to “region A” in FIG. 1) excluding the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence (included in that region) The total number of glutamine residues is a, and the sequence from the (A) n- motif located at the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and (A) the amino acid residues of all REPs excluding the n- motif When the total number of groups is b, the glutamine residue content is calculated as a / b.
　グルタミン残基含有率の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としているのは、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列」（ＲＥＰに相当する配列）には、フィブロインに特徴的な配列と相関性の低い配列が含まれることがあり、ｍが小さい場合（つまり、ドメイン配列が短い場合）、グルタミン残基含有率の算出結果に影響するので、この影響を排除するためである。 In the calculation of the glutamine residue content, “A sequence that is 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” The sequence (A) from the n motif to the C terminus of the domain sequence (sequence corresponding to REP) located on the side may include a sequence that is not highly correlated with the sequence characteristic of fibroin, where m is If it is small (that is, if the domain sequence is short), it affects the calculation result of the glutamine residue content, so this effect is eliminated.
　第２のフィブロインは、例えば、天然由来のフィブロインの遺伝子配列から、グルタミン残基含有率が９％以下になるように、ＲＥＰ中の１又は複数のグルタミン残基を欠失させること、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から、グルタミン残基含有率が９％以下になるように、ＲＥＰ中の１又は複数のグルタミン残基を欠失したこと、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数のグルタミン残基を欠失させること、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換することに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。アミノ酸残基の置換、欠失、挿入及び／又は付加を行う方法、並びに天然由来のフィブロインは、第１のフィブロインで説明したとおりである。 The second fibroin is, for example, deleting one or more glutamine residues in REP from the gene sequence of naturally occurring fibroin so that the glutamine residue content is 9% or less, and / or It can be obtained by replacing one or more glutamine residues in REP with other amino acid residues. In addition, for example, one or more glutamine residues in REP are deleted from the amino acid sequence of naturally occurring fibroin so that the glutamine residue content is 9% or less, and / or 1 in REP. Alternatively, it can be obtained by designing an amino acid sequence corresponding to substitution of a plurality of glutamine residues with other amino acid residues, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, 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. In addition to the modification corresponding to the substitution, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further performed. The method for substitution, deletion, insertion and / or addition of amino acid residues, and naturally-occurring fibroin are as described for the first fibroin.
　「他のアミノ酸残基」は、グルタミン残基以外のアミノ酸残基であればよいが、グルタミン残基よりも疎水性指標の大きいアミノ酸残基であることが好ましい。アミノ酸残基の疎水性指標については、公知の指標（Ｈｙｄｒｏｐａｔｈｙ　ｉｎｄｅｘ：Ｋｙｔｅ　Ｊ，＆Ｄｏｏｌｉｔｔｌｅ　Ｒ（１９８２）“Ａ　ｓｉｍｐｌｅ　ｍｅｔｈｏｄ　ｆｏｒ　ｄｉｓｐｌａｙｉｎｇ　ｔｈｅ　ｈｙｄｒｏｐａｔｈｉｃ　ｃｈａｒａｃｔｅｒ　ｏｆ　ａ　ｐｒｏｔｅｉｎ”，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１５７，ｐｐ．１０５－１３２）を使用する。具体的には、各アミノ酸の疎水性指標（ハイドロパシー・インデックス、以下「ＨＩ」とも記す。）は、下記表２に示すとおりである。 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. 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 2 below.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２に示すとおり、グルタミン残基よりも疎水性指標の大きいアミノ酸残基としては、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）アラニン（Ａ）、グリシン（Ｇ）、スレオニン（Ｔ）、セリン（Ｓ）、トリプトファン（Ｗ）、チロシン（Ｙ）、プロリン（Ｐ）及びヒスチジン（Ｈ）から選ばれるアミノ酸残基を挙げることができる。これらの中でも、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）から選ばれるアミノ酸残基であることがより好ましく、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）及びフェニルアラニン（Ｆ）から選ばれるアミノ酸残基であることが更に好ましい。 As shown in Table 2, 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. Among these, 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).
　第２のフィブロインは、ＲＥＰのアミノ酸配列中に、ＧＧＸモチーフ及びＧＰＧＸＸモチーフ（Ｇはグリシン残基、Ｐはフェニルアラニン残基、Ｘはグリシン残基以外のアミノ酸残基を示す。）から選ばれる少なくとも一つのモチーフが含まれていることが好ましい。ＲＥＰ中にこれらのモチーフが含まれることにより、第２のフィブロインの伸度を向上させることができる。 The second fibroin is at least one selected from the GGX motif and the GPGXX motif (G represents a glycine residue, P represents a phenylalanine residue, and X represents an amino acid residue other than a glycine residue) in the amino acid sequence of REP. Preferably, one motif is included. By including these motifs in REP, the elongation of the second fibroin can be improved.
　第２のフィブロインが、ＲＥＰ中にＧＰＧＸＸモチーフを含む場合、ＧＰＧＸＸモチーフ含有率は、通常１％以上であり、５％以上であってもよく、１０％以上であるのが好ましい。これにより、第２のフィブロインの伸度をより向上させることができる。ＧＰＧＸＸモチーフ含有率の上限に特に制限はなく、５０％以下であってよく、３０％以下であってもよい。 When the second fibroin contains a GPGXX motif in REP, the content ratio of the GPGXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more. Thereby, the elongation of the second fibroin can be further improved. There is no restriction | limiting in particular in the upper limit of GPGXX motif content rate, 50% or less may be sufficient and 30% or less may be sufficient.
　本明細書において、「ＧＰＧＸＸモチーフ含有率」は、以下の方法により算出される値である。式Ａ：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式Ｂ：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロインにおいて、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、その領域に含まれるＧＰＧＸＸモチーフの個数の総数を３倍した数（即ち、ＧＰＧＸＸモチーフ中のＧ及びＰの総数に相当）をｃとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｄとしたときに、ＧＰＧＸＸモチーフ含有率はｃ／ｄとして算出される。 In the present specification, the “GPGXX motif content” is a value calculated by the following method. Formula A: [(A) n motif-REP] m or Formula B: [(A) n motif-REP] m- (A) In fibroin containing a domain sequence represented by n motif, the most C-terminal side (A) In all REPs included in the sequence excluding the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence, the total number of GPGXX motifs included in the region is tripled (ie, (Corresponding to the total number of G and P in the GPGXX motif) as c, 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 n motif When the total number of amino acid residues of all the REPs removed is d, the GPGXX motif content is calculated as c / d.
　ＧＰＧＸＸモチーフ含有率の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。なお、ＲＥＰのＣ末端に「ＧＰＧＸＸモチーフ」が位置する場合、「ＸＸ」が例えば「ＡＡ」の場合であっても、「ＧＰＧＸＸモチーフ」として扱う。 In the calculation of the content ratio of the GPGXX motif, the reason why “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” is the same as the reason described above. It is. When the “GPGXX motif” is located at the C-terminus of REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.
　図１は、フィブロインのドメイン配列を示す模式図である。図１を参照しながらＧＰＧＸＸモチーフ含有率の算出方法を具体的に説明する。まず、図１に示したフィブロインのドメイン配列（「［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフ」タイプである。）では、全てのＲＥＰが「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」（図１中、「領域Ａ」で示した配列。）に含まれているため、ｃを算出するためのＧＰＧＸＸモチーフの個数は７であり、ｃは７×３＝２１となる。同様に、全てのＲＥＰが「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」（図１中、「領域Ａ」で示した配列。）に含まれているため、当該配列から更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数ｄは５０＋４０＋１０＋２０＋３０＝１５０である。次に、ｃをｄで除すことによって、ｃ／ｄ（％）を算出することができ、図１のフィブロインの場合２１／１５０＝１４．０％となる。 FIG. 1 is a schematic diagram showing the domain sequence of fibroin. The calculation method of the content ratio of GPGXX motif will be specifically described with reference to FIG. First, in the fibroin domain sequence shown in FIG. 1 (which is of the “[(A) n motif-REP] m- (A) n motif” type), all REPs are “most C-terminally located ( A) GPGXX for calculating c because it is included in the “sequence excluding the sequence from the n motif to the C-terminus of the domain sequence from the domain sequence” (the sequence indicated by “region A” in FIG. 1). The number of motifs is 7, and c is 7 × 3 = 21. Similarly, 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. 1). )), The total number d of amino acid residues of all REPs excluding (A) the n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, c / d (%) can be calculated by dividing c by d. In the case of fibroin in FIG. 1, 21/150 = 14.0%.
　第２のフィブロインは、ＲＥＰの疎水性度が、－０．８以上であることが好ましく、－０．７以上であることがより好ましく、０以上であることが更に好ましく、０．３以上であることが更により好ましく、０．４以上であることが特に好ましい。ＲＥＰの疎水性度の上限に特に制限はなく、１．０以下であってよく、０．７以下であってもよい。 In the second fibroin, 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. Even more preferably, it is particularly preferably 0.4 or more. There is no restriction | limiting in particular in the upper limit of the hydrophobicity of REP, It may be 1.0 or less and may be 0.7 or less.
　本明細書において、「ＲＥＰの疎水性度」は、以下の方法により算出される値である。
式Ａ：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式Ｂ：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロインにおいて、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列（図１の「領域Ａ」に相当する配列。）に含まれる全てのＲＥＰにおいて、その領域の各アミノ酸残基の疎水性指標の総和をｅとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｆとしたときに、ＲＥＰの疎水性度はｅ／ｆとして算出される。ＲＥＰの疎水性度の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, the “hydrophobicity of REP” is a value calculated by the following method.
Formula A: [(A) n motif-REP] m or Formula B: [(A) n motif-REP] m- (A) In fibroin containing a domain sequence represented by n motif, the most C-terminal side Positioned (A) In all REPs included in the sequence (sequence corresponding to “region A” in FIG. 1) obtained by removing the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence (each amino acid in the region) The sum of the hydrophobic indices of the residues is e, the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and (A) the total REP excluding the n motif When the total number of amino acid residues is f, the hydrophobicity of REP is calculated as e / f. In the calculation of the hydrophobicity of REP, the reason why “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” is the reason described above. It is the same.
　第２のフィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、第２のフィブロインの単離、固定化、検出及び可視化等が可能となる。タグ配列の具体例として、例えば、配列番号２５で示されるアミノ酸配列（Ｈｉｓタグを含むアミノ酸配列）が挙げられる。 The second 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 second fibroin. Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 25 (amino acid sequence containing a His tag).
　第２のフィブロインのより具体的な例として、（ｉｉｉ）配列番号１１～２４のいずれかで示されるアミノ酸配列（表３参照）、又は（ｉｖ）配列番号１１～２４のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むフィブロインを挙げることができる。 As a more specific example of the second fibroin, (iii) the amino acid sequence represented by any one of SEQ ID NOs: 11 to 24 (see Table 3) or (iv) the amino acid represented by any one of SEQ ID NOs: 11 to 24 Mention may be made of fibroin comprising an amino acid sequence having 90% or more sequence identity to the sequence.
　（ｉｉｉ）のフィブロインは、配列番号１１～２４のいずれかで示されるアミノ酸配列からなるものであってもよい。 (Iii) The fibroin may comprise an amino acid sequence represented by any of SEQ ID NOs: 11 to 24.
　（ｉｖ）のフィブロインは、配列番号１１～２４のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（ｉｖ）のフィブロインもまた、式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ２又は式３：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ３－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。（ｉｖ）のフィブロインは、グルタミン残基含有率が上述した範囲内にあることが好ましい。また、（ｉｖ）のフィブロインは、ＧＰＧＸＸモチーフ含有率が上述した範囲内にあることが好ましい。 The fibroin (iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 11 to 24. (Iv) Fibroin is also a protein comprising a domain sequence represented by Formula 2: [(A) n motif-REP] m2 or Formula 3: [(A) n motif-REP] m3- (A) n motif It is. The sequence identity is preferably 95% or more. The fibroin (iv) preferably has a glutamine residue content within the above-described range. Moreover, it is preferable that the fibroin of (iv) has the content ratio of GPGXX motif in the above-mentioned range.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（第３のフィブロイン）
　本発明に係る第３のフィブロインは、式４：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ４で表されるドメイン配列を含み、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｚとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｗとしたときに、ｚ／ｗが６．２％以上であるアミノ酸配列を有するフィブロインである（但し、第２のフィブロインに該当するフィブロインを除く。）。 (Third fibroin)
The third fibroin according to the present invention includes a domain sequence represented by Formula 4: [(A) n motif-REP] m4 , and is located on the most C-terminal side (A) from the n motif to the C terminal of the domain sequence. Z is the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs included in the sequence excluding the sequence up to the domain sequence. (A) When the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence is represented by w (6) z / w is 6. Fibroin having an amino acid sequence of 2% or more (excluding fibroin corresponding to the second fibroin).
　ここで、式４中、（Ａ）ｎモチーフは、４～２７アミノ酸残基から構成されるアミノ酸配列を示し、かつ（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が８０％以上であり、ＲＥＰは、１０～２００アミノ酸残基から構成されるアミノ酸配列を示し、ｍ４は、８～３００の整数を示す。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。 Here, in Formula 4, (A) n motif represents an amino acid sequence composed of 4 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 80%. In the above, REP represents an amino acid sequence composed of 10 to 200 amino acid residues, and m4 represents an integer of 8 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.
　第３のフィブロインは、ｚ／ｗが６．２％以上であればよく、７％以上であることが好ましく、１０％以上であることがより好ましく、２０％以上であることが更に好ましく、３０％以上であることが更により好ましい。ｚ／ｗの上限は、特に制限されないが、例えば、４５％以下であってもよい。 The third fibroin may have a z / w of 6.2% or more, preferably 7% or more, more preferably 10% or more, still more preferably 20% or more, and 30 It is still more preferable that it is% or more. The upper limit of z / w is not particularly limited, but may be 45% or less, for example.
