JP2023007506A - Fiber for artificial hair and manufacturing method therefor - Google Patents

Abstract

An object of the present invention is to provide an artificial hair fiber having a natural luster and suppressed reduction in strength, and a method for producing the same. The artificial hair fiber contains a recombinant structural protein and luster-suppressing particles incompatible with the recombinant structural protein, and the content of the luster-suppressing particles per 100 parts by mass of the recombinant structural protein is: It is more than 1 part by mass and 12 parts by mass or less, and the gloss suppressing particles are organic polymer particles or metal oxide particles having an average particle diameter of more than 0.2 μm and 10 μm or less. [Selection figure] None

Description

TECHNICAL FIELD The present invention relates to an artificial hair fiber and a method for producing the same.

Human hair has long been used as hair for headdresses such as wigs, hair for hair extensions, and hair substitutes. In recent years, synthetic resin fibers such as nylon and polyester, and regenerated collagen protein fibers are widely used as artificial hair from the viewpoint of declining quality of human hair and stable supply.

Artificial hair is required to have a wide variety of properties, but in particular, natural luster and texture similar to those of human hair are important properties in terms of the commercial value of artificial hair.

In order to give natural luster and texture to artificial hair, it is common practice to roughen the fiber surface by adding a certain amount or more of a matting agent that is different from the artificial hair material. It is

For example, in Patent Document 1, polyester resin is mixed with silica, titanium dioxide, or an inorganic substance of titanium dioxide covered with apatite to form fibers, and then alkaline edging is performed. Mixing an organic resin such as polyvinyl alcohol or carboxymethyl cellulose to form fibers, and Patent Document 3 discloses mixing regenerated collagen with a monofunctional epoxy compound and a metal aluminum salt to form fibers.

However, when a material (matting agent) different from the artificial hair material is added in a certain amount or more, there is a problem that the strength of the fiber itself produced by the addition is lowered.

Japanese Patent Application Laid-Open No. 2008-31565 JP 2009-138314 A Japanese Patent Application Laid-Open No. 2010-24586

An object of the present invention is to provide an artificial hair fiber having a natural luster and a suppressed reduction in strength, and a method for producing the same.

The inventors of the present invention conducted various studies to solve the above problems, and found that, surprisingly, by using gloss-suppressing particles with a specific particle size and blending ratio, natural gloss was obtained in structural protein fibers, In addition, it was found that not only the decrease in strength was suppressed, but also the knot elongation was improved.

The present invention has been completed based on such novel findings, and provides, for example, the following inventions.
[1]
comprising a recombinant structural protein and gloss-reducing particles incompatible with the recombinant structural protein;
The content of the gloss-suppressing particles is more than 1 part by mass and 12 parts by mass or less with respect to 100 parts by mass of the recombinant structural protein,
A fiber for artificial hair, wherein the luster-suppressing particles are organic polymer particles or metal oxide particles having an average particle size of more than 0.2 μm and not more than 10 μm.
[2]
The artificial hair fiber according to [1], wherein the recombinant structural protein is at least one selected from the group consisting of spider silk fibroin, silk fibroin and keratin.
[3]
The artificial hair fiber according to [1] or [2], wherein the recombinant structural protein is at least one selected from the group consisting of spider silk fibroin and keratin.
[4]
The organic polymer particles are particles made of at least one selected from the group consisting of acrylic resins, urethane resins, acrylic urethane composite resins, polyamide resins and silicone resins, and the metal oxide particles are titanium oxide and silicon dioxide. The artificial hair fiber according to any one of [1] to [3], which is a particle made of at least one selected from the group consisting of:
[5]
Any one of [1] to [4], wherein the organic polymer particles are at least one selected from the group consisting of acrylic resins, urethane resins and acrylic urethane composite resins, and the metal oxide particles are titanium oxide. The fiber for artificial hair according to .
[6]
The artificial hair fiber according to any one of [1] to [5], wherein the luster-suppressing particles are particles made of at least one selected from the group consisting of acrylic resins, urethane resins and acrylic-urethane composite resins.
[7]
The artificial hair fiber according to any one of [1] to [6], wherein the recombinant structural protein is spider silk fibroin.
[8]
The artificial hair fiber according to any one of [1] to [7], wherein the recombinant structural protein is modified spider silk fibroin.
[9]
The artificial hair fiber according to any one of [1] to [8], wherein the luster-suppressing particles have a glass transition temperature of 80° C. or lower.
[10]
The artificial hair fiber according to any one of [1] to [9], wherein the luster-suppressing particles are urethane resin particles.
[11]
A method for producing fibers for artificial hair, comprising:
extruding a dispersion comprising a recombinant structural protein, gloss-reducing particles incompatible with the recombinant structural protein, and an organic solvent from a spinneret, and then removing the solvent to form a structural protein fiber;
Organic polymer particles in which the content of the gloss-suppressing particles is more than 1 part by mass and not more than 12 parts by mass with respect to 100 parts by mass of the recombinant structural protein, and the gloss-suppressing particles have an average particle diameter of 0.2 μm or more and 10 μm or less. Or metal oxide particles, production method.
[12]
The method for producing an artificial hair fiber according to [11], wherein the recombinant structural protein is at least one selected from the group consisting of spider silk fibroin, silk fibroin and keratin.
[13]
The method for producing an artificial hair fiber according to [11] or [12], wherein the recombinant structural protein is at least one selected from the group consisting of spider silk fibroin and keratin.
[14]
The organic polymer particles are particles made of at least one selected from the group consisting of acrylic resins, urethane resins, acrylic urethane composite resins, silicone resins and polyamide resins, and the metal oxide particles are titanium oxide and silicon dioxide. The method for producing an artificial hair fiber according to any one of [11] to [13], wherein the particles are composed of at least one selected from the group consisting of:
[15]
[11] to [14], wherein the organic polymer particles are particles made of at least one selected from the group consisting of acrylic resins, urethane resins and acrylic urethane composite resins, and the metal oxide particles are titanium oxide; A method for producing an artificial hair fiber according to any one of .
[16]
Manufacture of artificial hair fibers according to any one of [11] to [15], wherein the luster-suppressing particles are particles made of at least one selected from the group consisting of acrylic resins, urethane resins, and acrylic-urethane composite resins. Method.
[17]
The method for producing an artificial hair fiber according to any one of [11] to [16], wherein the recombinant structural protein is spider silk fibroin.
[18]
The method for producing an artificial hair fiber according to any one of [11] to [17], wherein the recombinant structural protein is modified spider silk fibroin.
[19]
The method for producing fibers for artificial hair according to any one of [11] to [18], wherein the luster-suppressing particles have a glass transition temperature of 80° C. or lower.
[20]
The method for producing fibers for artificial hair according to any one of [11] to [19], wherein the organic solvent is at least one selected from the group consisting of formic acid and hexafluoroisopropanol.
[21]
The method for producing fibers for artificial hair according to any one of [11] to [20], wherein the organic solvent is formic acid.
[22]
The method for producing fibers for artificial hair according to any one of [11] to [21], wherein the luster-suppressing particles are urethane resin particles.
[23]
Artificial hair comprising the fiber for artificial hair according to any one of [1] to [10].
[24]
Artificial hair obtained using the fiber for artificial hair according to any one of [1] to [10].
[25]
The artificial hair of [23] or [24], which is a wig, extension, hair accessory, braid or doll hair.

According to the artificial hair fiber of the present invention, it is possible to provide an artificial hair fiber that has a natural luster, suppresses a decrease in strength, and improves the knot elongation. Further, according to the production method of the present invention, the artificial hair fibers having the characteristics described above can be easily produced.

FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. FIG. 4 is a diagram showing the distribution of z/w (%) values of naturally occurring fibroin. FIG. 4 is a diagram showing the distribution of x/y (%) values of naturally occurring fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. 1 shows images of dispersions obtained in Test Examples 1-12, 15 and 16 and formic acid dispersions obtained in Comparative Examples 1-12, 15 and 16. FIG. 1 is a scanning electron microscopy (SEM) image of side and cross sections of recombinant structural protein fibers obtained in Examples 1-6. 4 is a scanning electron microscope (SEM) image of side and cross sections of fibers obtained in Comparative Examples 24-25. It is an explanatory view showing an example of a spinning device roughly. FIG. 4 is a diagram showing an example of length change of raw material fibers due to contact with water.

[Artificial hair fibers]
The artificial hair fiber according to the present embodiment contains a recombinant structural protein and luster-suppressing particles incompatible with the recombinant structural protein, and the content of the luster-suppressing particles is 1 per 100 parts by mass of the recombinant structural protein. The content is more than 12 parts by mass and the gloss suppressing particles are organic polymer particles or metal oxide particles having an average particle size of more than 0.2 μm and 10 μm or less.

The artificial hair fiber means a fiber that can be used for artificial hair, and the artificial hair fiber may be used as it is for artificial hair, or the artificial hair fiber may be processed to obtain artificial hair. Processing to artificial hair includes, for example, processing such as coloring (dyeing), imparting a curled shape, and imparting a straight shape. The artificial hair fibers can be produced by the method for producing artificial hair fibers described below.

The fiber diameter of the fiber for artificial hair according to the present embodiment may be 40 μm to 120 μm, 45 μm to 120 μm, 48 μm to 120 μm, 50 μm to 120 μm, or 55 μm to 120 μm. 60 μm to 120 μm, 65 μm to 120 μm, 60 μm to 110 μm, 60 μm to 100 μm, 60 μm to 95 μm, 60 μm to 90 μm may be 60 μm to 85 μm, may be 60 μm to 80 μm, may be 55 μm to 110 μm, may be 55 μm to 100 μm, may be 55 μm to 95 μm, may be 55 μm to 90 μm, 55 μm to 85 μm, 55 μm to 80 μm, 50 μm to 110 μm, 50 μm to 100 μm, 50 μm to 95 μm, 50 μm to 90 μm, 50 μm to It may be 85 μm, and may be between 50 μm and 80 μm. By setting the fiber diameter to 40 μm or more, it is possible to reduce the sense of discomfort that occurs with human hair. By setting the fiber diameter to 120 μm or less, it is possible to more efficiently remove the solvent when forming the fibers.

