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WO2019151424A1 - Composition de fibroïne, solution de fibroïne, et procédé de fabrication de fibres de fibroïne - Google Patents

Composition de fibroïne, solution de fibroïne, et procédé de fabrication de fibres de fibroïne Download PDF

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Publication number
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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Japanese (ja)
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佑之介 安部
翔太 冨樫
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Spiber Inc
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Spiber Inc
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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

Definitions

  • 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.

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Abstract

L'invention concerne une composition de fibroïne qui contient une première fibroïne, et au moins une sorte de fibroïne choisie dans un groupe constitué d'une seconde et d'une troisième fibroïne. La première fibroïne possède une séquence de domaine dans laquelle la teneur en motif （Ａ）ｎ est réduite. La seconde fibroïne possède une séquence de domaine dans laquelle la teneur en résidu de glutamine est réduite. La troisième fibroïne possède une séquence de domaine contenant une région d'indice hydrophobe localement important.
PCT/JP2019/003456 2018-01-31 2019-01-31 Composition de fibroïne, solution de fibroïne, et procédé de fabrication de fibres de fibroïne Ceased WO2019151424A1 (fr)

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WO2021065812A1 (fr) * 2019-09-30 2021-04-08 Ｓｐｉｂｅｒ株式会社 Liquide dopant et procédé de production d'un article moulé en fibroïne modifiée l'utilisant
WO2021065851A1 (fr) * 2019-09-30 2021-04-08 Ｓｐｉｂｅｒ株式会社 Agent d'ajustement de la rétention d'eau pour cheveux artificiels et procédé d'ajustement de la rétention d'eau
CN114502782A (zh) * 2019-09-30 2022-05-13 丝芭博株式会社 一种接触冷感性及吸水速干性赋予剂、以及赋予物品接触冷感性及吸水速干性的方法
CN116538037A (zh) * 2023-05-06 2023-08-04 之江实验室 基于丝素蛋白的多响应性软体致动器、制备和调控方法

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WO2014002605A1 (fr) * 2012-06-28 2014-01-03 スパイバー株式会社 Fibre de protéine teintée dans la masse et procédé pour produire celle-ci
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WO2014002605A1 (fr) * 2012-06-28 2014-01-03 スパイバー株式会社 Fibre de protéine teintée dans la masse et procédé pour produire celle-ci
WO2017188434A1 (fr) * 2016-04-28 2017-11-02 Ｓｐｉｂｅｒ株式会社 Fibroïne modifiée
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021065812A1 (fr) * 2019-09-30 2021-04-08 Ｓｐｉｂｅｒ株式会社 Liquide dopant et procédé de production d'un article moulé en fibroïne modifiée l'utilisant
WO2021065851A1 (fr) * 2019-09-30 2021-04-08 Ｓｐｉｂｅｒ株式会社 Agent d'ajustement de la rétention d'eau pour cheveux artificiels et procédé d'ajustement de la rétention d'eau
CN114502782A (zh) * 2019-09-30 2022-05-13 丝芭博株式会社 一种接触冷感性及吸水速干性赋予剂、以及赋予物品接触冷感性及吸水速干性的方法
CN116538037A (zh) * 2023-05-06 2023-08-04 之江实验室 基于丝素蛋白的多响应性软体致动器、制备和调控方法

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