　ｚ／ｗは、以下の方法で算出される値である。算出には、ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列（以下、「配列Ａ」とする）を用いる。まず、配列Ａに含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値を算出する。疎水性指標の平均値は、連続する４アミノ酸残基に含まれる各アミノ酸残基のＨＩの総和を４（アミノ酸残基数）で除して求める。疎水性指標の平均値は、全ての連続する４アミノ酸残基について求める（各アミノ酸残基は、１～４回平均値の算出に用いられる。）。次いで、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域を特定する。あるアミノ酸残基が、複数の「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」に該当する場合であっても、領域中には１アミノ酸残基として含まれることになる。そして、当該領域に含まれるアミノ酸残基の総数がｚである。また、配列Ａに含まれるアミノ酸残基の総数がｗである。 z / w is a value calculated by the following method. For the calculation, a sequence (hereinafter referred to as “sequence A”) obtained by removing the sequence from the domain sequence to the C-terminal side of the domain sequence (A) n motif to the C-terminus of the domain sequence is used. First, in all REPs included in the sequence 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 z. The total number of amino acid residues contained in sequence A is w.
　例えば、「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が２０カ所抽出された場合（重複はなし）、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域には、連続する４アミノ酸残基（重複はなし）が２０含まれることになり、ｚは２０×４＝８０である。また、例えば、２つの「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が１アミノ酸残基だけ重複して存在する場合、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域には、７アミノ酸残基含まれることになる（ｚ＝２×４－１＝７。「－１」は重複分の控除である。）。例えば、図２に示したドメイン配列の場合、「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が重複せずに７つ存在するため、ｚは７×４＝２８となる。また、例えば、図２に示したドメイン配列の場合、ｗは４＋５０＋４＋４０＋４＋１０＋４＋２０＋４＋３０＝１７０である（Ｃ末端側の最後に存在する（Ａ）ｎモチーフは含めない）。次に、ｚをｗで除すことによって、ｚ／ｗ（％）を算出することができる。図２の場合２８／１７０＝１６．４７％となる。 For example, when 20 “four consecutive amino acid residues with an average value of hydrophobicity index of 2.6 or more” are extracted (no overlap), the average value of the hydrophobicity index of four consecutive amino acid residues is 2 The region of .6 or more contains 20 consecutive 4 amino acid residues (no overlap), and z is 20 × 4 = 80. In addition, for example, when two “four consecutive amino acid residues having an average value of hydrophobicity index of 2.6 or more” overlap by one amino acid residue, the hydrophobicity index of four consecutive amino acid residues In the region where the average value of is 2.6 or more, 7 amino acid residues are included (z = 2 × 4-1 = 7, where “−1” is a deduction of duplicates). For example, in the case of the domain sequence shown in FIG. 2, since there are seven non-overlapping “four consecutive amino acid residues with an average hydrophobicity index of 2.6 or more”, z is 7 × 4 = 28. For example, in the case of the domain sequence shown in FIG. 2, w is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (the (A) n motif present at the end on the C-terminal side is not included). Next, z / w (%) can be calculated by dividing z by w. In the case of FIG. 2, 28/170 = 16.47%.
　アミノ酸残基の疎水性指標については、公知の指標（Ｈｙｄｒｏｐａｔｈｙ　ｉｎｄｅｘ：Ｋｙｔｅ　Ｊ，＆Ｄｏｏｌｉｔｔｌｅ　Ｒ（１９８２）“Ａ　ｓｉｍｐｌｅ　ｍｅｔｈｏｄ　ｆｏｒ　ｄｉｓｐｌａｙｉｎｇ　ｔｈｅ　ｈｙｄｒｏｐａｔｈｉｃ　ｃｈａｒａｃｔｅｒ　ｏｆ　ａ　ｐｒｏｔｅｉｎ”，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１５７，ｐｐ．１０５－１３２）を使用する。具体的には、表２に示すとおりである。 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, it is as shown in Table 2.
　第３のフィブロインは、例えば、天然由来のフィブロインのアミノ酸配列を、上記のｚ／ｗの条件を満たすように、ＲＥＰ中の１又は複数の親水性アミノ酸残基（例えば、疎水性指標がマイナスであるアミノ酸残基）を疎水性アミノ酸残基（例えば、疎水性指標がプラスであるアミノ酸残基）に置換すること、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入することにより、局所的に疎水性指標の大きい領域を含むアミノ酸配列に改変することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から上記のｚ／ｗの条件を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当する改変を行ってもよい。アミノ酸残基の置換、欠失、挿入及び／又は付加を行う方法、並びに天然由来のフィブロインは、第１のフィブロインで説明したとおりである。 The third fibroin is, for example, one or more hydrophilic amino acid residues in the REP (for example, the hydrophobicity index is negative so that the amino acid sequence of naturally derived fibroin satisfies the above z / w condition). By replacing a certain amino acid residue) with a hydrophobic amino acid residue (eg, an amino acid residue having a positive hydrophobicity index) and / or by inserting one or more hydrophobic amino acid residues in a REP It can be obtained by locally modifying the amino acid sequence including a region having a large hydrophobicity index. Alternatively, for example, an amino acid sequence satisfying the above z / w condition 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. In any case, compared to naturally occurring fibroin, 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. In addition to modifications corresponding to insertion of amino acid residues having a large hydrophobicity index, modifications corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. . The method for substitution, deletion, insertion and / or addition of amino acid residues, and naturally-occurring fibroin are as described for the first fibroin.
　疎水性指標の大きいアミノ酸残基としては、特に制限はないが、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）が好ましく、バリン（Ｖ）、ロイシン（Ｌ）及びイソロイシン（Ｉ）がより好ましい。 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.
　第３のフィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、第３のフィブロインの単離、固定化、検出及び可視化等が可能となる。タグ配列の具体例として、例えば、配列番号２５で示されるアミノ酸配列（Ｈｉｓタグを含むアミノ酸配列）が挙げられる。 The third 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 third fibroin. Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 25 (amino acid sequence containing a His tag).
　第３のフィブロインのより具体的な例として、（ｖ）配列番号２７～３２のいずれかで示されるアミノ酸配列（表４参照）、又は（ｖｉ）配列番号２７～３２のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むフィブロインを挙げることができる。 As a more specific example of the third fibroin, (v) the amino acid sequence represented by any one of SEQ ID NOs: 27 to 32 (see Table 4) or (vi) the amino acid represented by any one of SEQ ID NOs: 27 to 32 Mention may be made of fibroin comprising an amino acid sequence having 90% or more sequence identity to the sequence.
　（ｖ）のフィブロインは、配列番号２７～３２のいずれかで示されるアミノ酸配列からなるものであってもよい。 (V) The fibroin may consist of an amino acid sequence represented by any of SEQ ID NOs: 27 to 32.
　（ｖｉ）のフィブロインは、配列番号２７～３２のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（ｖｉ）のフィブロインもまた、式４：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ４で表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。（ｖｉ）のフィブロインは、ｚ／ｗが上述した範囲内にあることが好ましい。 The fibroin (vi) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 27 to 32. The fibroin in (vi) is also a protein containing a domain sequence represented by Formula 4: [(A) n motif-REP] m4 . The sequence identity is preferably 95% or more. The fibroin (vi) preferably has z / w within the above-mentioned range.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（フィブロインの製造）
　第１のフィブロイン、第２のフィブロイン、及び第３のフィブロインは、特許文献２（国際公開第２０１７／１８８４３４号）に記載した方法により取得することができる。すなわち、本実施形態に係るフィブロインをコードする核酸を当該核酸配列に作動可能に連結された１又は複数の調節配列とを有する発現ベクターで宿主を形質転換し、当該宿主において、本発明に係る核酸を発現させることにより、本実施形態に係るフィブロインを生産することができる。また、特許文献２（国際公開第２０１７／１８８４３４号）に記載した方法により、精製されたフィブロインを取得することができる。 (Manufacture of fibroin)
The first fibroin, the second fibroin, and the third fibroin can be obtained by the method described in Patent Document 2 (International Publication No. 2017/188434). That is, a host is transformed with an expression vector having one or more regulatory sequences operably linked to the nucleic acid sequence encoding the fibroin according to the present embodiment, and the nucleic acid according to the present invention is transformed in the host. Can be produced to produce the fibroin according to the present embodiment. Further, purified fibroin can be obtained by the method described in Patent Document 2 (International Publication No. 2017/188434).
（フィブロイン組成物）
　本発明に係るフィブロイン組成物における、第１のフィブロイン、並びに第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種の含有比率は特に制限されるものではなく、フィブロイン組成物の用途等に応じて、適宜設定することができる。第１のフィブロインは、応力及び伸度に関して優れた性質を有しており、第２及び第３のフィブロインは耐水性に関して優れた性質を有している。したがって、フィブロイン組成物における第１のフィブロイン、並びに第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種の含有比率は、応力及び伸度、又は耐水性のいずれを強化したいかにより、適宜決定すればよく、例えば応力及び伸度を重視する場合には第１のフィブロインの割合を多くすればよく、第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選ばれる少なくとも１種との含有比率を９．９：０．１～５．０：５．０（重量比）の範囲にすることが好ましい。また、耐水性を重視する場合には第２のフィブロイン及び第３のフィブロインから選ばれる少なくとも１種の割合を多くすればよく、第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選ばれる少なくとも１種との含有比率を５．０：５．０～０．１：９．９の範囲にすることが好ましい。さらに応力及び伸度、並びに耐水性をバランスよく発揮させる場合には、第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選ばれる少なくとも１種との含有比率を５．０：５．０としてもよい。 (Fibroin composition)
In the fibroin composition according to the present invention, the content ratio of at least one selected from the first fibroin and the second fibroin and the third fibroin is not particularly limited, and is suitable for use of the fibroin composition, etc. Accordingly, it can be set as appropriate. The first fibroin has excellent properties with respect to stress and elongation, and the second and third fibroin have excellent properties with respect to water resistance. Therefore, the content ratio of at least one selected from the first fibroin and the second fibroin and the third fibroin in the fibroin composition depends on whether stress or elongation or water resistance is enhanced. For example, when stress and elongation are emphasized, the ratio of the first fibroin may be increased, and at least one selected from the first fibroin, the second fibroin and the third fibroin The content ratio is preferably in the range of 9.9: 0.1 to 5.0: 5.0 (weight ratio). In addition, when importance is attached to water resistance, the ratio of at least one selected from the second fibroin and the third fibroin may be increased, and the first fibroin, the second fibroin and the third fibroin are selected. The content ratio of at least one selected from the above is preferably in the range of 5.0: 5.0 to 0.1: 9.9. Furthermore, in the case where stress, elongation, and water resistance are exhibited in a balanced manner, the content ratio of the first fibroin and at least one selected from the second fibroin and the third fibroin is 5.0: 5. It may be 0.
　本発明に係るフィブロイン組成物は、粉末状、ペースト状、液状（例えば、懸濁液、後述のフィブロイン溶液）のいずれの形態であってもよい。また、本発明に係るフィブロイン組成物は、原料組成物の形態の他、当該フィブロイン組成物を含む、又は当該フィブロイン組成物からなる成形体（例えば、後述のフィブロイン繊維等の製品）の形態であってもよい。 The fibroin composition according to the present invention may be in any form of powder, paste, or liquid (for example, a suspension or a fibroin solution described later). In addition to the form of the raw material composition, the fibroin composition according to the present invention is in the form of a molded body containing the fibroin composition or made of the fibroin composition (for example, a product such as fibroin fiber described later). May be.
　本発明に係るフィブロイン組成物は、その形態等に応じて、第１のフィブロイン、並びに第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種以外の成分を含むものであってもよい。 The fibroin composition according to the present invention may contain a component other than the first fibroin and at least one selected from the second fibroin and the third fibroin depending on the form and the like.
〔製品〕
　本発明に係るフィブロイン組成物は、当該フィブロイン組成物を含む、又は当該フィブロイン組成物からなる製品であってもよい。製品としては、例えば、繊維、糸、フィルム、発泡体、球体、ナノフィブリル、ゲル（ヒドロゲル等）及び樹脂が挙げられる。これらは、特開２００９－５０５６６８号公報、特許第５６７８２８３号公報、特許第４６３８７３５号公報等に記載の方法に準じて製造することができる。 [Product]
The fibroin composition according to the present invention may be a product containing or consisting of the fibroin composition. Examples of products include fibers, yarns, films, foams, spheres, nanofibrils, gels (hydrogels, etc.) and resins. These can be produced according to the methods described in JP-A-2009-505668, JP-A-5678283, JP-A-4638735 and the like.
　本発明に係るフィブロイン組成物から形成されたフィブロイン繊維は、繊維（長繊維、短繊維、マルチフィラメント、又はモノフィラメント等）又は糸（紡績糸、撚糸、仮撚糸、加工糸、混繊糸、混紡糸等）として、織物、編物、組み物、不織布等に応用できる。また、ロープ、手術用縫合糸、電気部品用の可撓性止め具、さらには移植用生理活性材料（例えば、人工靭帯及び大動脈バンド）等の高強度用途にも応用できる。 The fibroin fiber formed from the fibroin composition according to the present invention is a fiber (long fiber, short fiber, multifilament, monofilament, etc.) or yarn (spun yarn, twisted yarn, false twisted yarn, processed yarn, blended yarn, blended yarn). Etc.) and can be applied to woven fabrics, knitted fabrics, braided fabrics, non-woven fabrics and the like. It can also be applied to high-strength applications such as ropes, surgical sutures, flexible stops for electrical components, and bioactive materials for transplantation (eg, artificial ligaments and aortic bands).
〔フィブロイン溶液〕
　本発明に係るフィブロイン溶液は、本発明に係るフィブロイン組成物が、溶媒に溶解してなるものである。当該フィブロイン溶液は、例えば、ドープ液として、フィブロイン繊維、フィブロインフィルム等の成形に用いることができる。 [Fibroin solution]
The fibroin solution according to the present invention is obtained by dissolving the fibroin composition according to the present invention in a solvent. The fibroin solution can be used, for example, as a dope solution for forming fibroin fibers, fibroin films and the like.