(recombinant structural protein)
Structural proteins refer to proteins that form biological structures or proteins derived from them. A recombinant structural protein is a structural protein produced by genetic recombination technology. The recombinant structural protein may be a naturally-occurring structural protein, or a modified structural protein in which a part of the amino acid sequence is modified based on the amino acid sequence of the naturally-occurring structural protein.

Recombinant structural proteins include, for example, any structural protein that is preferably produced on an industrial scale. Specific examples include structural proteins that can be used for industrial purposes, structural proteins that can be used for medical purposes, and the like. can. Specific examples of structural proteins that can be used for industrial or medical purposes include sparingly soluble proteins such as fibroin, collagen, resilin, elastin and keratin, and proteins derived from these. may be The fibroin may be, for example, one or more selected from the group consisting of silk fibroin (silk fibroin), spider silk fibroin (spider silk protein), and hornet silk fibroin.

The fibroin according to this embodiment includes naturally occurring fibroin and modified fibroin. As used herein, "naturally occurring fibroin" means fibroin having the same amino acid sequence as naturally occurring fibroin, and "modified fibroin" means fibroin having an amino acid sequence different from that of naturally occurring fibroin. do. As used herein, “modified fibroin” means artificially produced fibroin (artificial fibroin). The modified fibroin may be fibroin whose domain sequence differs from the amino acid sequence of naturally occurring fibroin, or may be fibroin whose domain sequence is identical to the amino acid sequence of naturally occurring fibroin.

The fibroin according to the present embodiment is preferably spider silk fibroin (spider silk protein). Spider silk fibroin includes natural spider silk fibroin and modified spider silk fibroin derived from natural spider silk fibroin. Natural spider silk fibroin includes, for example, spider silk proteins produced by spiders.

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

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically corresponding to the REP of the amino acid sequence). ) and is represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif means an array. Here, (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. (A) The number of amino acid residues in the _n motif may be an integer of 2-20, 4-27, 4-20, 8-20, 10-20, 4-16, 8-16, or 10-16 . In addition, (A) the ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed only of alanine residues. REP indicates an amino acid sequence composed of 2-200 amino acid residues. REP may be an amino acid sequence composed of 10-200 amino acid residues, 10-40, 10-60, 10-80, 10-100, 10-120, 10-140, 10-160, or It may be an amino acid sequence composed of 10 to 180 amino acid residues. m represents an integer of 2 to 300, It may be an integer of 20-140 or 20-120. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

Naturally occurring fibroin is a protein comprising a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. There are, specifically, fibroin produced by insects or arachnids.

Examples of fibroin produced by insects include, for example, Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, and Pilosa ), silk proteins produced by silkworms such as Samia cynthia, Caligura japonica, Antheraea mylitta, Antheraea assama, and the larvae of the wasp (Vespa simillima xantoptera) Hornet silk protein.

More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

Examples of fibroin produced by spiders include, for example, spiders belonging to the genus Araneus such as Araneus spiders, Araneus spiders, Red spiders, Green spiders, and Beetle spiders; Spiders belonging to the genus Pronus, spiders belonging to the genus Pronus such as spiders belonging to the Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius such as the genus Ordgarius and the spiders belonging to the genus Ordgarius, spiders belonging to the genus Argiope such as the argiope spider, the argiope spider, and the argiope spider, and the like Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas, such as spiders, spiders belonging to the genus Cytophora, such as spiders, spiders, and spiders Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as spiders belonging to the genus Cyclosa, spiders belonging to ), spiders belonging to the genus Cyclosa, and spiders belonging to the genus Chorizopes, such as spiders and spiders, Spiders belonging to the genus Tetragnatha such as the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucauge, such as the genus Leucauge, and the genus Nephila Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as golden spiders, spiders belonging to the genus Dyschiriognatha such as the brown spider, widow spiders such as the black widow spider, the redback spider, the gray widow spider and the western widow spider Spiders belonging to the genus Latrodectus and spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops Spider silk proteins can be mentioned. Spider silk proteins include, for example, MaSp (MaSp1 and MaSp2), dragline proteins such as ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of spider silk proteins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence ), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (derived from Nephila clavata) No. AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidropin 2 [Euprosthenops australispin] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys crentata] (GenBank Accession and Section No. ABR37278.1 (amino acid sequence).

More specific examples of naturally occurring fibroin include fibroin whose sequence information is registered in NCBI GenBank. For example, among sequence information registered in NCBI GenBank, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION from among sequences containing INV as DIVISION. It can be confirmed by extracting the specific product character string from the sequence, CDS, and the sequence described with the specific character string from SOURCE to TISSUE TYPE.

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

Modified fibroin is obtained by modifying the amino acid sequence by, for example, substituting, deleting, inserting and/or adding one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. can be obtained with 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 directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983).

The modified fibroin may be, for example, modified fibroin derived from silk protein produced by silkworms (modified silk fibroin), or modified fibroin derived from spider silk protein produced by spiders (modified spider silk fibroin). good too.

Specific examples of modified fibroin include a modified fibroin (first modified fibroin) derived from the dragline silk protein produced in the major pituitary gland of spiders, and a domain sequence with reduced content of glycine residues. (second modified fibroin), (A) modified fibroin having a domain sequence with reduced content of _n motifs (third modified fibroin), content of glycine residues, and (A) _n Modified fibroin with reduced motif content (fourth modified fibroin), modified fibroin having a domain sequence containing a region with a locally large hydrophobicity index (fifth modified fibroin), and content of glutamine residues modified fibroin having a reduced domain sequence (sixth modified fibroin).

A first modified fibroin includes a protein comprising a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In the first modified fibroin, in Formula 1, n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, even more preferably an integer of 10 to 20, 4 Integers from ˜16 are even more preferred, integers from 8 to 16 are particularly preferred, and integers from 10 to 16 are most preferred. In the first modified fibroin, the number of amino acid residues constituting REP in formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, 20 to 100 residues and even more preferably 20 to 75 residues. In the first modified fibroin, the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m is amino acid residue It is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more of the total number.

The first modified fibroin comprises an amino acid sequence unit represented by Formula 1: [(A) _n motif-REP] _m , and an amino acid sequence whose C-terminal sequence is shown in any one of SEQ ID NOs: 1 to 3; Alternatively, it may be a polypeptide, which is an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOS: 1-3.

The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of the C-terminal 50 amino acids of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 has the sequence It is identical to the amino acid sequence shown in No. 1 with 20 residues removed from the C-terminus, and the amino acid sequence shown in SEQ ID No. 3 has 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID No. 1. Identical to the amino acid sequence.

As more specific examples of the first modified fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroins comprising amino acid sequences with greater than % sequence identity can be mentioned. Preferably, the sequence identity is 95% or greater.

The amino acid sequence shown by SEQ ID NO: 4 is an amino acid sequence (SEQ ID NO: 5) consisting of an initiation codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus of ADF3. The 13th repeat region was increased to approximately double and mutated so that translation stops at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown by SEQ ID NO:4 is identical to the amino acid sequence shown by SEQ ID NO:3.

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

The second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to the naturally-occurring fibroin. The second modified fibroin can be said to have an amino acid sequence corresponding to at least one or more glycine residues in REP being replaced with another amino acid residue, as compared with naturally occurring fibroin. .

The second modified fibroin has a domain sequence of GGX and GPGXX (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine) in REP compared to naturally occurring fibroin. In at least one motif sequence selected from ), it has an amino acid sequence corresponding to at least one or more glycine residues in the motif sequence being replaced with another amino acid residue may

In the second modified fibroin, the percentage of the motif sequence in which the glycine residue is substituted with another amino acid residue may be 10% or more of the entire motif sequence.

The second modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the domain sequence, the (A) _n motif located most C-terminal to the domain sequence Let z be the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus of the domain sequence When w is the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence, z/w is 30% or more, It may have an amino acid sequence that is 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

The second modified fibroin preferably has an increased content of the amino acid sequence consisting of XGX by substituting one glycine residue in the GGX motif with another amino acid residue. 6. In the second modified fibroin, the content of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, even more preferably 10% or less. % or less, even more preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z/w) of the amino acid sequence consisting of XGX below.

A method for calculating z/w will be described in more detail. First, in a fibroin (modified fibroin or naturally occurring fibroin) containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , the (A) _n located on the most C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50×3=150. In addition, for example, when there is an X (central X) contained in two XGX, as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues be). w is the total number of amino acid residues contained in the domain sequence, excluding the sequence from the (A) _n motif positioned most on the C-terminal side to the C-terminus of the domain sequence. For example, for the domain sequence shown in Figure 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the most C-terminal (A) _n motif). Then z/w (%) can be calculated by dividing z by w.

Here, z/w in naturally occurring fibroin will be explained. First, as described above, fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, and 663 types of fibroin (among them, 415 types of arachnid-derived fibroin) were extracted. Naturally derived fibroin containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m and having an amino acid sequence consisting of GGX in fibroin content of 6% or less among all the extracted fibroin z/w was calculated from the amino acid sequence of fibroin in the above-described calculation method. The results are shown in FIG. The horizontal axis of FIG. 2 indicates z/w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the z/w for naturally occurring fibroin is all less than 50.9% (the highest is 50.86%).

In the second modified fibroin, z/w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, and 70% or more. and even more preferably 80% or more. Although the upper limit of z/w is not particularly limited, it may be, for example, 95% or less.

The second modified fibroin is obtained, for example, by substituting at least part of the nucleotide sequence encoding the glycine residue from the cloned naturally-derived fibroin gene sequence so as to encode another amino acid residue. Obtainable. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be substituted so that z/w is 50.9% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the alteration corresponding to replacing the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , insertions and/or additions may be made to the amino acid sequence.

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

More specific examples of the second modified fibroin include (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (2-ii) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Mention may be made of modified fibroin.