　溶媒としては、例えば、ヘキサフルオロイソプロパノール（ＨＦＩＰ）、ヘキサフルオロアセトン（ＨＦＡ）、ジメチルスルホキシド（ＤＭＳＯ）、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）、ギ酸、並びに尿素、グアニジン、ドデシル硫酸ナトリウム（ＳＤＳ）、臭化リチウム、塩化カルシウム及びチオシアン酸リチウム等を含む水溶液等を挙げることができる。これらの溶媒は、１種単独で使用してもよく、２種以上を混合して使用してもよい。 Examples of the solvent include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and urea, guanidine, sodium dodecyl sulfate (SDS), Examples include an aqueous solution containing lithium bromide, calcium chloride, lithium thiocyanate, and the like. These solvents may be used alone or in combination of two or more.
　本実施形態に係るフィブロイン溶液には、必要に応じて無機塩を添加してもよい。無機塩としては、例えば、以下に示すルイス酸とルイス塩基とからなる無機塩が挙げられる。ルイス塩基としては、例えば、オキソ酸イオン（硝酸イオン、過塩素酸イオン等）、金属オキソ酸イオン（過マンガン酸イオン等）、ハロゲン化物イオン、チオシアン酸イオン、シアン酸イオン等が挙げられる。ルイス酸としては、例えば、アルカリ金属イオン、アルカリ土類金属イオン等の金属イオン、アンモニウムイオン等の多原子イオン、錯イオン等が挙げられる。ルイス酸とルイス塩基とからなる無機塩の具体例としては、塩化リチウム、臭化リチウム、ヨウ化リチウム、硝酸リチウム、過塩素酸リチウム、及びチオシアン酸リチウム等のリチウム塩、塩化カルシウム、臭化カルシウム、ヨウ化カルシウム、硝酸カルシウム、過塩素酸カルシウム、及びチオシアン酸カルシウム等のカルシウム塩、塩化鉄、臭化鉄、ヨウ化鉄、硝酸鉄、過塩素酸鉄、及びチオシアン酸鉄等の鉄塩、塩化アルミニウム、臭化アルミニウム、ヨウ化アルミニウム、硝酸アルミニウム、過塩素酸アルミニウム、及びチオシアン酸アルミニウム等のアルミニウム塩、塩化カリウム、臭化カリウム、ヨウ化カリウム、硝酸カリウム、過塩素酸カリウム、及びチオシアン酸カリウム等のカリウム塩、塩化ナトリウム、臭化ナトリウム、ヨウ化ナトリウム、硝酸ナトリウム、過塩素酸ナトリウム、及びチオシアン酸ナトリウム等のナトリウム塩、塩化亜鉛、臭化亜鉛、ヨウ化亜鉛、硝酸亜鉛、過塩素酸亜鉛、及びチオシアン酸亜鉛等の亜鉛塩、塩化マグネシウム、臭化マグネシウム、ヨウ化マグネシウム、硝酸マグネシウム、過塩素酸マグネシウム、及びチオシアン酸マグネシウム等のマグネシウム塩、塩化バリウム、臭化バリウム、ヨウ化バリウム、硝酸バリウム、過塩素酸バリウム、及びチオシアン酸バリウム等のバリウム塩、並びに塩化ストロンチウム、臭化ストロンチウム、ヨウ化ストロンチウム、硝酸ストロンチウム、過塩素酸ストロンチウム、及びチオシアン酸ストロンチウム等のストロンチウム塩が挙げられる。 An inorganic salt may be added to the fibroin solution according to the present embodiment as necessary. As an inorganic salt, the inorganic salt which consists of the following Lewis acid and Lewis base is mentioned, for example. Examples of the Lewis base include oxo acid ions (nitrate ions, perchlorate ions, etc.), metal oxo acid ions (permanganate ions, etc.), halide ions, thiocyanate ions, cyanate ions, and the like. Examples of the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, complex ions, and the like. Specific examples of inorganic salts composed of a Lewis acid and a Lewis base include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate, calcium chloride, calcium bromide. Calcium salts such as calcium iodide, calcium nitrate, calcium perchlorate and calcium thiocyanate, iron salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate and iron thiocyanate, Aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate, potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and potassium thiocyanate Potassium salt, sodium chloride, sodium bromide, etc. Sodium salts such as sodium uride, sodium nitrate, sodium perchlorate and sodium thiocyanate, zinc salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, zinc perchlorate and zinc thiocyanate, chloride Magnesium salts such as magnesium, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium thiocyanate, barium chloride, barium bromide, barium iodide, barium nitrate, barium perchlorate, and barium thiocyanate And strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate.
　本実施形態に係るフィブロイン溶液は、第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種とが混合（以下、「混合フィブロイン」と呼ぶこともある。）して溶解した溶液を調製することにより得ることができる。混合は第１のフィブロインを溶媒に溶解させた溶解液と、第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種を溶媒に溶解させた溶解液を混合してもよく、第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種と、を同一の溶媒中で混合して溶解させてもよい。 In the fibroin solution according to this embodiment, the first fibroin and at least one selected from the second fibroin and the third fibroin are mixed (hereinafter also referred to as “mixed fibroin”). It can be obtained by preparing a dissolved solution. The mixing may be performed by mixing a solution obtained by dissolving the first fibroin in a solvent and a solution obtained by dissolving at least one selected from the second fibroin and the third fibroin in the solvent. Fibroin and at least one selected from the second fibroin and the third fibroin may be mixed and dissolved in the same solvent.
　本実施形態に係るフィブロイン溶液の調製時に、３０～９０℃に加温してもよい。使用する溶媒及びフィブロインの種類等に応じて溶解可能な温度を適時設定すればよい。溶解を促進するために振盪、撹拌してもよい。 In the preparation of the fibroin solution according to this embodiment, it may be heated to 30 to 90 ° C. What is necessary is just to set the temperature which can be melt | dissolved timely according to the kind of solvent to be used, and fibroin. It may be shaken and stirred to promote dissolution.
　本実施形態に係るフィブロイン溶液の粘度は、フィブロイン溶液の用途等に応じて適宜設定してよい。例えば、本実施形態に係るフィブロイン溶液を紡糸液（ドープ液）として使用する場合、その粘度は、紡糸方法に応じて適宜設定してよく、例えば、３５℃において１００～１５，０００ｃＰ（センチポイズ）、４０℃において１００～３０，０００ｃＰ（センチポイズ）等に設定すればよい。紡糸液の粘度は、例えば京都電子工業社製の商品名“ＥＭＳ粘度計”を使用して測定することができる。 The viscosity of the fibroin solution according to this embodiment may be appropriately set according to the use of the fibroin solution. For example, when the fibroin solution according to the present embodiment is used as a spinning solution (dope solution), the viscosity may be appropriately set according to the spinning method, for example, 100 to 15,000 cP (centipoise) at 35 ° C., What is necessary is just to set to 100-30,000 cP (centipoise) etc. in 40 degreeC. The viscosity of the spinning solution can be measured using, for example, a trade name “EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.
　本実施形態に係るフィブロイン溶液における、第１のフィブロイン、並びに第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種の含有比率は特に制限されるものではなく、フィブロイン溶液の用途等に応じて、適宜設定することができる。具体的には、例えば応力及び伸度を重視する場合には、第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選ばれる少なくとも１種との含有比率を９．９：０．１～５．０：５．０（重量比）の範囲にすることが好ましい。また、耐水性を重視する場合には、第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選ばれる少なくとも１種との含有比率を５．０：５．０～０．１：９．９の範囲にすることが好ましい。さらに応力及び伸度、並びに耐水性をバランスよく発揮させる場合には、第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選ばれる少なくとも１種との含有比率を５．０：５．０としてもよい。 In the fibroin solution according to the present embodiment, the content ratio of at least one selected from the first fibroin and the second fibroin and the third fibroin is not particularly limited and depends on the use of the fibroin solution and the like. And can be set as appropriate. Specifically, for example, when stress and elongation are emphasized, the content ratio of the first fibroin and at least one selected from the second fibroin and the third fibroin is 9.9: 0.1. It is preferably in the range of up to 5.0: 5.0 (weight ratio). When water resistance is important, the content ratio of the first fibroin and at least one selected from the second fibroin and the third fibroin is 5.0: 5.0 to 0.1: 9. Is preferably in the range of .9. Furthermore, in the case where stress, elongation, and water resistance are exhibited in a balanced manner, the content ratio of the first fibroin and at least one selected from the second fibroin and the third fibroin is 5.0: 5. It may be 0.
〔フィブロイン繊維の製造方法〕
　本発明に係るフィブロイン繊維は、本発明に係るフィブロイン溶液を紡糸液（ドープ液）として使用し、フィブロインの紡糸に通常使用されている方法で紡糸することにより、得ることができる。 [Production method of fibroin fiber]
The fibroin fiber according to the present invention can be obtained by using the fibroin solution according to the present invention as a spinning solution (dope solution) and spinning it by a method usually used for spinning fibroin.
　すなわち、例えば、本発明に係るフィブロイン繊維の製造方法は、本発明に係るフィブロイン溶液をドープ液とし、当該ドープ液を口金から凝固液に押し出し、未延伸糸を得る工程を含むものとすることができる。 That is, for example, the method for producing the fibroin fiber according to the present invention may include a step of using the fibroin solution according to the present invention as a dope liquid and extruding the dope liquid from a die into a coagulating liquid to obtain an undrawn yarn.
　紡糸液の溶媒としては、フィブロイン溶液の説明で例示した溶媒と同種のものを挙げることができる。紡糸液には、必要に応じて無機塩を添加してもよい。無機塩としては、フィブロイン溶液の説明で例示した無機塩と同種のものを挙げることができる。 Examples of the solvent for the spinning solution include the same solvents as those exemplified in the description of the fibroin solution. An inorganic salt may be added to the spinning solution as necessary. As an inorganic salt, the same kind as the inorganic salt exemplified in the description of the fibroin solution can be exemplified.
　紡糸液における第１のフィブロイン、並びに第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種の含有比率は、フィブロイン溶液で説明したとおりである。第１のフィブロインと、第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種とを適切に配合することにより、第１のフィブロインのみで紡糸したフィブロイン繊維より強度、伸度の優れたフィブロイン繊維（混合フィブロイン繊維）を得ることができる。また、第２のフィブロイン及び第３のフィブロインから選択される少なくとも１種のみで紡糸したフィブロイン繊維より耐水性の優れたフィブロイン繊維（混合フィブロイン繊維）を得ることができる。いずれにおいても、混合フィブロインを紡糸することにより、それぞれ単独でフィブロインを紡糸する場合より応力及び伸度又は耐水性に優れた混合フィブロイン繊維を得ることができる。 The content ratio of at least one selected from the first fibroin and the second fibroin and the third fibroin in the spinning solution is as described in the fibroin solution. By appropriately blending the first fibroin and at least one selected from the second fibroin and the third fibroin, fibroin superior in strength and elongation to the fibroin fiber spun only with the first fibroin. Fiber (mixed fibroin fiber) can be obtained. Further, it is possible to obtain a fibroin fiber (mixed fibroin fiber) superior in water resistance to a fibroin fiber spun with at least one selected from the second fibroin and the third fibroin. In any case, by spinning the mixed fibroin, it is possible to obtain mixed fibroin fibers that are superior in stress and elongation or water resistance compared to the case of spinning fibroin alone.
　具体的には、例えば、第１のフィブロインとしてＰＲＴ７９９（配列番号９）タンパク質とを用い、第２のフィブロインとしてＰＲＴ９１８（配列番号２２）タンパク質を用いた場合、第１のフィブロイン：第２のフィブロイン＝７：３の比率で混合溶解させた紡糸液（ドープ液）を使用して紡糸した混合フィブロイン繊維は、ＰＲＴ７９９（配列番号９）タンパク質のみを含む紡糸液を使用して紡糸したフィブロイン繊維より強度及び伸度が優れた混合フィブロイン繊維となる。また、第１のフィブロイン：第２のフィブロイン＝３：７の比率で混合溶解させた紡糸液を使用して紡糸した混合フィブロイン繊維は、ＰＲＴ９１８（配列番号２２）タンパク質のみを含む紡糸液を使用して紡糸したフィブロイン繊維より耐水性が優れた混合フィブロイン繊維となる。 Specifically, for example, when PRT799 (SEQ ID NO: 9) protein is used as the first fibroin and PRT918 (SEQ ID NO: 22) protein is used as the second fibroin, the first fibroin: second fibroin = The mixed fibroin fiber spun using a spinning solution (dope solution) mixed and dissolved at a ratio of 7: 3 is stronger than the fibroin fiber spun using a spinning solution containing only PRT799 (SEQ ID NO: 9) protein. It becomes a mixed fibroin fiber excellent in elongation. The mixed fibroin fiber spun using the spinning solution mixed and dissolved in the ratio of first fibroin: second fibroin = 3: 7 uses a spinning solution containing only the PRT918 (SEQ ID NO: 22) protein. The mixed fibroin fiber has better water resistance than the spun fibroin fiber.
　紡糸方法としては、本発明に係る混合フィブロインを紡糸できる方法であれば特に制限されず、例えば、乾式紡糸、溶融紡糸、湿式紡糸等を挙げることができる。好ましい紡糸方法としては、湿式紡糸を挙げることができる。 The spinning method is not particularly limited as long as it is a method capable of spinning the mixed fibroin according to the present invention, and examples thereof include dry spinning, melt spinning, and wet spinning. A preferred spinning method includes wet spinning.
　湿式紡糸では、混合フィブロインを溶解させた溶媒（紡糸液）を紡糸口金（ノズル）から凝固液（凝固液槽）の中に押出して、凝固液中で混合フィブロインを固めることにより糸の形状の未延伸糸を得ることができる。凝固液としては、脱溶媒できる溶液であればよく、例えば、メタノール、エタノール及び２－プロパノール等の炭素数１～５の低級アルコール、並びにアセトン等を挙げることができる。凝固液には、適宜水を加えてもよい。凝固液の温度は、０～３０℃であることが好ましい。紡糸口金として、直径０．１～０．６ｍｍのノズルを有するシリンジポンプを使用する場合、押し出し速度（吐出量）は１ホール当たり、０．００１～０．５０ｍＬ／分であってよく、０．０１～０．５０ｍＬ／分であることが好ましく、０．０１～０．４０ｍＬ／分であることがより好ましく、０．０１～０．３５ｍＬ／分であることがさらに好ましく、０．０２～０．３５ｍＬ／分であることが特に好ましい。凝固液槽の長さは、脱溶媒が効率的に行える長さがあればよく、例えば、２００～５００ｍｍである。未延伸糸の引き取り速度は、例えば、０．００１～１００ｍ／分であってよく、０．０１～１００ｍ／分であってよく、０．１～８０ｍ／分であってよく、０．１～６０ｍ／分であってよく、０．１～４０ｍ／分であってよく、０．１～３０ｍ／分であってよく、０．１～２５ｍ／分であってよく、０．１～２０ｍ／分であってよく、０．１～１５ｍ／分であってよく、１～１５ｍ／分であってよく、１～１３ｍ／分であってよく、１～１０ｍ／分であってよく、１～５ｍ／分であってよく、１～３ｍ／分であってよい。引き取り速度が０．００１ｍ／分より小さい場合は、充分な生産性を得ることができない。引き取り速度が１００ｍ／分を超える場合は、溶媒の液体飛散が著しくなるため好ましくない。滞留時間は、未延伸糸中からドープ溶媒が除去される時間であればよく、例えば、０．０１～３分であってよく、０．０１～１．５分であることが好ましく、０．０１～０．２分であることがより好ましく、０．０３～０．２分であることがさらに好ましく、０．０５～０．１５分であることが特に好ましい。また、凝固液中で延伸（前延伸）をしてもよい。低級アルコールの蒸発を抑えるため凝固液を低温に維持し、未延伸糸の状態で引き取ってもよい。凝固液槽は多段設けてもよく、また延伸は必要に応じて、各段、又は特定の段で行ってもよい。 In wet spinning, a solvent in which mixed fibroin is dissolved (spinning liquid) is extruded from a spinneret (nozzle) into a coagulating liquid (coagulating liquid tank), and the mixed fibroin is solidified in the coagulating liquid, whereby the shape of the yarn is not yet formed. A drawn yarn can be obtained. The coagulation liquid 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. Water may be appropriately added to the coagulation liquid. The temperature of the coagulation liquid is preferably 0 to 30 ° C. When a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm is used as the spinneret, the extrusion speed (discharge amount) may be 0.001 to 0.50 mL / min per hole. It is preferably from 01 to 0.50 mL / min, more preferably from 0.01 to 0.40 mL / min, further preferably from 0.01 to 0.35 mL / min, and from 0.02 to 0 Particularly preferred is .35 mL / min. The length of the coagulating liquid tank is not limited as long as the solvent can be efficiently removed, and is, for example, 200 to 500 mm. The undrawn yarn take-up speed may be, for example, 0.001 to 100 m / min, 0.01 to 100 m / min, 0.1 to 80 m / min, 0.1 to It may be 60 m / min, 0.1 to 40 m / min, 0.1 to 30 m / min, 0.1 to 25 m / min, 0.1 to 20 m / min Minutes, 0.1 to 15 m / min, 1 to 15 m / min, 1 to 13 m / min, 1 to 10 m / min, 1 to It may be 5 m / min and may be 1 to 3 m / min. When the take-up speed is less than 0.001 m / min, sufficient productivity cannot be obtained. When the take-up speed exceeds 100 m / min, liquid scattering of the solvent becomes remarkable, which is not preferable. The residence time may be any time as long as the dope solvent is removed from the undrawn yarn. For example, the residence time may be 0.01 to 3 minutes, preferably 0.01 to 1.5 minutes. More preferably, the time is 01 to 0.2 minutes, more preferably 0.03 to 0.2 minutes, and particularly preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulation liquid. In order to suppress evaporation of the lower alcohol, the coagulation liquid may be kept at a low temperature and taken up in an undrawn yarn state. The coagulating liquid tank may be provided in multiple stages, and the stretching may be performed at each stage or a specific stage as required.
　上記の方法で得られた未延伸糸（又は前延伸糸）は、延伸工程を経て延伸糸（混合フィブロイン繊維）とすることができる。延伸方法としては、湿熱延伸、乾熱延伸等をあげることができる。 The undrawn yarn (or predrawn yarn) obtained by the above method can be made into a drawn yarn (mixed fibroin fiber) through a drawing process. Examples of the stretching method include wet heat stretching and dry heat stretching.
　湿熱延伸は、温水中、温水に有機溶剤等を加えた溶液中、スチーム加熱中で行うことができる。温度としては、例えば、５０～９０℃であってよく、７５～８５℃が好ましい。湿熱延伸では、未延伸糸（又は前延伸糸）を、例えば、１～１０倍延伸することができ、２～８倍延伸することが好ましい。 Wet and hot stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, and in steam heating. The temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C. In wet heat drawing, undrawn yarn (or predrawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.
　乾熱延伸は、電気管状炉、乾熱板等を使用して行うことができる。温度としては、例えば、１４０℃～２７０℃であってよく、１６０℃～２３０℃が好ましい。乾熱延伸では、未延伸糸（又は前延伸糸）を、例えば、０．５～８倍延伸することができ、１～４倍延伸することが好ましい。 Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, or the like. The temperature may be, for example, 140 ° C. to 270 ° C., and preferably 160 ° C. to 230 ° C. In dry heat drawing, an undrawn yarn (or predrawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
　湿熱延伸及び乾熱延伸はそれぞれ単独で行ってもよく、またこれらを多段で、又は組み合わせて行ってもよい。すなわち、一段目延伸を湿熱延伸で行い、二段目延伸を乾熱延伸で行う、又は一段目延伸を湿熱延伸行い、二段目延伸を湿熱延伸行い、更に三段目延伸を乾熱延伸で行う等、湿熱延伸及び乾熱延伸を適宜組み合わせて行うことができる。 Wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and the third stage stretching is performed by dry heat stretching. For example, wet heat stretching and dry heat stretching can be appropriately combined.
　延伸工程における最終的な延伸倍率は、未延伸糸（又は前延伸糸）に対して、例えば、５～２０倍であり、６～１１倍であることが好ましい。 The final draw ratio in the drawing step is, for example, 5 to 20 times, preferably 6 to 11 times that of the undrawn yarn (or predrawn yarn).
　本発明に係る混合フィブロインは、延伸して混合フィブロイン繊維とした後、混合フィブロイン繊維内のポリペプチド分子間で化学的に架橋させてもよい。架橋させることができる官能基は、例えば、アミノ基、カルボキシル基、チオール基及びヒドロキシ基等が挙げられる。例えば、ポリペプチドに含まれるリジン側鎖のアミノ基は、グルタミン酸又はアスパラギン酸側鎖のカルボキシル基と脱水縮合によりアミド結合で架橋できる。真空加熱下で脱水縮合反応を行なうことにより架橋してもよいし、カルボジイミド等の脱水縮合剤により架橋させてもよい。 The mixed fibroin according to the present invention may be drawn into a mixed fibroin fiber and then chemically cross-linked between polypeptide molecules in the mixed fibroin fiber. Examples of functional groups that can be crosslinked include amino groups, carboxyl groups, thiol groups, and hydroxy groups. For example, the amino group of the lysine side chain contained in the polypeptide can be crosslinked 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 by a dehydration condensation agent such as carbodiimide.
　ポリペプチド分子間の架橋は、カルボジイミド、グルタルアルデヒド等の架橋剤を用いて行ってもよく、トランスグルタミナーゼ等の酵素を用いて行ってもよい。カルボジイミドは、一般式Ｒ１Ｎ＝Ｃ＝ＮＲ２（但し、Ｒ１及びＲ２は、それぞれ独立に、炭素数１～６のアルキル基、シクロアルキル基を含む有機基を示す。）で示される化合物である。カルボジイミドの具体例として、１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩（ＥＤＣ）、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミド（ＤＣＣ）、１－シクロヘキシル－３－（２－モルホリノエチル）カルボジイミド、ジイソプロピルカルボジイミド（ＤＩＣ）等が挙げられる。これらの中でも、ＥＤＣ及びＤＩＣはポリペプチド分子間のアミド結合形成能が高く、架橋反応し易いことから好ましい。 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 a C 1-6 alkyl group or a cycloalkyl group). A compound. Specific examples of carbodiimide include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N, N′-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide And diisopropylcarbodiimide (DIC). Among these, 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.
　架橋処理は、混合フィブロイン繊維に架橋剤を付与して真空加熱乾燥で架橋するのが好ましい。架橋剤は純品を混合フィブロイン繊維に付与してもよいし、炭素数１～５の低級アルコール及び緩衝液等で０．００５～１０質量％の濃度に希釈したものを混合フィブロイン繊維に付与してもよい。架橋処理は、温度２０～４５℃で３～４２時間行うのが好ましい。架橋処理により、混合フィブロイン繊維に更に高い応力（強度）を付与することができる。 The cross-linking treatment is preferably performed by adding a cross-linking agent to the mixed fibroin fiber and performing cross-linking by vacuum heating and drying. As the cross-linking agent, a pure product may be imparted to the mixed fibroin fiber, or a diluted product of 0.005 to 10% by mass with a lower alcohol having 1 to 5 carbon atoms or a buffer solution may be imparted to the mixed fibroin fiber. May be. The crosslinking treatment is preferably performed at a temperature of 20 to 45 ° C. for 3 to 42 hours. By the crosslinking treatment, a higher stress (strength) can be applied to the mixed fibroin fiber.
（混合フィブロイン繊維の物性評価）
　以下のようにして、混合フィブロイン繊維の物性を測定し、評価することができる。
（ａ）光学顕微鏡を用いて繊維の直径を求める。
（ｂ）温度２０℃、相対湿度６５％の条件で引張り試験機（ＩＮＳＴＲＯＮ３３４２）を用いて繊維の応力、初期弾性率、伸度（破断点変位、変位）、を測定する。引張試験では１０ｍ秒間隔で測定することが好ましい。例えば、混合フィブロイン繊維のサンプルを厚紙で作製した型枠に貼り付け、つかみ具間距離を２０ｍｍ、引張り速度を１０ｍｍ／分として行えばよい。ロードセル容量１０Ｎ、つかみ冶具はクリップ式でよい。測定値は、例えば、サンプル数ｎ＝５の平均値として算出することが好ましい。 (Evaluation of physical properties of mixed fibroin fiber)
The physical properties of the mixed fibroin fiber can be measured and evaluated as follows.
(A) The fiber diameter is determined using an optical microscope.
(B) The fiber stress, initial elastic modulus and elongation (displacement at break, displacement) are measured using a tensile tester (INSTRON 3342) under conditions of a temperature of 20 ° C. and a relative humidity of 65%. In the tensile test, it is preferable to measure at intervals of 10 milliseconds. For example, a mixed fibroin fiber sample may be attached to a mold made of cardboard, and the distance between the grippers may be 20 mm, and the pulling speed may be 10 mm / min. The load cell capacity is 10N, and the gripping jig may be a clip type. The measured value is preferably calculated as an average value of the number of samples n = 5, for example.
（混合フィブロイン繊維の収縮性評価）
　水等との接触によるフィブロイン繊維の長さの変化の例を図３に示す。フィブロイン繊維は、沸点未満の水に接触（湿潤）させることにより収縮する（一次収縮）特性を有する。一次収縮後、乾燥させると更に収縮する（二次収縮）。二次収縮後、再度沸点未満の水に接触させると二次収縮前の長さにまで膨張し、以後乾燥と湿潤を繰り返すと、二次収縮と同程度の幅（図３の「伸縮率」）で、収縮と膨張を繰り返す（図３）。フィブロイン繊維において、このような収縮が少ない程好ましいが、特にフィブロイン繊維からなる織物等の製品においては、この二次収縮が少ないことが好ましい。 (Evaluation of shrinkage of mixed fibroin fiber)
An example of the change in the length of the fibroin fiber due to contact with water or the like is shown in FIG. Fibroin fibers have the property of shrinking (primary shrinkage) by contact (wetting) with water below the boiling point. After the primary shrinkage, when it is dried, it further shrinks (secondary shrinkage). After the secondary shrinkage, when it is again brought into contact with water below the boiling point, it expands to the length before the secondary shrinkage, and after that, when drying and wetting are repeated, the width is about the same as the secondary shrinkage (“stretch rate” in FIG. 3). ), The contraction and the expansion are repeated (FIG. 3). In the fibroin fiber, the smaller the shrinkage, the better. However, in the product such as a woven fabric made of fibroin fiber, the secondary shrinkage is preferably small.
　二次収縮は、以下の方法で求められる二次収縮率を指標として評価することができる。
＜二次収縮率＞
　長さ約３０ｃｍの複数本のフィブロイン繊維を束ね、繊度１５０デニールの繊維束とする。この繊維束に０．８ｇの鉛錘を取り付け、その状態で繊維束を４０℃の水に１０分間浸漬し一次収縮させ、水中で繊維束の長さを測定する。
　一次収縮した繊維束を水中から取り出し、０．８ｇの鉛錘を取り付けたまま室温で２時間おいて乾燥させる。乾燥後、繊維束の長さを測定する。再度、湿潤、乾燥を少なくとも３回繰り返し、湿潤時の平均の長さ（Ｌｗｅｔ）、乾燥時の平均の長さ（Ｌｄｒｙ）を求める。二次収縮率は下記式に従って算出される。
　式：二次収縮率（％）＝（１－（Ｌｄｒｙ／Ｌｗｅｔ））＊１００ Secondary shrinkage can be evaluated using the secondary shrinkage rate obtained by the following method as an index.
<Secondary shrinkage>
A plurality of fibroin fibers having a length of about 30 cm are bundled to form a fiber bundle having a fineness of 150 denier. A 0.8 g lead weight is attached to the fiber bundle, and in this state, the fiber bundle is immersed in water at 40 ° C. for 10 minutes to undergo primary shrinkage, and the length of the fiber bundle is measured in water.
The primary-shrinked 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, wetting and drying are repeated at least three times to determine the average length (Lwet) when wet and the average length (Ldry) when dry. The secondary shrinkage rate is calculated according to the following formula.