The modified fibroin of (2-i) will be explained. The amino acid sequence shown by SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence shown by SEQ ID NO: 10 corresponding to naturally occurring fibroin with GQX. The amino acid sequence shown by SEQ ID NO: 7 is obtained by deleting every two (A) _n motifs from the amino acid sequence shown by SEQ ID NO: 6 from the N-terminal side to the C-terminal side, and furthermore, in front of the C-terminal sequence. [(A) _n motif-REP] is inserted into the . The amino acid sequence shown in SEQ ID NO: 8 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a partial glutamine (Q) residue. It is obtained by substituting serine (S) residues and deleting some amino acids on the C-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and a His tag sequence are added to the C-terminus of a sequence in which is repeated four times.

The z/w value in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z/w values in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are each 58.7%, 70.1%, 66.1% and 70.0%. In addition, the value of x/y in the jagged ratio (described later) of 1:1.8 to 11.3 of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is 15.0%, 15.0%, 93.4%, 92.7% and 89.8% respectively.

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

The modified fibroin (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and XGX contained in REP ( where X represents an amino acid residue other than glycine). is preferably 50.9% or more.

The second modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection, visualization, and the like of modified fibroin.

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

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

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

Furthermore, a tag sequence that can be cleaved by a specific protease can also be used. The modified fibroin from which the tag sequence has been cut off can also be recovered by treating the protein adsorbed via the tag sequence with protease.

More specific examples of modified fibroin containing a tag sequence include: (2-iii) amino acids represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) sequence, or (2-iv) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

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

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

The modified fibroin of (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(2-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is contained in REP ( where X represents an amino acid residue other than glycine). is preferably 50.9% or more.

The second modified fibroin may contain a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A third modified fibroin has an amino acid sequence in which the domain sequence has a reduced content of (A) _n motifs compared to the naturally occurring fibroin. The domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to deletion of at least one or more (A) _n motifs compared to the naturally occurring fibroin.

A third modified fibroin may have an amino acid sequence corresponding to 10-40% deletion of the (A) _n motif from the naturally-occurring fibroin.

A third modified fibroin has a domain sequence at least one (A) _n motif for every 1-3 (A) _n motifs from the N-terminal side to the C-terminal side as compared to the naturally-occurring fibroin. may have an amino acid sequence corresponding to the deletion of

The third modified fibroin has at least two consecutive (A) _n motif deletions from the N-terminal side to the C-terminal side and one (A ) may have an amino acid sequence corresponding to deletion of _n motifs repeated in this order.

The third modified fibroin may have an amino acid sequence corresponding to deletion of at least every two (A) _n motifs from the N-terminal side to the C-terminal side of the domain sequence. .

A third modified fibroin comprises a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , with two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -REP] unit sequentially compares the number of amino acid residues of REP, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11. When the maximum value of the total sum of the numbers of amino acid residues of two adjacent [(A) _n motif-REP] units equal to 3 is x, and the total number of amino acid residues of the domain sequence is y Furthermore, it may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

A method for calculating x/y will be described in more detail with reference to FIG. FIG. 1 shows the domain sequence of the modified fibroin with the N-terminal and C-terminal sequences removed. The domain sequence is, from the N-terminal side (left side), (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n Motif-third REP (10 amino acid residues)-(A) _n motif-fourth REP (20 amino acid residues)-(A) _n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called _n motif.

Two adjacent [(A) _n -motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, unselected [(A) _n motif-REP] units may be present. In FIG. 1, pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP and the fourth REP), pattern 2 (comparison of the first REP and the second REP, and comparison of the fourth REP and the fifth REP), pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP and the fifth REP), pattern 4 (comparison of the first REP and A second REP comparison) was shown. Note that there are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two adjacent [(A) _n motif-REP] units selected is compared. Comparison is carried out by determining the ratio of the number of amino acid residues to the number of amino acid residues of the other. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the second REP is The ratio of amino acid residue numbers is 100/50=2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with fewer amino acid residues is set to 1, the fifth REP is 30/20=1.5.

In FIG. 1, a set of [(A) _n motif-REP] units having a ratio of 1.8 to 11.3 for the number of amino acid residues of the other is set to 1 for the one with the smaller number of amino acid residues. It is indicated by a solid line. Such a ratio is hereinafter referred to as a serration ratio. A set of [(A) _n motif-REP] units in which the other amino acid residue number ratio is less than 1.8 or greater than 11.3 when the number of amino acid residues is less than 1 is indicated by a dashed line. Indicated.

In each pattern, the numbers of all amino acid residues of two adjacent [(A) _n motif-REP] units indicated by solid lines are summed up (not only REP but also the number of amino acid residues of (A) _n motifs). be.). Then, the added total values are compared, and the total value (maximum total value) of the pattern with the maximum total value is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the largest.

Then x/y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

In the third modified fibroin, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, and even more preferably 70% or more. Preferably, 75% or more is even more preferable, and 80% or more is particularly preferable. The upper limit of x/y is not particularly limited, and may be, for example, 100% or less. When the serration ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the serration ratio is 1:1.8 to 3.4, x /y is preferably 77.1% or more, and when the serration ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and the serration ratio is 1 : 1.9 to 4.1, x/y is preferably 64.2% or more.

When the third modified fibroin is a modified fibroin in which at least 7 of the (A) _n motifs present in the domain sequence are composed only of alanine residues, x/y is 46.4% or more. is preferably 50% or more, more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and 80% or more It is particularly preferred to have The upper limit of x/y is not particularly limited as long as it is 100% or less.

Here, x/y in naturally occurring fibroin will be explained. First, as described above, fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, and 663 types of fibroin (among them, 415 types of arachnid-derived fibroin) were extracted. Of all the extracted fibroin, from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , x/y was calculated. FIG. 3 shows the results when the serration ratio is 1:1.9 to 4.1.

The horizontal axis in FIG. 3 indicates x/y (%), and the vertical axis indicates frequency. As is clear from FIG. 3, the x/y ratios for naturally occurring fibroin are all less than 64.2% (64.14% being the highest).

The third modified fibroin, for example, deletes one or more of the (A) _n motif-encoding sequences from the cloned naturally occurring fibroin gene sequence such that x/y is 64.2% or more. can be obtained by Alternatively, for example, an amino acid sequence corresponding to deletion of one or more (A) _n motifs is designed such that x/y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence described above. 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 substituted, deleted, inserted and/or added. Alterations in the amino acid sequence corresponding to what has been done may be made.

More specific examples of the third modified fibroin include (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Mention may be made of modified fibroin.

The modified fibroin of (3-i) will be explained. The amino acid sequence represented by SEQ ID NO: 17 is every two (A) _n The motif was deleted and one [(A) _n motif-REP] was inserted in front of the C-terminal sequence. The amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified fibroin.

The x/y value of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) is 15.0% at the jagged ratio of 1:1.8 to 11.3. The x/y values of the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%. The z/w values in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.0%, respectively. 1% and 70.0%.

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

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and from the N-terminal side to the C-terminal side , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Amino acid residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (Giza ratio is 1:1.8 to 11.3) It is preferable that x/y is 64.2% or more, where x is the maximum sum of the cardinal numbers and y is the total number of amino acid residues in the domain sequence.

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

As more specific examples of modified fibroin containing a tag sequence, (3-iii) the amino acid shown in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) sequence, or (3-iv) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 have SEQ ID NO: 11 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in (including His tag sequence and hinge sequence) is added.

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

(3-iv) modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(3-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Let x be the maximum value of the total sum of the amino acid residue numbers of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3. , x/y is preferably 64.2% or more, where y is the total number of amino acid residues in the domain sequence.

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

The fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) _n motifs and a reduced content of glycine residues compared to naturally occurring fibroin. have. The domain sequence of the fourth modified fibroin is that at least one or more (A) _n motifs are deleted, and at least one or more glycine residues in REP are deleted compared to the naturally occurring fibroin. It can be said to have an amino acid sequence corresponding to substitution with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having both the features of the above-described second modified fibroin and the third modified fibroin. Specific aspects and the like are as described in the second modified fibroin and the third modified fibroin.

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

A fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a larger hydrophobicity index than in naturally occurring fibroin, and/or REP It may have an amino acid sequence that includes regions of high local hydrophobicity index corresponding to the insertion of one or more amino acid residues with high hydrophobicity index therein.

A region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

The amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). A residue is more preferred.

A fifth modified fibroin is obtained by replacing one or more amino acid residues in REP with amino acid residues having a higher hydrophobicity index than in naturally occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, one or more amino acid residues are substituted, deleted, inserted and / or added as compared to naturally occurring fibroin There may be alterations in the amino acid sequence corresponding to what has been done.

For the fifth modified fibroin, for example, one or more hydrophilic amino acid residues (eg, amino acid residues with a negative hydrophobicity index) in REP from the cloned naturally occurring fibroin gene sequence are replaced with hydrophobic amino acid residues. by substituting a group (for example, an amino acid residue with a positive hydrophobicity index) and/or by inserting one or more hydrophobic amino acid residues into REP. Further, for example, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to the insertion of and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acids in REP In addition to modifications corresponding to residue insertions, amino acid sequence modifications corresponding to substitutions, deletions, insertions and/or additions of one or more amino acid residues may also be made.

The fifth modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence Let p be the total number of amino acid residues contained in a region where the average value of the hydrophobic index of four consecutive amino acid residues is 2.6 or more in all REPs contained in the sequences excluding the sequences from the domain sequence, p/q is 6, where q is the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. It may have an amino acid sequence that is greater than or equal to .2%.

Hydropathy indexes of amino acid residues are known (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 157). 105-132). Specifically, the hydropathic index (hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

A method for calculating p/q will be described in more detail. For the calculation, the sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence (hereinafter referred to as “sequence A”) is used. First, in all REPs contained in sequence A, the average value of the hydrophobic index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the 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 to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to multiple "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

For example, if 20 “continuous 4 amino acid residues with an average hydrophobic index of 2.6 or more” are extracted (no overlap), the average hydrophobic index of 4 consecutive amino acid residues is 2 A region that is 0.6 or greater will contain 20 consecutive 4-amino acid residues (no overlap), and p is 20×4=80. Further, for example, when two "consecutive 4 amino acid residues having an average hydrophobicity index of 2.6 or more" exist overlapping by one amino acid residue, the hydrophobic index of the consecutive 4 amino acid residues A region with an average of 2.6 or more contains 7 amino acid residues (p=2×4−1=7, where “−1” is duplicate subtraction). For example, in the case of the domain sequence shown in FIG. 4, there are seven non-overlapping “continuous 4 amino acid residues with an average hydrophobicity index of 2.6 or more”, so p is 7 × 4 = 28. For example, in the case of the domain sequence shown in FIG. 4, q is 4+50+4+40+4+10+4+20+4+30=170 (not including the (A) _n motif present at the end of the C-terminal side). Then p/q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170=16.47%.