Formula: Secondary shrinkage (%) = (1− (Ldry / Lwet)) * 100
　天然由来のフィブロインを紡糸したフィブロイン繊維は、通常、二次収縮率は１１～２０％であるが、本発明に係る第２のフィブロインを単独で紡糸したフィブロイン繊維（例えば、後述の参考例１１）は、二次収縮率が８％以下に低減された繊維である。本実施形態に係る混合フィブロイン繊維は、第２のフィブロインを単独で紡糸したフィブロイン繊維と同等又はより低減された二次収縮率を示す。 A fibroin fiber spun from naturally derived fibroin usually has a secondary shrinkage of 11 to 20%, but a fibroin fiber obtained by spinning the second fibroin according to the present invention alone (for example, Reference Example 11 described later). Is a fiber whose secondary shrinkage is reduced to 8% or less. The mixed fibroin fiber which concerns on this embodiment shows the secondary shrinkage rate equivalent or more reduced than the fibroin fiber which spun only the 2nd fibroin.
〔フィブロインフィルム〕
　本発明に係るフィブロインフィルムは、本発明に係る混合フィブロイン溶液をドープ溶液として使用し、当該ドープ溶液を基材表面にキャスト成形し、乾燥及び／又は脱溶媒することにより得ることができる。 [Fibroin film]
The fibroin film according to the present invention can be obtained by using the mixed fibroin solution according to the present invention as a dope solution, casting the dope solution on a substrate surface, drying and / or removing the solvent.
　ドープ溶液の溶媒としては、フィブロイン溶液の説明で例示した溶媒と同種のものを挙げることができる。また、溶媒としては、ギ酸、ヘキサフルオロ－２－プロパノール（ＨＦＩＰ）、又はジメチルスルホキシド等の極性溶媒が好ましい。ドープ溶液には、必要に応じて無機塩を添加してもよい。無機塩としては、フィブロイン溶液の説明で例示した無機塩と同種のものを挙げることができる。 Examples of the solvent for the dope solution include the same solvents as those exemplified in the description of the fibroin solution. The solvent is preferably a polar solvent such as formic acid, hexafluoro-2-propanol (HFIP), or dimethyl sulfoxide. An inorganic salt may be added to the dope solution as necessary. As an inorganic salt, the same kind as the inorganic salt exemplified in the description of the fibroin solution can be exemplified.
　フィブロインフィルムを形成する際のドープ溶液の粘度は１５～８０ｃＰ（センチポアズ）であることが好ましく、２０～７０ｃＰであることがより好ましい。 The viscosity of the dope solution when forming the fibroin film is preferably 15 to 80 cP (centipoise), more preferably 20 to 70 cP.
　また、ドープ溶液全量を１００質量％としたとき、本発明に係る混合フィブロインの濃度は３～５０質量％であることが好ましく、３．５～３５質量％であることがより好ましく、４．２～１５．８質量％であることがさらに好ましい。 When the total amount of the dope solution is 100% by mass, the concentration of the mixed fibroin according to the present invention is preferably 3 to 50% by mass, more preferably 3.5 to 35% by mass, and 4.2. More preferably, it is ˜15.8% by mass.
　基材は、樹脂基板、ガラス基板、金属基板等であってよい。基材は、キャスト成形後のフィルムを容易に剥離できる観点から、好ましくは樹脂基板である。樹脂基板としては、例えば、ポリエチレンテレフタレート（ＰＥＴ）フィルム、ポリテトラフルオロエチレン等のフッ素樹脂フィルム、ポリプロピレン（ＰＰ）フィルム、又はこれらのフィルム表面にシリコーン化合物を固定化させた剥離フィルムであってよい。基材は、ＨＦＩＰ、ＤＭＳＯ溶媒等に対して安定であり、ドープ溶液を安定してキャスト成形でき、成形後のフィルムを容易に剥離できる観点から、ＰＥＴフィルム又はＰＥＴフィルム表面にシリコーン化合物を固定化させた剥離フィルムであることがより好ましい。 The base material may be a resin substrate, a glass substrate, a metal substrate, or the like. The base material is preferably a resin substrate from the viewpoint that the film after cast molding can be easily peeled off. The resin substrate may be, for example, a polyethylene terephthalate (PET) film, a fluororesin film such as polytetrafluoroethylene, a polypropylene (PP) film, or a release film in which a silicone compound is immobilized on the surface of these films. The base material is stable to HFIP, DMSO solvent, etc., the dope solution can be stably cast-molded, and the silicone compound is immobilized on the PET film or PET film surface from the viewpoint that the film after molding can be easily peeled off. It is more preferable that it is a peeled film.
　具体的な手順を説明すると、まずドープ液を基材表面に流延し、アプリケーター、ナイフコーター、バーコーター等の膜厚制御手段を使用して、所定の厚さ（例えば、乾燥及び／又は脱溶媒後の厚さで１～１０００μｍ）の濡れ膜を作製する。 A specific procedure is explained. First, a dope solution is cast on the surface of a substrate, and a predetermined thickness (for example, drying and / or removing) using a film thickness control means such as an applicator, a knife coater, or a bar coater. A wet film having a thickness of 1 to 1000 μm after the solvent is prepared.
　乾燥及び／又は脱溶媒は、乾式又は湿式で行うことができる。乾式で行う方法としては、真空乾燥、熱風乾燥、風乾等を挙げることができる。湿式で行う方法としては、キャストフィルムを脱溶媒液（凝固液とも言う）に浸漬して溶媒を脱離する方法等を挙げることができる。脱溶媒液として、水、メタノール、エタノール、２－プロパノール等の炭素数１～５の低級アルコール等のアルコール液、水とアルコールとの混合液等を挙げることができる。脱溶媒液（凝固液）の温度は０～９０℃であることが好ましい。 Drying and / or desolvation can be performed dry or wet. Examples of the dry method include vacuum drying, hot air drying, and air drying. Examples of the wet method include a method in which a cast film is immersed in a solvent removal liquid (also referred to as a coagulation liquid) to remove the solvent. Examples of the solvent removal liquid include water, alcohol liquids such as lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and mixed liquids of water and alcohol. The temperature of the solvent removal liquid (coagulation liquid) is preferably 0 to 90 ° C.
　乾燥及び／又は脱溶媒後の未延伸フィルムは、水中で１軸延伸又は２軸延伸することができる。２軸延伸は、逐次延伸でも同時２軸延伸でもよい。２段以上の多段延伸をしてもよい。延伸倍率は、縦、横ともに、好ましくは１．０１～６倍、より好ましくは１．０５～４倍である。この範囲であると応力－歪のバランスがとりやすい。水中延伸は、２０～９０℃の水温で行われることが好ましい。延伸後のフィルムは、５０～２００℃の乾熱で５～６００秒間熱固定することが好ましい。この熱固定により、常温における寸法安定性が得られる。なお、１軸延伸したフィルムは１軸配向フィルムとなり、２軸延伸したフィルムは２軸配向フィルムとなる。 The unstretched film after drying and / or desolvation can be uniaxially stretched or biaxially stretched in water. Biaxial stretching may be sequential stretching or simultaneous biaxial stretching. Multi-stage stretching of two or more stages may be performed. The stretching ratio is preferably 1.01 to 6 times, more preferably 1.05 to 4 times in both length and width. Within this range, it is easy to balance stress-strain. The stretching in water is preferably performed at a water temperature of 20 to 90 ° C. The stretched film is preferably heat-set for 5 to 600 seconds with a dry heat of 50 to 200 ° C. This heat setting provides dimensional stability at room temperature. A uniaxially stretched film becomes a uniaxially oriented film, and a biaxially stretched film becomes a biaxially oriented film.
（フィルムの耐水性評価）
　フィルムの耐水性は、塩類の飽和水溶液を用いた飽和塩法を利用し、高湿度下での吸湿の度合いを測定することにより評価することができる。 (Water resistance evaluation of film)
The water resistance of the film can be evaluated by measuring the degree of moisture absorption under high humidity using a saturated salt method using a saturated aqueous solution of salts.
　塩類としては、硫酸カリウム、塩化カリウム、塩化ナトリウム、臭化ナトリウム、炭酸カリウム、塩化マグネシウム等を挙げることができる。 Examples of salts include potassium sulfate, potassium chloride, sodium chloride, sodium bromide, potassium carbonate, magnesium chloride and the like.
　フィルムの耐水性は、例えば、硫酸カリウムの飽和水溶液を入れたファルコンチューブ等の密閉容器に、適当な大きさに切断したフィルムを、水溶液に浸からないように設置し、例えば、相対湿度９８％のような高湿度で平衡状態にある空気中で、２０～４８時間静置し、フィルムの重量及び水分含量を測定し、重量あたりの水分含量より水分率をもとめることにより、評価することができる。 The water resistance of the film is set, for example, in a sealed container such as a falcon tube containing a saturated aqueous solution of potassium sulfate so that the film cut to an appropriate size is not immersed in the aqueous solution. It can be evaluated by allowing the film to stand for 20 to 48 hours in air at high humidity and in an equilibrium state, measuring the weight and moisture content of the film, and determining the moisture content from the moisture content per weight. .
　以下、実施例に基づいて本発明をより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
〔（１）フィブロインをコードする核酸の合成、及び発現ベクターの構築〕
　第１のフィブロインとして、配列番号１～１０で示されるアミノ酸配列を有するフィブロイン、及び第２のフィブロインとして、配列番号１１～２４で示されるアミノ酸配列を有するフィブロインを設計した。これらのフィブロインは、天然由来のフィブロインであるＮｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ（ＧｅｎＢａｎｋアクセッション番号：Ｐ４６８０４．１、ＧＩ：１１７４４１５）の塩基配列及びアミノ酸配列に基づき、設計したものである。 [(1) Synthesis of nucleic acid encoding fibroin and construction of expression vector]
Fibroin having the amino acid sequence shown by SEQ ID NO: 1 to 10 was designed as the first fibroin, and fibroin having the amino acid sequence shown by SEQ ID NO: 11 to 24 was designed as the second fibroin. These fibroins are designed based on the base sequence and amino acid sequence of Nephila clavies (GenBank accession number: P46804.1, GI: 1174415), which is a fibroin derived from nature.
　配列番号６～１０で示されるアミノ酸配列は、それぞれ配列番号１～５で示されるアミノ酸配列のＮ末端に配列番号２５で示されるアミノ酸配列（タグ配列及びヒンジ配列）を付加したものである。配列番号１８～２４で示されるアミノ酸配列は、それぞれ配列番号１１～１７で示されるアミノ酸配列のＮ末端に配列番号２５で示されるアミノ酸配列（タグ配列及びヒンジ配列）を付加したものである。 The amino acid sequences shown in SEQ ID NOs: 6 to 10 are obtained by adding the amino acid sequence shown in SEQ ID NO: 25 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NOs: 1 to 5, respectively. The amino acid sequences represented by SEQ ID NOs: 18 to 24 are obtained by adding the amino acid sequence represented by SEQ ID NO: 25 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequences represented by SEQ ID NOs: 11 to 17, respectively.
　配列番号１～１０で示されるアミノ酸配列を有するフィブロインは、本発明に係る第１のフィブロインに該当する（表１参照）。配列番号１１～２４で示されるアミノ酸配列を有するフィブロインは、本発明に係る第２のフィブロインに該当する（表３参照）。 The fibroin having the amino acid sequence represented by SEQ ID NOs: 1 to 10 corresponds to the first fibroin according to the present invention (see Table 1). Fibroin having the amino acid sequence shown by SEQ ID NO: 11 to 24 corresponds to the second fibroin according to the present invention (see Table 3).
　配列番号１で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ３９９）は、上記天然由来のフィブロインの（Ａ）ｎモチーフ中のアラニン残基が連続するアミノ酸配列をアラニン残基が連続する数を５つになるよう欠失したアミノ酸配列に対し、Ｎ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフ（（Ａ）５）を欠失させ、更にＣ末端配列の手前に［（Ａ）ｎモチーフ－ＲＥＰ］を１つ挿入したものである。 The amino acid sequence represented by SEQ ID NO: 1 (Met-PRT399) is such that the number of consecutive alanine residues is five in the amino acid sequence in which the alanine residues in the (A) n motif of the naturally derived fibroin are continuous. (A) n motif ((A) 5 ) is deleted from the deleted amino acid sequence every two from the N-terminal side to the C-terminal side, and [(A) n is inserted before the C-terminal sequence. A motif-REP] is inserted.
　配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）は、配列番号１で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ３９９）のＲＥＰ中の全てのＧＧＸをＧＱＸに置換したものである。 The amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) is obtained by replacing all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 1 (Met-PRT399) with GQX.
　配列番号３で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ５８７）は、上記天然由来のフィブロインのアミノ酸配列に対して、生産性の向上を目的としてアミノ酸残基の置換、挿入及び欠失を施したものである。 The amino acid sequence represented by SEQ ID NO: 3 (Met-PRT587) is obtained by performing substitution, insertion and deletion of amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of the natural fibroin. .
　配列番号４で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ７９９）は、上記天然由来のフィブロインのアミノ酸配列に対して、生産性の向上を目的としてアミノ酸残基の置換、挿入及び欠失を施したものである。 The amino acid sequence represented by SEQ ID NO: 4 (Met-PRT799) is obtained by performing substitution, insertion and deletion of amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of the natural fibroin. .
　配列番号５で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４６８）は、配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）の各（Ａ）ｎモチーフのＣ末端側に２つのアラニン残基を挿入し、更に一部のグルタミン（Ｑ）残基をセリン（Ｓ）残基に置換し、配列番号２の分子量とほぼ同じとなるようにＮ末端側の一部のアミノ酸を欠失させたものである。 The amino acid sequence represented by SEQ ID NO: 5 (Met-PRT468) 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: 2 (Met-PRT410), and Some glutamine (Q) residues are substituted with serine (S) residues, and some amino acids on the N-terminal side are deleted so as to be approximately the same as the molecular weight of SEQ ID NO: 2.