In the fifth modified fibroin, p/q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, and 20% or more. Even more preferably, it is still more preferably 30% or more. Although the upper limit of p/q is not particularly limited, it may be, for example, 45% or less.

For the fifth modified fibroin, for example, the amino acid sequence of the cloned naturally occurring fibroin is modified so as to satisfy the p/q conditions described above, so that one or more hydrophilic amino acid residues in REP (eg, hydrophobicity index). negative amino acid residue) with a hydrophobic amino acid residue (e.g., an amino acid residue with a positive hydrophobicity index), and/or inserting one or more hydrophobic amino acid residues in REP can be obtained by locally modifying an amino acid sequence containing a region with a large hydrophobicity index. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above p/q conditions from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more amino acid residues in REP have been replaced with amino acid residues having a higher hydrophobicity index compared to naturally-occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, further modification corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues may be performed. .

Amino acid residues with a large hydrophobicity index are not particularly limited, but areoleucine (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.

As more specific examples of the fifth modified fibroin, (5-i) an amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665), or SEQ ID NO: 21 (Met-PRT666); or (5-ii) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified fibroin of (5-i) will be explained. The amino acid sequence shown by SEQ ID NO: 19 is an amino acid sequence consisting of 3 amino acid residues every other REP except for the terminal domain sequence on the C-terminal side with respect to the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410). The sequence (VLI) was inserted at two locations, and further, some glutamine (Q) residues were substituted with serine (S) residues, and some amino acids on the C-terminal side were deleted. The amino acid sequence represented by SEQ ID NO: 20 is the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) in which an amino acid sequence (VLI) consisting of three amino acid residues is inserted at every other REP. be. The amino acid sequence shown by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 8.

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

The modified fibroin (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

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

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

The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are added to the N-terminals of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, where the amino acid sequence shown in SEQ ID NO: 11 (His tag sequence and hinge sequence) are added.

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

The modified fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

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

A sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues compared to naturally occurring fibroin.

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

When the sixth modified fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.

As used herein, the "GPGXX motif content" is a value calculated by the following method.

Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total by three (that is, the total number of G and P in the GPGXX motif) is defined as s, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is taken from the domain sequence. The GPGXX motif content rate is calculated as s/t, where t is the total number of amino acid residues in all REPs excluding (A) _n motifs.

In the calculation of the GPGXX motif content rate, the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is "the most C-terminal side (A) The sequence from the _n motif to the C-terminus of the domain sequence” (the sequence corresponding to REP) may include sequences that are poorly correlated with sequences characteristic of fibroin, and m is small If the domain sequence is short (that is, if the domain sequence is short), it affects the calculation result of the GPGXX motif content rate, so this effect is to be eliminated. When a "GPGXX motif" is located at the C-terminus of REP, it is treated as a "GPGXX motif" even if "XX" is, for example, "AA".

FIG. 5 is a schematic diagram showing the domain sequence of fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. First, in the fibroin domain sequence (“[(A) _n motif-REP] _m -(A) _n motif” type) shown in FIG. A) GPGXX for calculating s because it is included in the "sequence obtained by removing the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence" (sequence shown in "region A" in FIG. 5) The number of motifs is 7, and s is 7×3=21. Similarly, all REPs are "sequences obtained by removing the sequences from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequences from the domain sequences" (in FIG. 5, the sequence indicated by "region A" .), the total number of amino acid residues t of all REPs further excluding the (A) _n motif from the sequence is 50+40+10+20+30=150. Next, s/t (%) can be calculated by dividing s by t, which is 21/150=14.0% for fibroin in FIG.

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

As used herein, "glutamine residue content" is a value calculated by the following method.

Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence (the sequence corresponding to "region A" in FIG. 5). REP, the total number of glutamine residues contained in the region is u, the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and (A) _n The glutamine residue content is calculated as u/t, where t is the total number of amino acid residues in all REPs excluding motifs. In the calculation of the glutamine residue content, the target is the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because of the reasons described above. It is the same.

A sixth modified fibroin corresponds to a domain sequence that lacks one or more glutamine residues in REP or replaces them with other amino acid residues as compared to the naturally-occurring fibroin. It may have an amino acid sequence.

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

As shown in Table 1, amino acid residues having a higher 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); can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

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

As used herein, the "hydrophobicity of REP" is a value calculated by the following method.

Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is v, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v/t, where t is the total number of amino acid residues in all REPs excluding _n motifs. In calculating the hydrophobicity of REP, the reason for targeting "the sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.

A sixth modified fibroin has a domain sequence lacking one or more glutamine residues in REP and/or one or more glutamine residues in REP compared to naturally-occurring fibroin In addition to the modification corresponding to the substitution of another amino acid residue, there may be modifications of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. .

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

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

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

The amino acid sequence shown by SEQ ID NO:30 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:8 (Met-PRT525) with VL. The amino acid sequence shown by SEQ ID NO: 31 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 8 with VL, and replacing the remaining Q with I.

The amino acid sequence represented by SEQ ID NO: 32 consists of 20 domain sequence regions present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) that are repeated twice, and all QQ in the sequence are replaced with VF, And the remaining Q is replaced with I.

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

SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all glutamine residues The group content is below 9% (Table 2).

The modified fibroin of (6-i) is represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 It may consist of the amino acid sequence shown.

The modified fibroin of (6-ii) is represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 Amino acid sequences having 90% or greater sequence identity with the indicated amino acid sequences are included. The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

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

The sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection, visualization, and the like of modified fibroin.

More specific examples of the sixth modified fibroin containing a tag sequence include (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), Modified fibroin comprising an amino acid sequence represented by SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028) , or (6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the sequence can be mentioned.

The amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are SEQ ID NO: 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 at the N-terminus of SEQ ID NO: 11 The amino acid sequence (including the His-tag sequence and hinge sequence) is added. Since the tag sequence was only added to the N-terminus, there was no change in the glutamine residue content, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It may consist of the amino acid sequence shown.

The modified fibroin of (6-iv) is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 Amino acid sequences having 90% or greater sequence identity with the indicated amino acid sequences are included. The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

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

The sixth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

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

Modified fibroin may be hydrophilic modified fibroin or hydrophobic modified fibroin. As used herein, the term "hydrophobic modified fibroin" refers to a value obtained by obtaining the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin, and then dividing the sum by the total number of amino acid residues. (Average HI) greater than 0 is a modified fibroin. The hydrophobicity index is as shown in Table 1. Further, "modified hydrophilic fibroin" is modified fibroin having an average HI of 0 or less. Hydrophilic modified fibroin is preferable as the modified fibroin from the viewpoint of excellent combustion resistance.

Examples of hydrophobically modified fibroin include amino acid sequences represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43, SEQ ID NO: 35, A modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44 is included.

Examples of modified hydrophilic fibroin include the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 12, and SEQ ID NO: 14. or the amino acid sequence represented by SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, the amino acid represented by SEQ ID NO: 17, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15 A modified fibroin comprising the amino acid sequence shown in the sequence SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 is included.

The modified fibroin according to this embodiment may contain only one type of modified fibroin, or may contain two or more types of modified fibroin in combination.
Moreover, it may contain a combination of modified fibroin and a structural protein other than modified fibroin.

The structural protein may be a polypeptide derived from the native structural protein described above, ie a recombinant polypeptide.

As a collagen-derived structural protein, for example, a protein comprising a domain sequence represented by Formula 3: [REP2] _p (here, in Formula 3, p represents an integer of 5 to 300. REP2 is Gly-X- represents an amino acid sequence composed of Y, where X and Y represent any amino acid residue other than Gly.A plurality of REP2s present may have the same amino acid sequence or different amino acid sequences.) can. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO:45 can be mentioned. The amino acid sequence represented by SEQ ID NO: 45 corresponds to the repeat portion and motif of the partial sequence of human collagen type 4 obtained from the NCBI database (NCBI GenBank accession number: CAA56335.1, GI: 3702452). The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 11 was added to the N-terminal of the amino acid sequence from the 301st to 540th residues.

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

Structural proteins derived from elastin include, for example, proteins having amino acid sequences of NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine), and the like. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO:47 can be mentioned. The amino acid sequence shown by SEQ ID NO: 47 is the amino acid sequence shown by SEQ ID NO: 11 (tag sequence and hinge sequence) are added.

Structural proteins derived from keratin include, for example, type I keratin of Capra hircus. Specifically, a protein comprising the amino acid sequence represented by SEQ ID NO: 48 (amino acid sequence of accession number ACY30466 in GenBank of NCBI) can be mentioned.

(Recombinant expression of structural proteins)
A recombinant structural protein can be expressed, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the structural protein and one or more regulatory sequences operably linked to the nucleic acid sequence. can be produced by

A method for producing a nucleic acid encoding a structural protein is not particularly limited. For example, using a gene encoding a structural protein such as natural fibroin, a method of amplifying and cloning by polymerase chain reaction (PCR) or the like, and modifying it by genetic engineering techniques as necessary, or chemically synthesizing The nucleic acid can be produced by the method described above. The method for chemically synthesizing the nucleic acid is not particularly limited, and for example, based on the amino acid sequence information of the protein obtained from the NCBI web database, etc., with AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.). A gene can be chemically synthesized by a method of linking automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and/or confirmation of the recombinant structural protein, a nucleic acid encoding a structural protein consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence is prepared. may be synthesized.

A regulatory sequence is a sequence that controls expression of a recombinant structural protein in a host (eg, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected according to the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the recombinant structural protein may be used. An inducible promoter is a promoter whose transcription can be controlled by physical factors such as the presence of an inducer (expression inducer), the absence of a repressor molecule, or an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected from plasmid vectors, virus vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, etc., depending on the type of host. Expression vectors that are capable of autonomous replication in host cells or that are capable of integration into host chromosomes and that contain a promoter at a position at which a nucleic acid encoding a recombinant structural protein can be transcribed are preferably used.

As hosts, both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammonium philum. Microorganisms belonging to the genus Brevibacterium include, for example, Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

When prokaryotes are used as hosts, examples of vectors for introducing nucleic acids encoding structural proteins include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569) and the like.

Eukaryotic hosts include, for example, yeast and filamentous fungi (such as molds). Examples of yeast include yeast belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces, and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.

When a eukaryotic host is used, examples of vectors for introducing nucleic acids encoding structural proteins include YEp13 (ATCC37115) and YEp24 (ATCC37051). Any method for introducing DNA into the host cell can be used as the method for introducing the expression vector into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like.

As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning, Second Edition. .

A recombinant structural protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. The method of culturing the host in the culture medium can be carried out according to a method commonly used for culturing the host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the host so that the host can be efficiently cultured. Either a natural medium or a synthetic medium may be used as long as the medium is used.

Any carbon source can be used as long as it can be assimilated by the above-mentioned transformed microorganism. Examples include carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch and starch hydrolysates, acetic acid and propionic acid, and the like. Organic acids and alcohols such as ethanol and propanol can be used. Nitrogen sources include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolysates, soybean meal and soybean meal hydrolysates, various fermented cells and their digests can be used. Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

Prokaryotes such as E. coli or eukaryotes such as yeast can be cultured under aerobic conditions such as shaking culture or deep aeration stir culture. The culture temperature is, for example, 15-40°C. Culture time is usually 16 hours to 7 days. The pH of the culture medium during cultivation is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium during the culture, if necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using a lac promoter, and indole acrylic when culturing a microorganism transformed with an expression vector using a trp promoter. Acids and the like may be added to the medium.

(Method for Collecting Solubilized Fraction Containing Recombinant Structural Protein)
The expressed recombinant structural protein can be recovered as a soluble fraction. Collection as a solubilized fraction enables the removal or reduction of host cells and/or host cell-derived contaminants, and is therefore preferably performed before step (A).

The method for collecting the solubilized fraction is, for example, when the recombinant structural protein is expressed in a dissolved state in host cells, the host cells are first disrupted by physical or chemical treatment, and the host cell lysate is obtained. get Examples of physical treatment include ultrasonic treatment and crushing treatment with a homogenizer, and examples of chemical treatment mainly include a method of treating with a solvent that dissolves the target recombinant structural protein but does not dissolve host cells. and hexafluoroisopropanol (HFIP) as a solvent. Next, a solubilized fraction containing the recombinant structural protein of interest is recovered from the host cell lysate. Methods for collecting the solubilized fraction containing the recombinant structural protein of interest include common methods such as centrifugation and filtration through filters such as drum filters and press filters. In the case of filter filtration, a method using a Teflon filter, a method using a filter aid such as celite or diatomaceous earth and a pre-coating agent, etc., can more efficiently recover the soluble fraction containing the target recombinant structural protein. .

In addition, when the recombinant structural protein forms an insoluble form in the cells and is expressed, the host cells are similarly disrupted by physical or chemical treatment, and then centrifuged to obtain the recombinant structural protein as a precipitate fraction. An insoluble protein is recovered. The recovered insoluble recombinant structural protein can be solubilized with a protein denaturant. After this manipulation, a solubilized fraction containing the recombinant structural protein can be obtained by the same method as described above. When the recombinant structural protein is extracellularly secreted, a solubilized fraction containing the recombinant structural protein can be recovered from the culture supernatant. That is, a solubilized fraction containing the recombinant structural protein can be obtained by treating the culture by centrifugation, filtration through a drum filter, a press filter, or the like. In the case of filter filtration, a method using a Teflon filter, a method using a filter aid such as celite or diatomaceous earth, a pre-coating agent, or the like can more efficiently collect the soluble fraction containing the recombinant structural protein.

(Crude purification of recombinant structural protein)
Methods commonly used to isolate and purify proteins from soluble fractions containing recombinant structural proteins, namely, solvent extraction, salting-out with ammonium sulfate, desalting, precipitation with organic solvents, diethylaminoethyl (DEAE) - Anion exchange chromatography using resins such as Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), Butyl Sepharose , Hydrophobic chromatography using resins such as phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing alone or in combination. can be used to obtain crude preparations of recombinant structural proteins.

(Method for preparing recombinant structural protein solution)
The recombinant structural protein solution is not particularly limited as long as it is a solution in which the recombinant structural protein is dissolved in a solvent for dissolution. For example, it can be obtained by dissolving a crude preparation of the recombinant structural protein described above in a solvent for dissolution, or a soluble fraction containing the recombinant structural protein described above can be obtained as it is, or by substituting or adding a solvent to the solvent for dissolution. Alternatively, it can be obtained by dissolving the host cell expressing the recombinant structural protein (or host cell lysate or lysate) in a dissolution solvent. After dissolving the recombinant structural protein by adding a solvent for dissolution to the host cells used as the recombinant structural protein or to the lysate or lysate of the host cells, the recombinant structural protein solution is collected as a solubilized fraction, Preferably, cells and/or host cell-derived contaminants are removed or reduced. The method for collecting the solubilized fraction is as described above.

The conditions for dissolving the recombinant structural protein in the dissolving solvent can be appropriately set according to the type of dissolving solvent, the type and concentration of salt to be added, the type of recombinant structural protein, and the like. In general, the recombinant structural protein can be dissolved in the dissolution solvent by appropriately setting the dissolution conditions.

The dissolution temperature is preferably such that the recombinant structural protein dissolves, but the contaminants derived from the host cell expressing the recombinant structural protein do not dissolve, and the temperature is maintained for a predetermined period of time. The temperature for dissolution may be determined according to the type of dissolution solvent, the type and concentration of salt to be added, the type of recombinant structural protein, etc. Examples include temperatures of 30 to 100°C and 40 to 60°C. be able to. For example, the upper temperature limit for dissolution may be 100°C, 90°C, 80°C, or 70°C, and the lower temperature limit for dissolution may be 30°C, 40°C, and 50°C. The time for dissolution is not particularly limited as long as the recombinant structural protein is sufficiently dissolved and contaminants are less dissolved. ~60 minutes is more preferable, and 10 to 30 minutes is even more preferable.

When the recombinant structural protein is fibroin, collagen, resilin, elastin, keratin, and proteins derived from these, for example, the following conditions can be mentioned. The amount of the solvent for dissolution added to the host expressing the structural protein is preferably 100 to 300 times the weight (wt) of the recombinant structural protein as a ratio of solvent (vol)/recombinant structural protein weight (wt). ~250 times is more preferable, and 175 to 225 times is even more preferable. As the salt to be added to the solvent for dissolution, lithium chloride, calcium chloride and sodium trifluoroacetate are preferable, and sodium trifluoroacetate is more preferable. In addition, the concentration when adding a salt is preferably more than 0 M and 1.0 M or less, more preferably more than 0 M and 0.6 M or less, further preferably more than 0 M and 0.5 M or less, based on the total amount of the dissolving solvent, and more than 0 M. It may be 0.01M or less.

When the recombinant structural protein is fibroin (including modified fibroin), for example, the following conditions can be mentioned. The amount of the dissolution solvent added to the host cells expressing the recombinant structural protein is 100 to 300 times the weight of the recombinant structural protein (wt) as the ratio of solvent for dissolution (vol)/weight of the recombinant structural protein (wt). is preferred, 150 to 250 times is more preferred, and 175 to 225 times is even more preferred. As the salt to be added to the solvent for dissolution, lithium chloride, calcium chloride and sodium trifluoroacetate are preferable, and sodium trifluoroacetate is more preferable. In addition, the concentration when adding a salt is preferably more than 0 M and 1.0 M or less, more preferably more than 0 M and 0.6 M or less, further preferably more than 0 M and 0.5 M or less, based on the total amount of the dissolving solvent, and more than 0 M. It may be 0.01M or less. Temperature conditions include temperatures of 30 to 100° C. and 40 to 60° C. using the above solvents. For example, the upper temperature limit for dissolution may be 100°C, 90°C, 80°C, or 70°C, and the lower temperature limit for dissolution may be 30°C, 40°C, and 50°C. The time for dissolution is, for example, preferably 10 to 120 minutes, more preferably 10 to 60 minutes, even more preferably 10 to 30 minutes.

When the recombinant structural protein solution is obtained by dissolving host cells expressing the recombinant structural protein (or host cell lysate or lysate, etc.) in a solvent for dissolution, the recombinant structural protein solution is collected and Insolubles may be separated to remove or reduce host cells and/or host cell-derived contaminants. Recovery of the structural protein solution and separation of the insoluble matter are not particularly limited as long as the solution and the sediment (aggregate) can be separated. Separation by filtration and/or centrifugation is preferred for ease of handling. Separation by filtration can be performed using, for example, filter paper, a filter membrane, or the like. Conditions for centrifugation are not particularly limited. For example, it can be carried out at room temperature (20±5° C.) at 8000×g to 15000×g for 5 to 20 minutes. The separation of the insoluble matter may be performed twice or more.

(gloss suppressing particles)
The luster-suppressing particles according to the present embodiment are organic polymer particles or metal oxide particles that are incompatible with the recombinant structural protein and have an average particle size of more than 0.2 μm and 10 μm or less. The term “gloss suppression” refers to suppressing (matting) the gloss of the fiber by adding unevenness to the surface of the fiber by adding the gloss suppressing particles. By appropriately selecting the particle size and amount of particles used according to the use of the fiber, the gloss of the fiber can be suppressed (matted) to a desired level, and the gloss can be controlled. Incompatible refers to the property of being incompatible or partially incompatible with the recombinant structural protein.

Any of organic polymer particles and metal oxides having a structure that is incompatible or partially incompatible with the recombinant structural protein, which is the main component, can be used as the luster-suppressing particles. One or more types of gloss-suppressing particles may be used in combination.