　配列番号１１で示されるアミノ酸配列（Ｍ＿ＰＲＴ８８８）は、配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）のＲＥＰ中のＱＱを全てＶＬに置換したものである。 The amino acid sequence represented by SEQ ID NO: 11 (M_PRT888) is obtained by replacing all QQs in REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with VL.
　配列番号１２で示されるアミノ酸配列（Ｍ＿ＰＲＴ９６５）は、配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）のＲＥＰ中のＱＱを全てＴＳに置換し、かつ残りのＱをＡに置換したものである。 The amino acid sequence represented by SEQ ID NO: 12 (M_PRT965) is obtained by substituting all QQs in REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with TS and substituting the remaining Q with A. .
　配列番号１３で示されるアミノ酸配列（Ｍ＿ＰＲＴ８８９）は、配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）のＲＥＰ中のＱＱを全てＶＬに置換し、かつ残りのＱをＩに置換したものである。 The amino acid sequence represented by SEQ ID NO: 13 (M_PRT889) is obtained by replacing all QQs in the REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with VL and replacing the remaining Q with I. .
　配列番号１４で示されるアミノ酸配列（Ｍ＿ＰＲＴ９１６）は、配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）のＲＥＰ中のＱＱを全てＶＩに置換し、かつ残りのＱをＬに置換したものである。 The amino acid sequence represented by SEQ ID NO: 14 (M_PRT916) is obtained by substituting all QQs in REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with VI and replacing the remaining Q with L. .
　配列番号１５で示されるアミノ酸配列（Ｍ＿ＰＲＴ９１８）は、配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）のＲＥＰ中のＱＱを全てＶＦに置換し、かつ残りのＱをＩに置換したものである。 The amino acid sequence represented by SEQ ID NO: 15 (M_PRT918) is obtained by replacing all QQs in the REP of the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410) with VF and replacing the remaining Q with I. .
　配列番号１６で示されるアミノ酸配列（Ｍ＿ＰＲＴ６９９）は、配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）に対し、各（Ａ）ｎモチーフ（Ａ５）のＣ末端側に２つのアラニン残基を挿入し、配列番号２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）の分子量とほぼ同じになるよう、Ｃ末端側のドメイン配列２つを欠失させ、かつグルタミン残基（Ｑ）１３箇所をセリン残基（Ｓ）又はプロリン残基（Ｐ）に置換したアミノ酸配列（Ｍ＿ＰＲＴ５２５：配列番号２６）を得た後、更に配列番号２６で示されるアミノ酸配列（Ｍ＿ＰＲＴ５２５）のＲＥＰ中のＱＱを全てＶＬに置換したものである。 The amino acid sequence represented by SEQ ID NO: 16 (M_PRT699) has two alanine residues on the C-terminal side of each (A) n motif (A 5 ) with respect to the amino acid sequence represented by SEQ ID NO: 2 (Met-PRT410). The two C-terminal domain sequences were deleted so that the molecular weight of the amino acid sequence shown in SEQ ID NO: 2 (Met-PRT410) was approximately the same, and 13 glutamine residues (Q) remained as serine residues. After obtaining the amino acid sequence (M_PRT525: SEQ ID NO: 26) substituted with the group (S) or proline residue (P), QQ in the REP of the amino acid sequence (M_PRT525) represented by SEQ ID NO: 26 is further substituted with VL. It is what.
　配列番号１７で示されるアミノ酸配列（Ｍ＿ＰＲＴ６９８）は、配列番号２６で示されるアミノ酸配列（Ｍ＿ＰＲＴ５２５）のＲＥＰ中のＱＱを全てＶＬに置換し、かつ残りのＱをＩに置換したものである。 The amino acid sequence represented by SEQ ID NO: 17 (M_PRT698) is obtained by replacing all QQs in the REP of the amino acid sequence represented by SEQ ID NO: 26 (M_PRT525) with VL and replacing the remaining Q with I.
　設計した配列番号６～１０及び配列番号１８～２４で示されるアミノ酸配列を有するタンパク質をコードする核酸をそれぞれ合成した。当該核酸には、５’末端にＮｄｅＩサイト、終止コドン下流にＥｃｏＲＩサイトを付加した。これら５種類の核酸をクローニングベクター（ｐＵＣ１１８）にクローニングした。その後、同核酸をＮｄｅＩ及びＥｃｏＲＩで制限酵素処理して切り出した後、タンパク質発現ベクターｐＥＴ－２２ｂ（＋）に組換えて発現ベクターを得た。 The nucleic acids encoding the proteins having the amino acid sequences shown by SEQ ID NOs: 6 to 10 and SEQ ID NOs: 18 to 24 were synthesized. The nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon. These five types of nucleic acids were cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
〔（２）タンパク質の発現〕
　配列番号６～１０及び配列番号１８～２４で示されるアミノ酸配列を有するタンパク質をコードする核酸を含むｐＥＴ２２ｂ（＋）発現ベクターで、大腸菌ＢＬＲ（ＤＥ３）を形質転換した。当該形質転換大腸菌を、アンピシリンを含む２ｍＬのＬＢ培地で１５時間培養した。当該培養液をアンピシリンを含む１００ｍＬのシード培養用培地（表５）にＯＤ６００が０．００５となるように添加した。培養液温度を３０℃に保ち、ＯＤ６００が５になるまでフラスコ培養を行い（約１５時間）、シード培養液を得た。 [(2) Protein expression]
Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequence shown in SEQ ID NO: 6-10 and SEQ ID NO: 18-24. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium (Table 5) containing ampicillin so that the OD 600 was 0.005. The culture temperature was kept at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours) to obtain a seed culture solution.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　当該シード培養液を５００ｍＬの生産培地（表６）を添加したジャーファーメンターにＯＤ６００が０．０５となるように添加して形質転換大腸菌を植菌した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持するようにした。 The seed culture was added to a jar fermenter to which 500 mL of production medium (Table 6) was added so that the OD 600 was 0.05, and transformed E. coli was inoculated. 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.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　生産培地中のグルコースが完全に消費された直後に、フィード液（グルコース４５５ｇ／１Ｌ、Ｙｅａｓｔ　Ｅｘｔｒａｃｔ　１２０ｇ／１Ｌ）を１ｍＬ／分の速度で添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持するようにし、２０時間培養を行った。その後、１Ｍのイソプロピル－β－チオガラクトピラノシド（ＩＰＴＧ）を培養液に対して終濃度１ｍＭになるよう添加し、目的のタンパク質を発現誘導させた。ＩＰＴＧ添加後２０時間経過した時点で、培養液を遠心分離し、菌体を回収した。ＩＰＴＧ添加前とＩＰＴＧ添加後の培養液から調製した菌体を用いてＳＤＳ－ＰＡＧＥを行い、ＩＰＴＧ添加に依存した目的とするタンパク質サイズのバンドの出現により、目的とするタンパク質の発現を確認した。 Immediately after the glucose in the production medium was completely consumed, a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and cultured for 20 hours. Thereafter, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target protein. At the time when 20 hours passed after the addition of IPTG, the culture solution was centrifuged, and the cells were collected. SDS-PAGE was performed using cells prepared from the culture solution before and after the addition of IPTG, and the expression of the target protein was confirmed by the appearance of a band of the desired protein size depending on the addition of IPTG.
〔（３）タンパク質の精製〕
　ＩＰＴＧを添加してから２時間後に回収した菌体を２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｂｕｆｆｅｒ（ｐＨ７．４）で洗浄した。洗浄後の菌体を約１ｍＭのＰＭＳＦを含む２０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）に懸濁させ、高圧ホモジナイザー（ＧＥＡ　Ｎｉｒｏ　Ｓｏａｖｉ社）で細胞を破砕した。破砕した細胞を遠心分離し、沈殿物を得た。得られた沈殿物を、高純度になるまで２０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）で洗浄した。洗浄後の沈殿物を１００ｍｇ／ｍＬの濃度になるように８Ｍ　グアニジン緩衝液（８Ｍグアニジン塩酸塩、１０ｍＭリン酸二水素ナトリウム、２０ｍＭ　ＮａＣｌ、１ｍＭ　Ｔｒｉｓ－ＨＣｌ、ｐＨ７．０）で懸濁し、６０℃で３０分間、スターラーで撹拌し、溶解させた。溶解後、透析チューブ（三光純薬株式会社製のセルロースチューブ３６／３２）を用いて水で透析を行った。透析後に得られた白色の凝集タンパク質を遠心分離により回収し、凍結乾燥機で水分を除き、凍結乾燥粉末を回収した。 [(3) Protein purification]
The cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. 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 (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, the water was removed with a freeze dryer, and the lyophilized powder was recovered.
　得られた凍結乾燥粉末における目的タンパク質の精製度は、粉末のポリアクリルアミドゲル電気泳動の結果をＴｏｔａｌｌａｂ（ｎｏｎｌｉｎｅａｒ　ｄｙｎａｍｉｃｓ　ｌｔｄ．）を用いて画像解析することにより確認した。 The degree of purification of the target protein in the obtained lyophilized powder was confirmed by image analysis of the result of polyacrylamide gel electrophoresis of the powder using Totallab (nonlinear dynamics ltd.).
〔（４）フィブロイン繊維の作製及び収縮性評価〕
　上記で調製した第１のフィブロイン又は第２のフィブロイン単独でフィブロイン繊維を作製し、収縮性を評価した。 [(4) Production of fibroin fiber and evaluation of shrinkage]
Fibroin fibers were produced using the first fibroin prepared above or the second fibroin alone, and the contractility was evaluated.
（４－１）紡糸液（ドープ液）の調製
　４質量％になるように塩化リチウムを溶解したＤＭＳＯを溶媒として用い、上記で調製したＰＲＴ４１０（配列番号７：参考例１）、ＰＲＴ８８８（配列番号１８：参考例２）、ＰＲＴ９６５（配列番号１９：参考例３）、ＰＲＴ８８９（配列番号２０：参考例４）、ＰＲＴ９１６（配列番号２１：参考例５）及びＰＲＴ９１８（配列番号２２：参考例６）タンパク質の凍結乾燥粉末を、それぞれ濃度２４質量％となるように、溶媒に添加した。９０℃のアルミブロックヒーターで１時間溶解させた後、不溶物と泡を取り除き、紡糸液（ドープ液）とした。 (4-1) Preparation of spinning solution (dope solution) PRT410 (SEQ ID NO: 7: Reference Example 1), PRT888 (SEQ ID NO: 1) prepared above using DMSO in which lithium chloride is dissolved to 4% by mass as a solvent. 18: Reference Example 2), PRT965 (SEQ ID NO: 19: Reference Example 3), PRT889 (SEQ ID NO: 20: Reference Example 4), PRT916 (SEQ ID NO: 21: Reference Example 5) and PRT918 (SEQ ID NO: 22: Reference Example 6) Protein freeze-dried powders were added to the solvent to a concentration of 24% by mass, respectively. After dissolving with an aluminum block heater at 90 ° C. for 1 hour, insoluble matters and bubbles were removed to obtain a spinning solution (dope solution).
（４－２）紡糸
　紡糸液をリザーブタンクに充填し、０．１又は０．２ｍｍ径のモノホールノズルからギアポンプを用い１００質量％メタノール凝固浴槽中へ吐出させた。吐出量は０．０１～０．０８ｍＬ／分に調整した。凝固後、１００質量％メタノール洗浄浴槽で洗浄及び延伸を行った。洗浄及び延伸後、乾熱板を用いて乾燥させ、得られた原糸（フィブロイン繊維）を巻き取った。 (4-2) Spinning The spinning solution was filled in a reserve tank and discharged from a monohole nozzle having a diameter of 0.1 or 0.2 mm into a 100 mass% methanol coagulation bath using a gear pump. The discharge rate was adjusted to 0.01 to 0.08 mL / min. After solidification, washing and stretching were performed in a 100 mass% methanol washing bath. After washing and drawing, it was dried using a dry heat plate, and the obtained raw yarn (fibroin fiber) was wound up.
（４－３）フィブロイン繊維の収縮性評価
　得られた原糸を長さ約３０ｃｍに揃えて、束ね、繊度１５０デニールのフィブロイン繊維束とした。各フィブロイン繊維束に０．８ｇの鉛錘を取り付け、その状態でフィブロイン繊維束を４０℃の水に１０分間浸漬して一次収縮させ、水中でフィブロイン繊維束の長さを測定した。一次収縮したフィブロイン繊維束を水中から取り出し、０．８ｇの鉛錘を取り付けたまま、室温で２時間おいて乾燥させた。乾燥後、各フィブロイン繊維束の長さを測定した。この湿潤及び乾燥の操作を３回繰り返し、湿潤時の平均の長さ（Ｌｗｅｔ：単位ｃｍ）、乾燥時の平均の長さ（Ｌｄｒｙ：単位ｃｍ）を求め、下記式に従って二次収縮率を算出した。結果を表７に示す。
　二次収縮率（％）＝｛１－（Ｌｄｒｙ／Ｌｗｅｔ）｝×１００
Figure JPOXMLDOC01-appb-T000007
 (4-3) Evaluation of shrinkage of fibroin fiber The obtained raw yarn was aligned to a length of about 30 cm and bundled into a fibroin fiber bundle having a fineness of 150 denier. A 0.8 g lead weight was attached to each fibroin fiber bundle, and in that state, the fibroin fiber bundle was immersed in water at 40 ° C. for 10 minutes to cause primary shrinkage, and the length of the fibroin fiber bundle was measured in water. The primary contracted fibroin fiber bundle was 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 each fibroin fiber bundle was measured. This wet and dry operation is repeated three times to determine the average length when wet (Lwet: unit cm) and the average length when dry (Ldry: unit cm), and calculate the secondary shrinkage rate according to the following formula. did. The results are shown in Table 7.