Examples of organic polymer particles include particles made of at least one selected from the group consisting of acrylic resins, urethane resins, acrylic urethane composite resins, silicone resins, polyamide resins, crosslinked polystyrene resins, polyarylates, fluororesins, and cellulose. It may be particles made of at least one selected from the group consisting of acrylic resins, urethane resins, acrylic urethane composite resins, silicone resins, polyamide resins and cellulose.

As the organic polymer particles, particles made of at least one selected from the group consisting of acrylic resins, urethane resins, and acrylic urethane composite resins from the viewpoint of better maintenance of fiber properties (more prevention of deterioration in fiber properties). is preferred, particles made of at least one selected from the group consisting of urethane resins and acrylic urethane composite resins are more preferred, and urethane resin particles are particularly preferred. A crosslinked acrylic resin is preferable as the acrylic resin.

The metal oxide particles may be, for example, particles made of at least one selected from the group consisting of titanium oxide and silicon dioxide, and may be titanium oxide particles.

The particles described above can be produced by known methods, but are also commercially available. For example, acrylic resin particles (GM-0449S-2 and GM-0630H manufactured by Aica Kogyo Co., Ltd.), urethane particles (GU-0700P manufactured by Aica Kogyo Co., Ltd., and C-800 transparent (Tg = -13) manufactured by Negami Kogyo Co., Ltd. ° C.), B-800T, BP-892T, BP-1092T, BP-1592T and CE-800T), acrylic urethane composite particles (TE-812T manufactured by Negami Kogyo Co., Ltd.) cellulose particles (cellulose beads manufactured by Negami Kogyo Co., Ltd. ), silk particles (Silkgen G powder manufactured by Ichimaru Farcos Co., Ltd.), soy protein particles (New Fujipro SHE and Fujipro E manufactured by Fuji Oil Co., Ltd.), titanium oxide particles (SA-1 manufactured by Sakai Chemical Industry Co., Ltd.), etc. be done. These may be used individually by 1 type, and may be used in combination of 2 or more type.

The average particle size of the gloss suppressing particles according to the present embodiment is more than 0.2 μm and 10 μm or less. The average particle size is the average value of diameters assuming that the particles are spherical particles. The average particle size can be measured by a laser diffraction/scattering method, a Coulter counter method, or the like, and calculated as a median size or an arithmetic mean size. The "average particle size" defined in the present embodiment is the median size of the particle size distribution measured using a laser diffraction particle size distribution analyzer. When the average particle size is 10 µm or less, the voids generated at the particle interface in the fiber can be further reduced. When the average particle size is more than 0.2 μm, it is possible to obtain a more luster-suppressing (matting) effect of the fibers, and it is possible to bring the natural luster closer to that of human hair. Gloss control particles having different average particle sizes may be used in combination.

The average particle size of the gloss-suppressing particles may be 1.5 μm or more and 10 μm or less, may be 1.5 μm or more and 9.0 μm or less, may be 1.5 μm or more and 9.5 μm or less, or may be 1.5 μm or more. 8.5 μm or less, 1.5 μm or more and 8.0 μm or less, 1.5 μm or more and 7.5 μm or less, or 1.5 μm or more and 7.0 μm or less; may be 5 μm or more and 6.5 μm or less, may be 1.5 μm or more and 6.0 μm or less, may be 1.5 μm or more and 5.5 μm or less, may be 1.5 μm or more and 5.0 μm or less, may be 1.5 μm or more and 4.5 μm or less, may be 1.5 μm or more and 4.0 μm or less, may be 2.0 μm or more and 10 μm or less, may be 2.0 μm or more and 9.0 μm or less, It may be 2.0 μm or more and 9.5 μm or less, may be 2.0 μm or more and 8.5 μm or less, may be 2.0 μm or more and 8.0 μm or less, or may be 2.0 μm or more and 7.5 μm or less. well, it may be 2.0 μm or more and 7.0 μm or less, it may be 2.0 μm or more and 6.5 μm or less, it may be 2.0 μm or more and 6.0 μm or less, and it may be 2.0 μm or more and 5.5 μm or less. may be 2.0 μm or more and 5.0 μm or less, may be 2.0 μm or more and 4.5 μm or less, may be 2.0 μm or more and 4.0 μm or less, or may be 2.5 μm or more and 10 μm or less may be 2.5 μm or more and 9.0 μm or less, may be 2.5 μm or more and 9.5 μm or less, may be 2.5 μm or more and 8.5 μm or less, or may be 2.5 μm or more and 8.0 μm may be 2.5 μm or more and 7.5 μm or less, may be 2.5 μm or more and 7.0 μm or less, may be 2.5 μm or more and 6.5 μm or less, or may be 2.5 μm or more and 6.5 μm or less; 0 μm or less, 2.5 μm or more and 5.5 μm or less, 2.5 μm or more and 5.0 μm or less, or 2.5 μm or more and 4.5 μm or less, or 2.5 μm 4.0 μm or less, 3.0 μm or more and 10 μm or less, 3.0 μm or more and 9.0 μm or less, 3.0 μm or more and 9.5 μm or less, or 3.0 μm 3.0 μm or more and 8.5 μm or less, may be 3.0 μm or more and 8.0 μm or less, may be 3.0 μm or more and 7.5 μm or less, or may be 3.0 μm or more and 7.0 μm or less; 0 μm or more and 6.5 μm or less, and 3.0 μm or more. μm or more and 6.0 μm or less, may be 3.0 μm or more and 5.5 μm or less, may be 3.0 μm or more and 5.0 μm or less, may be 3.0 μm or more and 4.5 μm or less, It may be 3.0 μm or more and 4.0 μm or less.

The glass transition temperature of the gloss-suppressing particles according to the present embodiment may be 200° C. or lower, 150° C. or lower, 100° C. or lower, preferably 80° C. or lower, and 70° C. or lower. is more preferably 60° C. or lower, more preferably 50° C. or lower, even more preferably 40° C. or lower, and particularly preferably 30° C. or lower. The lower the glass transition temperature, the easier it is for the particles to soften, and the more voids can be produced at the particle interface during fiber stretching.

In the artificial hair fiber of the present embodiment, the content of the luster-suppressing particles is more than 1 part by mass and 12 parts by mass or less, preferably 2 parts by mass or more and 10 parts by mass or less, relative to 100 parts by mass of the recombinant structural protein. is 3 to 10 parts by mass, 3 to 8 parts by mass, 3 to 7 parts by mass, 3 to 6 parts by mass, and 2 parts by mass ~8 parts by mass, may be 2 parts by mass to 7.5 parts by mass, may be 2 parts by mass to 7 parts by mass, may be 2 parts by mass to 6.5 parts by mass, 2 It may be from 2 parts by mass to 6 parts by mass, may be from 2 parts by mass to 5.5 parts by mass, may be from 2.5 parts by mass to 5.5 parts by mass, and may be from 4 parts by mass to 6 parts by mass. It may be 4 parts by mass to 5 parts by mass. By setting the content of the gloss-suppressing particles to more than 1 part by mass with respect to 100 parts by mass of the recombinant structural protein, the gloss-suppressing effect can be further enhanced. By setting the content of the luster-suppressing particles to 12 parts by mass or less with respect to 100 parts by mass of the recombinant structural protein, it is possible to further reduce the feeling of foreign matter when the fiber is touched.

[Method for producing fiber for artificial hair]
In the method for producing an artificial hair fiber according to the present embodiment, a dispersion containing a recombinant structural protein, luster-suppressing particles incompatible with the recombinant structural protein, and an organic solvent is ejected from a spinneret, and then the solvent is removed. forming a structural protein fiber (spinning step), the content of the gloss-suppressing particles is more than 1 part by mass and not more than 12 parts by mass with respect to 100 parts by mass of the recombinant structural protein, and the gloss-suppressing particles are It is characterized by organic polymer particles or metal oxide particles having an average particle size of 0.2 μm or more and 10 μm or less. Recombinant structural proteins and methods for their production, and gloss-reducing particles are described above.

(dispersion liquid)
The dispersion liquid according to the present embodiment contains a recombinant structural protein, gloss-suppressing particles incompatible with the recombinant structural protein, and an organic solvent, and the content of the gloss-suppressing particles is 1 mass with respect to 100 parts by mass of the recombinant structural protein. and the gloss suppressing particles are organic polymer particles or metal oxide particles having an average particle diameter of more than 0.2 µm and not more than 10 µm. The dispersion is used as a dope (spinning dope) for spinning.

Any organic solvent that can disperse or dissolve the recombinant structural protein can be used as the organic solvent according to the present embodiment. DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N -methylmorpholine N-oxide (NMO) and formic acid, and the like. From the viewpoint of better solubility of the recombinant structural protein, HFIP, DMSO and formic acid are more preferable, HFIP and formic acid are more preferable, and formic acid is particularly preferable as the solvent for dissolving the recombinant structural protein. These organic solvents may contain water. These solvents may be used singly or in combination of two or more.

The dispersion may further contain a solubility enhancer. Examples of the dissolution accelerator include inorganic salts composed of a Lewis acid and a Lewis base shown below. Lewis bases include, for example, oxoacid ions (nitrate ion, perchlorate ion, etc.), metal oxoacid ions (permanganate ion, etc.), halide ions, thiocyanate ions, cyanate ions, and the like. Examples of Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions. 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, and 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 salts such as sodium chloride, sodium bromide, sodium iodide, sodium nitrate, sodium perchlorate and sodium thiocyanate, zinc chloride, zinc bromide, zinc iodide, zinc nitrate, perchloric acid Zinc and zinc salts such as zinc thiocyanate, magnesium salts such as magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate and magnesium thiocyanate, barium chloride, barium bromide, barium iodide, Barium salts such as 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.

The content of the dissolution accelerator is 1.0 parts by mass or more, 1.5 parts by mass or more, 2.0 parts by mass or more, 2.5 parts by mass or more, or 3.0 parts by mass with respect to the total amount of 100 parts by mass of the protein. It may be more than part. The content of the dissolution accelerator may be 10 parts by mass or less, 7.0 parts by mass or less, or 5.0 parts by mass or less with respect to 100 parts by mass of the total amount of protein.