Secondary shrinkage (%) = {1- (Ldry / Lwet)} × 100
Figure JPOXMLDOC01-appb-T000007
　ＰＲＴ４１０（参考例１）タンパク質で紡糸した繊維は、１２％の二次収縮率を示したが、ドメイン内のグルタミン残基（Ｑ）を別のアミノ酸残基に置換することにより、グルタミン残基含有率を低減させた（６．３％）ＰＲＴ８８８（配列番号１８）では、顕著な二次収縮率の低減効果が認められた（参考例２）。この二次収縮率の低減効果は、グルタミン残基含有率を更に低下（０％）させることにより、ＲＥＰの疎水性度をＰＲＴ８８８のように高めなくても認められた（参考例３）。また、グルタミン残基含有率を更に低下（０％）させると共に、さらに疎水性度の高いアミノ酸残基に置換することにより、二次収縮率の低減効果がより顕著に認められた（参考例４～６）。 The fiber spun with PRT410 (Reference Example 1) protein showed a secondary contraction rate of 12%, but it contained a glutamine residue by replacing the glutamine residue (Q) in the domain with another amino acid residue. In PRT888 (SEQ ID NO: 18) with a reduced rate (6.3%), a remarkable effect of reducing the secondary contraction rate was observed (Reference Example 2). This effect of reducing the secondary contraction rate was recognized without further increasing the hydrophobicity of REP as in PRT888 by further reducing (0%) the glutamine residue content (Reference Example 3). In addition, the content of glutamine residues was further reduced (0%), and the effect of reducing the secondary contraction rate was more remarkably observed by substituting amino acid residues with higher hydrophobicity (Reference Example 4). To 6).
〔（５）フィブロインフィルムの作製及び耐水性評価〕
　上記で調製した第１のフィブロイン又は第２のフィブロイン単独でフィブロインフィルムを作製し、耐水性を評価した。 [(5) Production of fibroin film and evaluation of water resistance]
A fibroin film was produced using the first fibroin prepared above or the second fibroin alone, and the water resistance was evaluated.
（５－１）フィルム作製用ドープ液の調製
　ＰＲＴ４１０（配列番号７：参考例７）、ＰＲＴ６９９（配列番号２３：参考例８）及びＰＲＴ６９８（配列番号２４：参考例９）タンパク質の凍結乾燥粉末を９９％ヘキサフルオロ－２－プロパノール（ＨＦＩＰ）に濃度１０質量％となるように添加し、５５℃、４００ｒｐｍの条件で２０分間振盪し、溶解させ、ドープ液とした。 (5-1) Preparation of Dope Solution for Film Production PRT410 (SEQ ID NO: 7: Reference Example 7), PRT699 (SEQ ID NO: 23: Reference Example 8) and PRT698 (SEQ ID NO: 24: Reference Example 9) It was added to 99% hexafluoro-2-propanol (HFIP) to a concentration of 10% by mass, shaken for 20 minutes at 55 ° C. and 400 rpm, and dissolved to obtain a dope solution.
（５－２）フィルムキャスト成形
　厚さ７５μｍのポリエチレンテレフタレート（ＰＥＴ）フィルム表面にシリコーン化合物を固定化させた離形フィルム（三井化学東セロ株式会社製、商品番号“ＳＰ－ＰＥＴ－０１－７５－ＢＵ”）を基板として使用した。バッチ式塗工機（井元製作所製）を使用して、送り速度２０ｍｍ／秒、スリット幅０．１８ｍｍの条件で、上記で調製したドープ液を基板の表明にキャスト成形し、濡れ膜を作製した。 (5-2) Film Cast Molding A release film in which a silicone compound is fixed on the surface of a 75 μm thick polyethylene terephthalate (PET) film (manufactured by Mitsui Chemical Tosero Co., Ltd., product number “SP-PET-01-75-BU”). )) Was used as the substrate. Using a batch type coating machine (manufactured by Imoto Seisakusho), the dope solution prepared above was cast and formed on the substrate surface under the conditions of a feed rate of 20 mm / second and a slit width of 0.18 mm, to prepare a wet film. .
（５－３）乾燥及び脱溶媒
　成形した濡れ膜を、５５℃の恒温槽（ｅｓｐｅｃ社製）中で１２時間静置し、乾燥した。その後、乾燥フィルムを基板から剥離し、メタノールに１２時間浸漬した。浸漬後、再度６０℃の恒温槽（ｅｓｐｅｃ社製）中で１２時間静置し、乾燥した。得られたフィルムを３０ｍｍ角に切断し、以下の耐水性評価を行った。 (5-3) Drying and desolvation The molded wet film was allowed to stand for 12 hours in a constant temperature bath (manufactured by espec) at 55 ° C. and dried. Thereafter, the dried film was peeled off from the substrate and immersed in methanol for 12 hours. After immersion, it was allowed to stand again in a constant temperature bath (manufactured by espec) for 60 hours and dried. The obtained film was cut into 30 mm squares and subjected to the following water resistance evaluation.
（５－４）フィルムの耐水性評価〕
　硫酸カリウム（Ｋ２ＳＯ４・Ｈ２Ｏ）の飽和水溶液を入れたファルコンチューブに、３０ｍｍ角に切断したフィルムを、水溶液に浸からないように設置し、９８％の高湿度下で４８時間静置した。静置後のフィルムの水分含量を、カールフィッシャー（京都電子工業株式会社製）で吸湿の程度を測定することで水分率（％）として求めた。結果を表８に示す。
Figure JPOXMLDOC01-appb-T000008
 (5-4) Water resistance evaluation of film]
A film cut to 30 mm square was placed on a falcon tube containing a saturated aqueous solution of potassium sulfate (K 2 SO 4 .H 2 O) so as not to be immersed in the aqueous solution, and allowed to stand for 48 hours at 98% high humidity. I put it. The moisture content of the film after standing was determined as a moisture content (%) by measuring the degree of moisture absorption with Karl Fischer (Kyoto Electronics Industry Co., Ltd.). The results are shown in Table 8.
Figure JPOXMLDOC01-appb-T000008
　グルタミン残基含有率の低下に伴い、フィルムの吸水性が低下することが確認され、耐水性が向上することが分かった。 It was confirmed that the water absorption of the film was reduced as the glutamine residue content was reduced, and the water resistance was improved.
〔（６）フィブロイン繊維の作製及び物性評価〕
　上記で調製した第１のフィブロイン及び第２のフィブロイン単独又は混合して、フィブロイン繊維を作製し、物性を評価した。 [(6) Preparation of fibroin fiber and evaluation of physical properties]
Fibroin fibers were prepared by mixing the first fibroin prepared above and the second fibroin alone or mixed, and the physical properties were evaluated.
（６－１）紡糸液（ドープ液）の調製
　ギ酸（９８％）を溶媒として用い、第１のフィブロインとして上記で調製したＰＲＴ７９９（配列番号９）タンパク質、及び第２のフィブロインとして上記で調製したＰＲＴ９１８（配列番号２２）タンパク質の凍結乾燥粉末を、それぞれ以下に示す比率で、総濃度が３０質量％となるように溶媒に添加した。７０℃のアルミブロックヒーターで１時間加温し、溶解させた後、不溶物と泡を取り除き、紡糸液（ドープ液）とした。
　ＰＲＴ７９９：ＰＲＴ９１８＝１０：０（参考例１０）
　ＰＲＴ７９９：ＰＲＴ９１８＝７：３（実施例１）
　ＰＲＴ７９９：ＰＲＴ９１８＝５：５（実施例２）
　ＰＲＴ７９９：ＰＲＴ９１８＝７：３（実施例３）
　ＰＲＴ７９９：ＰＲＴ９１８＝０：１０（参考例１１） (6-1) Preparation of spinning solution (dope solution) Formic acid (98%) was used as a solvent, PRT799 (SEQ ID NO: 9) protein prepared above as the first fibroin, and the above prepared as the second fibroin. The lyophilized powder of PRT918 (SEQ ID NO: 22) protein was added to the solvent in the following ratios so that the total concentration was 30% by mass. After heating and dissolving with a 70 ° C. aluminum block heater for 1 hour, insoluble matters and bubbles were removed to obtain a spinning solution (dope solution).
PRT799: PRT918 = 10: 0 (Reference Example 10)
PRT799: PRT918 = 7: 3 (Example 1)
PRT799: PRT918 = 5: 5 (Example 2)
PRT799: PRT918 = 7: 3 (Example 3)
PRT799: PRT918 = 0: 10 (Reference Example 11)
（６－２）紡糸
　紡糸液をリザーブタンクに充填し、０．１又は０．２ｍｍ径のモノホールノズルからギアポンプを用い１００質量％メタノール凝固浴槽中へ吐出させた。吐出量は０．０１～０．０８ｍＬ／分に調整した。凝固後、１００質量％メタノール洗浄浴槽で洗浄及び延伸を行った。洗浄及び延伸後、乾熱板を用いて乾燥させ、得られた原糸（フィブロイン繊維）を卓上の紡糸装置を用いて巻き取った。 (6-2) Spinning The spinning solution was filled in a reserve tank and discharged from a monohole nozzle having a diameter of 0.1 or 0.2 mm into a 100 mass% methanol coagulation bath using a gear pump. The discharge rate was adjusted to 0.01 to 0.08 mL / min. After solidification, washing and stretching were performed in a 100 mass% methanol washing bath. After washing and drawing, it was dried using a dry heat plate, and the obtained raw yarn (fibroin fiber) was wound up using a table-top spinning device.
（６－３）フィブロイン繊維の収縮性評価
　（６－２）で得られた原糸を長さ約３０ｃｍに揃えて、束ね、繊度１５０デニールのフィブロイン繊維束とした。各フィブロイン繊維束に０．８ｇの鉛錘を取り付け、その状態でフィブロイン繊維束を４０℃の水に１０分間浸漬して一次収縮させ、水中でフィブロイン繊維束の長さを測定した。一次収縮したフィブロイン繊維束を水中から取り出し、０．８ｇの鉛錘を取り付けたまま、室温で２時間おいて乾燥させた。乾燥後、各フィブロイン繊維束の長さを測定した。この湿潤及び乾燥の操作を３回繰り返し、湿潤時の平均の長さ（Ｌｗｅｔ：単位ｃｍ）、乾燥時の平均の長さ（Ｌｄｒｙ：単位ｃｍ）を求め、下記式に従って二次収縮率を算出した。
　二次収縮率（％）＝｛１－（Ｌｄｒｙ／Ｌｗｅｔ）｝×１００ (6-3) Evaluation of shrinkage of fibroin fiber The raw yarn obtained in (6-2) was aligned to a length of about 30 cm and bundled into a fibroin fiber bundle having a fineness of 150 denier. A 0.8 g lead weight was attached to each fibroin fiber bundle, and in that state, the fibroin fiber bundle was immersed in water at 40 ° C. for 10 minutes to cause primary shrinkage, and the length of the fibroin fiber bundle was measured in water. The primary contracted fibroin fiber bundle was 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 each fibroin fiber bundle was measured. This wet and dry operation is repeated three times to determine the average length when wet (Lwet: unit cm) and the average length when dry (Ldry: unit cm), and calculate the secondary shrinkage rate according to the following formula. did.
Secondary shrinkage (%) = {1- (Ldry / Lwet)} × 100
　各実施例及び参考例のフィブロイン繊維の二次収縮率を表９に示す。表９に示す二次収縮率は、参考例１０（ＰＲＴ７９９単独）のフィブロイン繊維の二次収縮率の値を１００としたときの相対値である。 Table 9 shows the secondary shrinkage rates of the fibroin fibers of the examples and reference examples. The secondary shrinkage shown in Table 9 is a relative value when the secondary shrinkage of the fibroin fiber of Reference Example 10 (PRT799 alone) is 100.
　ＰＲＴ７９９に対してＰＲＴ９１８を等量以下の割合で混合した混合フィブロイン繊維（実施例１）は、ＰＲＴ９１８単独のフィブロイン繊維（参考例１１）と比べて、二次収縮率の低減効果に加えて、応力及び伸度に優れており、予想されない秀逸した結果を得ることができた。また、ＰＲＴ７９９単独のフィブロイン繊維（参考例１０）を基準に見ると、ＰＲＴ９１８を混合した混合フィブロイン繊維は、ＰＲＴ９１８の割合が高いほど、応力及び伸度を有するＰＲＴ７９９単独のフィブロイン繊維に対して、更に二次収縮率の低減効果を付与可能であることが認められた（実施例１～３）。 The mixed fibroin fiber (Example 1) in which PRT918 was mixed at an equal ratio or less with respect to PRT799, compared to the fibroin fiber of PRT918 alone (Reference Example 11), in addition to the effect of reducing the secondary shrinkage rate, the stress Moreover, it was excellent in elongation, and an excellent and unexpected result could be obtained. Further, when looking at the fibroin fiber of PRT799 alone (Reference Example 10), the mixed fibroin fiber mixed with PRT918 is more in proportion to the fibroin fiber of PRT799 alone having higher stress and elongation as the proportion of PRT918 is higher. It was confirmed that the secondary shrinkage reduction effect could be imparted (Examples 1 to 3).
（６－４）フィブロイン繊維の物性測定による評価
　（６－２）で得られた原糸の物性を以下の方法で測定した。
（ａ）光学顕微鏡を用いてフィブロイン繊維の直径を求めた。
（ｂ）温度２０℃、相対湿度６５％の条件で引張り試験機（ＩＮＳＴＲＯＮ３３４２）を用いてフィブロイン繊維の応力、初期弾性率、伸度（破断点変位、変位）を測定した。引張試験では１０ｍ秒間隔で測定した。各サンプルは厚紙で作製した型枠に貼り付け、つかみ治具間距離は２０ｍｍ、引張り速度は１０ｍｍ／分とした。ロードセル容量１０Ｎ、つかみ冶具はクリップ式とした。測定値はサンプル数ｎ＝５の平均値とした。 (6-4) Evaluation by physical property measurement of fibroin fiber The physical property of the raw yarn obtained in (6-2) was measured by the following method.
(A) The diameter of the fibroin fiber was determined using an optical microscope.
(B) The stress, initial elastic modulus, and elongation (displacement at break) of the fibroin fiber were measured using a tensile tester (INSTRON 3342) under the conditions of a temperature of 20 ° C. and a relative humidity of 65%. In the tensile test, measurements were made at 10 msec intervals. Each sample was attached to a mold made of cardboard, the distance between the gripping jigs was 20 mm, and the pulling speed was 10 mm / min. The load cell capacity was 10N, and the gripping jig was a clip type. The measured value was an average value of the number of samples n = 5.
　各原糸の応力及び伸度を測定した結果を表９に示す。表９に示す応力及び伸度は、参考例１０（ＰＲＴ７９９単独）のフィブロイン繊維の応力及び伸度の値を１００としたときの相対値である。 The results of measuring the stress and elongation of each raw yarn are shown in Table 9. The stress and elongation shown in Table 9 are relative values when the value of stress and elongation of the fibroin fiber of Reference Example 10 (PRT799 alone) is 100.
　応力に関して、ＰＲＴ７９９に対してＰＲＴ９１８を等量以下の割合で混合した混合フィブロイン繊維（実施例１）は、ＰＲＴ７９９単独のフィブロイン繊維（参考例１０）と比べて、より応力が向上するという効果が認められ、予想されない秀逸した結果を得ることができた。また、ＰＲＴ９１８単独のフィブロイン繊維（参考例１１）を基準に見ると、ＰＲＴ７９９を混合した混合フィブロイン繊維は、ＰＲＴ７９９の割合が高いほど、応力を向上させることが可能であることが認められた（実施例１～３）。 Regarding the stress, the mixed fibroin fiber (Example 1) in which PRT918 was mixed with PRT799 at a ratio equal to or less than that of PRT799 has an effect that the stress is further improved as compared with the fibroin fiber of PRT799 alone (Reference Example 10). The result was an unexpected and excellent result. Further, when the fibroin fiber of PRT918 alone (Reference Example 11) was seen as a reference, it was found that the mixed fibroin fiber mixed with PRT799 can improve the stress as the proportion of PRT799 increases (implementation). Examples 1 to 3).
　伸度に関して、ＰＲＴ７９９に対してＰＲＴ９１８を等量以下の割合で混合した混合フィブロイン繊維（実施例１）は、ＰＲＴ７９９単独のフィブロイン繊維（参考例１０）と比べて、より伸度が向上するという効果が認められ、予想されない秀逸した結果を得ることができた。また、ＰＲＴ９１８単独のフィブロイン繊維（参考例１１）を基準に見ると、ＰＲＴ７９９を混合した混合フィブロイン繊維は、ＰＲＴ７９９の割合が高いほど、伸度を向上させることが可能であることが認められた（実施例１～３）。
Figure JPOXMLDOC01-appb-T000009
 Regarding the elongation, the mixed fibroin fiber (Example 1) obtained by mixing PRT918 at a ratio equal to or less than that of PRT799 has an effect that the elongation is further improved as compared with the fibroin fiber of PRT799 alone (Reference Example 10). Was recognized, and we were able to obtain unexpected and excellent results. Moreover, when the fibrin fiber of PRT918 alone (Reference Example 11) was seen as a reference, it was confirmed that the mixed fibroin fiber mixed with PRT799 can improve the elongation as the proportion of PRT799 increases ( Examples 1 to 3).
Figure JPOXMLDOC01-appb-T000009
　伸度及び二次収縮率低減効果（耐水性）を重視した配合である、ＰＲＴ７９９とＰＲＴ９１８を等量混合したドープ液を用い、直径０．１ｍｍのノズルを有するマルチホールノズルを用いて実機で紡糸した（実施例４）。比較として、ＰＲＴ９１８単独で同様に紡糸した（参考例１２）。得られた混合フィブロイン繊維（実施例４）及びフィブロイン繊維（参考例１２）の二次収縮率、応力及び伸度を上述した方法により測定した。 Spinning with an actual machine using a multi-hole nozzle having a nozzle with a diameter of 0.1 mm, using a dope solution in which PRT799 and PRT918 are mixed in equal amounts, which is a blend that emphasizes elongation and secondary shrinkage reduction effect (water resistance) (Example 4). As a comparison, PRT918 alone was similarly spun (Reference Example 12). The secondary shrinkage, stress and elongation of the obtained mixed fibroin fiber (Example 4) and fibroin fiber (Reference Example 12) were measured by the methods described above.
　表１０に二次収縮率、応力及び伸度の測定結果を示す。表１０に示す二次収縮率、応力及び伸度は、ＰＲＴ９１８単独のフィブロイン繊維（参考例１２）の値を１００としたときの相対値である。 Table 10 shows the measurement results of the secondary shrinkage rate, stress and elongation. The secondary shrinkage rate, stress, and elongation shown in Table 10 are relative values when the value of the fibroin fiber (Reference Example 12) of PRT918 alone is 100.
　二次収縮率、応力及び伸度いずれも、卓上の紡糸装置で得られた結果と同等の結果が得られた。
Figure JPOXMLDOC01-appb-T000010
 The secondary shrinkage, stress, and elongation were all the same as those obtained with the tabletop spinning apparatus.
Figure JPOXMLDOC01-appb-T000010

Claims (16)

  1. 　第１のフィブロインと、第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインと、を含むフィブロイン組成物であって、
    　前記第１のフィブロインが、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ１で表されるドメイン配列を含み、Ｎ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる前記隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、前記ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが５０％以上であるアミノ酸配列を有するフィブロインであり、
    　前記第２のフィブロインが、式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ２で表されるドメイン配列又は式３：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ３－（Ａ）ｎモチーフで表されるドメイン配列を含み、グルタミン残基含有率が９％以下であるアミノ酸配列を有するフィブロインであり、
    　前記第３のフィブロインが、式４：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ４で表されるドメイン配列を含み、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｚとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｗとしたときに、ｚ／ｗが６．２％以上であるアミノ酸配列を有するフィブロインである、フィブロイン組成物。
    ［式１、式２、式３及び式４中、（Ａ）ｎモチーフは、それぞれ独立に、４～２７アミノ酸残基から構成されるアミノ酸配列を示し、かつ（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が８０％以上であり、ＲＥＰは、それぞれ独立に、１０～２００アミノ酸残基から構成されるアミノ酸配列を示し、ｍ１、ｍ２、ｍ３及びｍ４は、それぞれ独立に、８～３００の整数を示す。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。］     A fibroin composition comprising: a first fibroin; and at least one fibroin selected from the group consisting of a second fibroin and a third fibroin,
    Said first fibroin, formula 1: comprising a domain sequence represented by [(A) n motif -rep] m1, toward the N-terminus to the C-terminal side, two [(A) n motif adjacent -REP] When the number of amino acid residues in the REP of the unit is sequentially compared, and the number of amino acid residues in the REP with a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to The maximum total value of the total number of amino acid residues of the two adjacent [(A) n motif-REP] units that becomes 11.3 is x, and the total number of amino acid residues of the domain sequence is y. A fibroin having an amino acid sequence in which x / y is 50% or more,
    The second fibroin is represented by a domain sequence represented by Formula 2: [(A) n motif-REP] m2 or Formula 3: [(A) n motif-REP] m3- (A) n motif A fibroin having an amino acid sequence comprising a domain sequence and having a glutamine residue content of 9% or less;
    The third fibroin contains a domain sequence represented by Formula 4: [(A) n motif-REP] m4 , and is located on the most C-terminal side (A) Sequence from the n motif to the C terminus of the domain sequence Z is the total number of amino acid residues contained in a region in which the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more in all REPs included in the sequence excluding from the domain sequence. (A) z / w is 6.2% or more when the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence is w A fibroin composition, which is a fibroin having an amino acid sequence of
    [In Formula 1, Formula 2, Formula 3 and Formula 4, (A) n motif independently represents an amino acid sequence composed of 4 to 27 amino acid residues, and (A) all amino acids in n motif The number of alanine residues with respect to the number of residues is 80% or more, REP independently represents an amino acid sequence composed of 10 to 200 amino acid residues, m1, m2, m3 and m4 are each independently An integer from 8 to 300 is indicated. 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. ]
  2. 　前記第２のフィブロインのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１若しくは複数のグルタミン残基を欠失したこと、又は他のアミノ酸残基に置換したことに相当する、グルタミン残基の含有量が低減されたアミノ酸配列である、請求項１に記載のフィブロイン組成物。 Glutamine, wherein the domain sequence of the second fibroin corresponds to deletion of one or more glutamine residues in REP or substitution with other amino acid residues as compared to naturally occurring fibroin The fibroin composition according to claim 1, which is an amino acid sequence having a reduced content of residues.
  3. 　前記第２のフィブロインにおける前記他のアミノ酸残基が、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）アラニン（Ａ）、グリシン（Ｇ）、スレオニン（Ｔ）、セリン（Ｓ）、トリプトファン（Ｗ）、チロシン（Ｙ）、プロリン（Ｐ）及びヒスチジン（Ｈ）からなる群より選択されるアミノ酸残基である、請求項１又は２に記載のフィブロイン組成物。 The other amino acid residues in the second fibroin are isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) alanine (A), glycine ( The amino acid residue selected from the group consisting of G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). A fibroin composition according to claim 1.
  4. 　前記第２のフィブロインは、ＲＥＰ中にＧＰＧＸＸ（但し、Ｘはグリシン残基以外のアミノ酸残基を示す。）モチーフを含み、ＧＰＧＸＸモチーフ含有率が１０％以上である、請求項１～３のいずれか一項に記載のフィブロイン組成物。 The second fibroin contains a GPGXX (where X represents an amino acid residue other than a glycine residue) motif in REP, and has a GPGXX motif content of 10% or more. A fibroin composition according to claim 1.
  5. 　前記第２のフィブロインは、ＲＥＰの疎水性度が－０．８以上である、請求項１～４のいずれか一項に記載のフィブロイン組成物。 The fibroin composition according to any one of claims 1 to 4, wherein the second fibroin has a hydrophobicity of REP of -0.8 or more.
  6. 　前記第１のフィブロインは、配列番号１～１０のいずれかで示されるアミノ酸配列、又は配列番号１～１０のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、請求項１～５のいずれか一項に記載のフィブロイン組成物。 The first fibroin comprises an amino acid sequence represented by any of SEQ ID NOs: 1 to 10, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 10. The fibroin composition according to any one of claims 1 to 5.
  7. 　前記第２のフィブロインは、配列番号１１～２４のいずれかで示されるアミノ酸配列、又は配列番号１１～２４のいずれかで示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、請求項１～６のいずれか一項に記載のフィブロイン組成物。 The second fibroin comprises an amino acid sequence represented by any of SEQ ID NOs: 11 to 24, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 11 to 24. The fibroin composition according to any one of claims 1 to 6.
  8. 　請求項１～７のいずれか一項に記載のフィブロイン組成物を含み、
    　繊維、糸、フィルム、発泡体、球体、ナノフィブリル、ゲル及び樹脂からなる群から選択される製品。     Comprising a fibroin composition according to any one of claims 1 to 7,
    A product selected from the group consisting of fibers, yarns, films, foams, spheres, nanofibrils, gels and resins.
  9. 　請求項１～７のいずれか一項に記載のフィブロイン組成物が、溶媒に溶解してなる、フィブロイン溶液。 A fibroin solution obtained by dissolving the fibroin composition according to any one of claims 1 to 7 in a solvent.
  10. 　前記溶媒が、ヘキサフルオロイソプロパノール（ＨＦＩＰ）、ヘキサフルオロアセトン（ＨＦＡ）、ジメチルスルホキシド（ＤＭＳＯ）、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）、ギ酸、尿素、グアニジン、ドデシル硫酸ナトリウム（ＳＤＳ）、臭化リチウム、塩化カルシウム及びチオシアン酸リチウム、並びにこれら２種以上の混合溶媒からなる群より選ばれる溶媒である、請求項９に記載のフィブロイン溶液。 The solvent is hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide The fibroin solution according to claim 9, which is a solvent selected from the group consisting of calcium chloride and lithium thiocyanate, and a mixed solvent of two or more of these.
  11. 　前記第１のフィブロインと、前記第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインとの含有比率が、重量基準で９．９：０．１～５．０：５．０である、請求項９又は１０に記載のフィブロイン溶液。 The content ratio of the first fibroin and at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 9.9: 0.1 to 5.0: on a weight basis. The fibroin solution according to claim 9 or 10, which is 5.0.
  12. 　前記第１のフィブロインと、前記第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインとの含有比率が、重量基準で５．０：５．０～０．１：９．９である、請求項９又は１０に記載のフィブロイン溶液。 The content ratio of the first fibroin to at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 5.0: 5.0 to 0.1: The fibroin solution according to claim 9 or 10, which is 9.9.
  13. 　前記第１のフィブロインと、前記第２のフィブロイン及び第３のフィブロインからなる群より選択される少なくとも１種のフィブロインとの含有比率が、重量基準で５．０：５．０である、請求項９又は１０に記載のフィブロイン溶液。 The content ratio of the first fibroin and at least one fibroin selected from the group consisting of the second fibroin and the third fibroin is 5.0: 5.0 on a weight basis. The fibroin solution according to 9 or 10.
  14. 　請求項９～１３のいずれか一項に記載のフィブロイン溶液を使用したフィブロイン繊維の製造方法であって、
    　前記フィブロイン溶液をドープ液とし、
    　前記ドープ液を口金から凝固液に押し出し、未延伸糸を得る工程を含む、フィブロイン繊維の製造方法。     A method for producing fibroin fiber using the fibroin solution according to any one of claims 9 to 13,
    The fibroin solution as a dope solution,
    A method for producing fibroin fibers, comprising a step of extruding the dope solution from a die into a coagulation solution to obtain an undrawn yarn.
  15. 　前記未延伸糸を延伸する工程を更に含む、請求項１４に記載のフィブロイン繊維の製造方法。 The method for producing fibroin fiber according to claim 14, further comprising a step of drawing the undrawn yarn.
  16. 　前記凝固液が、メタノール、エタノール及び２－プロパノールからなる群より選ばれる少なくとも１種である、請求項１４又は１５に記載のフィブロイン繊維の製造方法。 The method for producing a fibroin fiber according to claim 14 or 15, wherein the coagulation liquid is at least one selected from the group consisting of methanol, ethanol and 2-propanol.
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