When used as a spinning stock solution, the viscosity of the dispersion according to the present embodiment may be appropriately set according to the spinning method. 000~35,000mPa・sec, 5,000~35,000mPa・sec, 7,000~35,000mPa・sec, 5,000~30,000mPa・sec, 7,000~30,000mPa・sec, 10, 000 to 30,000 mPa·sec or the like. The viscosity of the spinning stock solution can be measured, for example, using a product name "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

(Spinning process)
The artificial hair fibers or raw fibers thereof can be produced by a known spinning method. For example, first, the above-described recombinant structural protein and luster-suppressing particles are added in a desired mixing ratio to the above-described organic solvent together with an inorganic salt as a dissolution accelerator as necessary, dispersed, and dispersed in the above-described dispersion (dope solution). ) are prepared. The order of addition of the recombinant structural protein and the luster-suppressing particles may be simultaneous or sequential. Preferably, the gloss-reducing particles are dispersed in an organic solvent prior to dissolving the recombinant structural protein. Then, using this dope solution, spinning is performed by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning to obtain the desired raw material fiber. Preferred spinning methods include wet spinning or dry-wet spinning. Here, the raw material fiber means the structural protein fiber immediately after the spinning process, and the raw material fiber may be used as the artificial hair fiber as it is.

FIG. 9 is an explanatory diagram schematically showing an example of a spinning device for producing raw fibers. A spinning device 10 shown in FIG. 9 is an example of a spinning device for dry-wet spinning, and includes an extrusion device 1, an undrawn yarn manufacturing device 2, a wet-heat drawing device 3, and a drying device 4.

A spinning method using the spinning apparatus 10 will be described. First, the dope liquid 6 stored in the storage tank 7 is extruded (discharged) from the spinneret (spinneret) 9 by the gear pump 8 . On a lab scale, the dope solution may be filled into a cylinder and pushed out through a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied through the air gap 19 into the coagulation liquid 11 of the coagulation liquid bath 20, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulum is formed. The fibrous coagulum is then fed into the hot water 12 in the drawing bath 21 and drawn. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14 . After that, the drawn fibrous coagulate is supplied to the drying device 4 and dried in the yarn path 22 to obtain the raw material fiber as the wound yarn 5 . 18a-18g are thread guides.

The coagulating liquid 11 may be any solvent that can remove the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulation liquid 11 may contain water as appropriate. The temperature of the coagulation liquid 11 is preferably 0 to 30.degree. When a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the nozzle 9, the extrusion rate is preferably 0.2 to 6.0 ml/hour, preferably 1.4 to 4.0 ml/hour. It can be time. The distance over which the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the thread guide 18a to the thread guide 18b) should be sufficient for efficient removal of the solvent, for example, 200 to 500 mm. is. The undrawn yarn take-up speed may be, for example, 1 to 20 m/min, or 1 to 3 m/min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, or 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulating liquid 11 . The coagulation liquid bath 20 may be provided in multiple stages, and stretching may be performed in each stage or in a specific stage as required.

In addition to the pre-stretching performed in the coagulation bath 20 and the wet-heat stretching performed in the stretching bath 21, dry-heat stretching may also be employed as the stretching performed when obtaining the raw material fiber.

The wet heat stretching can be carried out in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating. The temperature may be, for example, 50 to 90°C, preferably 75 to 85°C. In wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, preferably 2 to 8 times.

Dry heat drawing 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, preferably 160°C to 230°C. In the dry heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, preferably 1 to 4 times.

The wet heat stretching and the dry heat stretching may be carried out individually, or they may be carried out in multiple stages or in combination. That is, the first step drawing is performed by wet heat drawing, the second step drawing is performed by dry heat drawing, or the first step drawing is performed by wet heat drawing, the second step drawing is performed by wet heat drawing, and the third step drawing is performed by dry heat drawing. For example, wet heat stretching and dry heat stretching can be combined as appropriate.

The lower limit of the final draw ratio is preferably more than 1 times, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times with respect to the undrawn yarn (or pre-drawn yarn). 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less. , 12 times or less, 11 times or less, and 10 times or less. If the raw material fiber is a fiber spun at a draw ratio of 2 or more, the shrinkage rate becomes higher when the raw material fiber is brought into contact with water to make it wet.

The method for producing fibers for artificial hair according to the present embodiment may further include a shrinking step of shrinking the raw material fibers obtained above with water. The shrinking step may include, for example, a step of irreversibly shrinking the above-described raw fibers (raw fibers after spinning but before contact with water) by contacting with water (contacting step). The shrinking step may comprise, after the contacting step, drying the fibers to further shrink them (drying step).

FIG. 10 is a diagram showing an example of length change of raw material fibers due to contact with water. The raw material fiber according to the present embodiment has a property of shrinking (change in length indicated by “primary shrinkage” in FIG. 10) when it is brought into contact with water (wet). After primary shrinkage, it shrinks further when dried (change in length indicated by “secondary shrinkage” in FIG. 10). After the secondary shrinkage, when it is brought into contact with water again, it is stretched to the same or similar length as before the secondary shrinkage. Contraction and expansion are repeated with the width shown in the "expansion ratio".

The irreversible shrinkage of raw material fibers in the contact step (“primary shrinkage” in FIG. 10) is considered to occur for the following reasons, for example. That is, one reason is considered to be due to the secondary structure and tertiary structure of the raw material fiber, and another reason is that, for example, in the raw material fiber that has residual stress due to drawing in the manufacturing process, water is added to the fiber. It is considered that the residual stress is relieved by penetrating between or into the fibers. Therefore, it is conceivable that the shrinkage ratio of the raw material fiber in the shrinking step can be arbitrarily controlled, for example, according to the magnitude of the draw ratio in the manufacturing process of the raw material fiber described above.

In the contacting step, after spinning, the raw fibers before contacting with water are brought into contact with water to wet the raw fibers. A wet state means a state in which at least part of the raw material fibers is wet with water. As a result, the raw material fibers can be shrunk without depending on an external force. This contraction is irreversible (corresponding to "primary contraction" in FIG. 10).

The temperature of the water contacting the raw fibers in the contacting step may be below the boiling point. As a result, the handleability and the workability of the shrinking process are improved. From the viewpoint of sufficiently shortening the shrinkage time, the lower limit of the water temperature is preferably 10°C or higher, more preferably 40°C or higher, and even more preferably 70°C or higher. , more preferably 80° C. or higher, more preferably 90° C. or higher, and particularly preferably 95° C. or higher. The upper limit of the temperature of water is preferably below the boiling point.

In the contact step, the method of bringing the raw material fibers into contact with water is not particularly limited. Examples of such methods include a method of immersing the raw material fibers in water, a method of spraying water onto the raw material fibers at room temperature or in the form of heated steam, and a method of immersing the raw material fibers in a high-humidity environment filled with water vapor. A method of exposure and the like can be mentioned. Among these methods, in the contacting step, the method of immersing the raw material fibers in water is preferable because the contraction time can be effectively shortened and the processing equipment can be simplified.

In the contacting step, if the raw material fibers are brought into contact with water in a relaxed state, the raw material fibers may not only shrink, but may also wrinkle. In order to prevent the occurrence of such crimps, the contact step may be performed in a state in which the raw material fibers are not relaxed, such as by contacting the raw material fibers with water while tensioning (pulling) them in the fiber axis direction.

The method for producing fibers for artificial hair according to this embodiment may further include a drying step. The drying step is a step of drying and further shrinking the raw fibers that have passed through the contacting step (or the artificial hair fibers obtained through the contacting step) (corresponding to “secondary shrinkage” in FIG. 10). Drying may be, for example, natural drying or forced drying using drying equipment. As the drying equipment, any known contact-type or non-contact-type drying equipment can be used. Also, the drying temperature is not particularly limited as long as it is lower than the temperature at which the protein contained in the raw material fiber is decomposed or the raw material fiber is thermally damaged. The temperature is in the range of -150°C, preferably in the range of 50-100°C. This temperature range allows the fibers to dry more quickly and efficiently without thermal damage to the fibers or degradation of the proteins contained in the fibers. The drying time is appropriately set according to the drying temperature and the like. For example, a time that can eliminate the influence of overdrying on the quality and physical properties of the artificial hair fibers as much as possible is adopted.

The fiber for artificial hair according to the present embodiment can be stably supplied and can suppress the occurrence of discomfort with human hair, so that it is suitably used as artificial hair.
[Artificial hair]

The artificial hair in this embodiment is not particularly limited, but examples thereof include wigs (wigs, hairpieces), extensions, hair accessories, braids, doll hair (hair for dolls) and other headdresses. The wig may be a full wig, half wig, partial wig, cube wig, top cover, bangs wig, point wig, and the like.

EXAMPLES The present invention will now be described more specifically based on examples. However, the present invention is not limited to the following examples.

[Production of recombinant structural protein]
(1) Preparation of expression vector A recombinant structural protein having SEQ ID NO: 40 (hereinafter referred to as In the amino acid sequence shown by SEQ ID NO: 40, all QQ in the amino acid sequence shown by SEQ ID NO: 7 were replaced with VF, and the remaining Q was designed for the purpose of improving hydrophobicity. is replaced with I, and the amino acid sequence shown by SEQ ID NO: 11 is added to the N-terminus.

Next, a nucleic acid encoding a recombinant structural protein (spider silk fibroin) PRT966 having the designed amino acid sequence of SEQ ID NO:40 was synthesized. The nucleic acid was added with an NdeI site at the 5' end and an EcoRI site downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was treated with restriction enzymes NdeI and EcoRI, excised, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Expression of Recombinant Structural Protein Escherichia coli BLR (DE3) was transformed with the expression vector obtained in (1). The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin to an OD ₆₀₀ of 0.005. The temperature of the culture solution was kept at 30° C., and flask culture was performed until OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 5) to an OD ₆₀₀ of 0.05. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and culture was carried out for 20 hours. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the modified fibroin. After 20 hours from the addition of IPTG, the culture solution was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture medium before and after the addition of IPTG, and the appearance of the target recombinant structural protein band dependent on the addition of IPTG confirmed the presence of the target recombinant structural protein (spider silk). fibroin) was confirmed.

(3) Purification of Recombinant Structural Protein Cells collected 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 (manufactured by 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 precipitate after washing was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg/mL, and heated at 60°C. 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, water was removed with a freeze dryer, and the freeze-dried powder was recovered to obtain a recombinant structural protein (spider silk fibroin, PRT966).

(4) Evaluation of Dispersibility of Gloss-suppressing Particles (4-1) Evaluation of Dispersibility of Gloss-suppressing Particles in Organic Solvent The dispersibility of each gloss-suppressing particle shown in Table 6 in an organic solvent was evaluated. As organic polymer particles, acrylic resin particles (GM-0449S-2 and GM-0630H manufactured by Aica Kogyo Co., Ltd.), urethane particles (GU-0700P manufactured by Aica Kogyo Co., Ltd., and C-800 transparent manufactured by Negami Kogyo Co., Ltd. ( Tg = -13 ° C.), B-800T, BP-892T, BP-1092T, BP-1592T and CE-800T), acrylic urethane composite particles (TE-812T manufactured by Negami Kogyo Co., Ltd.) cellulose particles (Negami Kogyo Co., Ltd. cellulose beads manufactured by the company), silk particles (Silkgen G powder manufactured by Ichimaru Farcos Co., Ltd.), and soy protein particles (New Fujipro SHE and Fujipro E manufactured by Fuji Oil Co., Ltd.) were used. Titanium oxide particles (SA-1 manufactured by Sakai Chemical Industry Co., Ltd.) were used as the metal oxide particles.

Formic acid as an organic solvent and each particle were stirred at room temperature to prepare a formic acid dispersion. The content of formic acid relative to the total amount of the formic acid dispersion was 97% by mass, and the content of each particle was 3% by mass. After allowing the prepared formic acid dispersion to stand still for 3 hours, the dispersibility of the particles in the formic acid dispersion was visually evaluated. Table 6 shows the evaluation results. A photograph of each formic acid dispersion is shown in FIG. Dispersibility evaluation criteria are as follows.
◎: Particles are uniformly dispersed ○: Particles partially settle or aggregate and disperse unevenly △: Phase separation occurs, but particles are partially dispersed ×: Phase separation (particles settle at the bottom or separate at the top)
Dissolution: Particles dissolve

(4-2) Evaluation of dispersibility of luster-suppressing particles in dope solution The spider silk fibroin (PRT966) obtained in the above purification step was added to the formic acid dispersion prepared in (4-1) and stirred at room temperature. It was dissolved to prepare a dope solution. The content of spider silk fibroin with respect to the total amount of the dope solution was 30% by mass. After the prepared dope solution was allowed to stand for 8 hours, the dispersibility of the particles in the dope solution was visually evaluated. The dispersibility evaluation results are shown in Table 6, and the photograph of each dispersion is shown in FIG. Dispersibility evaluation criteria are as follows.
◎: Particles are uniformly dispersed ○: Particles partially settle or aggregate and disperse unevenly △: Phase separation occurs, but particles are partially dispersed ×: Phase separation (particles settle at the bottom or separate at the top)
Dissolution: Particles dissolve

As shown in Table 6 and FIG. 6, in the formic acid dispersion containing acrylic resin particles, urethane particles, acrylic urethane composite particles, silicone resin particles, cellulose particles, titanium oxide particles and silicone particles, sedimentation, aggregation and Phase separation occurred (Comparative Examples 1-17). In the formic acid solution to which silk particles and soybean protein particles were added, all the particles dissolved in formic acid to form a solution (Comparative Examples 18 to 23). On the other hand, in the dope solution to which spider silk fibroin was added, the particles remained even after 8 hours in all cases using acrylic resin particles, urethane particles, acrylic urethane composite particles, cellulose particles, titanium oxide particles, and silicone particles. They were uniformly dispersed in the dope solution (Test Examples 1 to 17). It was shown that spider silk fibroin served as a dispersing aid.

[Production of Structural Protein Fiber (Artificial Hair Fiber)]
(1) Preparation of dope liquid (dispersion liquid) The particles shown in Tables 7, 8 and 9 and formic acid (purity 99%) were mixed and stirred at room temperature. The recombinant structural protein powder (spider silk fibroin, PRT966) obtained in the above purification step was further added and dissolved by stirring at room temperature to prepare a dope solution (dispersion solution).

Organic polymer particles and metal oxide particles were used as the luster-suppressing particles. Examples of organic polymer particles include acrylic resin particles (GM-0449S-2 manufactured by Aica Kogyo Co., Ltd.), urethane particles (CE-800T (Tg = 34 ° C.) manufactured by Negami Kogyo Co., Ltd., BP-892T (Tg = −37° C.), BP-1092T (Tg=−37° C.) and BP-1592T (Tg=−37° C.)) and acrylic urethane composite particles (TE-812T manufactured by Neagari Kogyo Co., Ltd.) were used. As metal oxide particles, titanium oxide particles (SA-1, manufactured by Sakai Chemical Industry Co., Ltd.) were used.

The contents of formic acid and spider silk fibroin in the dope solution were 70 mass % (formic acid) and 30 mass % (spider silk fibroin), respectively, with the total of formic acid and spider silk fibroin being 100 mass %. The content of particles in the dope liquid is 0.1% by mass, 1.0% by mass, 3.0% by mass, 3.5% by mass, 5.3% by mass, and 7.0% by mass with respect to spider silk fibroin. %. For comparison, a dope solution for comparison was prepared in the same manner as above except that no particles were added.

(2) Production of Structural Protein Fiber (Artificial Hair Fiber) (2-1) Wet Spinning Using a known spinning device, the dope solution (dispersion) prepared in (1) above is introduced into the coagulation solution with a gear pump. It was discharged and wet-spun. The spinning conditions were as shown below. Structural protein fibers (spider silk fibroin fibers) were thereby obtained (Examples 1 to 14). The content of gloss-reducing particles in the structural protein fibers of Examples 1-6 was about 3.4% by weight (3.5% by weight added to the dope solution). For comparison, a fiber was obtained in the same manner as described above using the dope solution prepared in (1) above and containing no particles (Comparative Example 24).
(Spinning conditions)
Spinning nozzle hole diameter: 0.3 mm
Coagulation liquid: 18% by mass sodium sulfate aqueous solution Temperature of coagulation liquid: 40°C
Water wash bath temperature: 90°C
Stretch ratio in water washing bath: 3.4 times Hot roller temperature: 60°C

(2-2) Shrinkage Treatment The structural protein fibers obtained in (2-1) were subjected to shrinkage-proof treatment by the following procedure. The fibers were cut to a length of about 60 cm and bundled to form a fiber bundle having a fineness of 150 denier. This fiber bundle was immersed (wet) in water at 90° C. for 1 hour to remove residual stress in the fiber resulting from the spinning process, and allowed to stand overnight at room temperature to dry.

(3) Physical property evaluation The knot strength [gf / D], knot elongation [%] and elastic modulus [gf / D] of the structural protein fibers obtained in (2) above were measured using Instron It was carried out using a 3345 series tensile tester manufactured by Co., Ltd. The test conditions were a temperature of 20° C., a relative humidity of 65%, a test length of 50 mm, a test speed of 50 mm/min, and a load cell capacity of 10 N. Each measurement value of the recombinant structural protein fiber was calculated as an average value of n=5 samples, which were obtained by randomly extracting 5 monofilaments from the multifilament bundle. Tables 7, 8 and 9 show the evaluation results of physical properties. The values of knot strength [gf/D] and elastic modulus [gf/D] are obtained by multiplying the knot strength [gf/D] and elastic modulus [gf/D] values of the fiber containing no particles (Comparative Example 24) by 100%. It is shown as a relative value when

(4) Gloss Evaluation The gloss of the fibers obtained in (2) above was visually evaluated under a fluorescent lamp and under sunlight. The evaluation results are shown in Tables 7, 8 and 9. The criteria for gloss evaluation are as follows.
A: Gloss to the extent that no difference from human hair can be recognized even when carefully compared B: Gloss that can be judged to be more or less glossy than human hair when carefully compared C: Gloss that can be judged to be more or less glossy than human hair when compared with normal attention D: Clearly too glossy than human hair without contrast, or , too little gloss

As shown in Table 7, when compared with a fiber containing no gloss suppressing particles (Comparative Example 24) and a fiber containing gloss suppressing particles having an average particle size of 0.2 μm (Comparative Example 25), the In the structural protein fibers (Examples 1 to 6), even when gloss-suppressing particles of acrylic resin particles, urethane particles, and acrylic urethane composite particles (organic polymer particles) were used, a decrease in knot strength was suppressed, and elasticity was improved. It maintained its elasticity and exhibited a natural gloss. In addition, an unexpectedly excellent result was obtained that the knot elongation was improved. In particular, structural protein fibers using urethane particles with an average particle size of 5.8 μm were able to obtain luster comparable to that of human hair (Example 4). In addition, spinning stability was also obtained when highly vegetable urethane (Examples 4-6) was used for the gloss control particles.

As shown in Table 8, when compared with fibers containing no gloss-suppressing particles (Comparative Example 24) and fibers with the amount of gloss-suppressing particles added of 0.1 to 1.0% by mass (Comparative Examples 26 and 27), In the recombinant structural protein fibers (Examples 7 and 8) containing more than 1.0% by mass of acrylic resin particles obtained in (2), the decrease in knot strength and elastic modulus was suppressed, and natural gloss was exhibited. bottom. In addition, an unexpectedly excellent result was obtained that the knot elongation was improved. It was confirmed that a gloss suppressing (matting) effect can be obtained by adding more than 1.0% by mass of the gloss suppressing particles.

As shown in Table 9, the structural protein fibers containing the urethane particles obtained in (2) (Examples 9 to 14) exhibited knot strength and elasticity when compared with the fiber containing no luster-suppressing particles (Comparative Example 24). The decrease in the modulus was suppressed, and a natural luster was exhibited. In addition, an unexpectedly excellent result was obtained that the knot elongation was improved. Furthermore, it was confirmed that the spinning stability was also excellent.

(5) Observation of the surface and cross section of the fiber The surface and cross section of the fiber obtained in (2) above were observed using SEM (Phenome ProX Desktop SEM, manufactured by Thermo Fisher Scientific) at a magnification of 1000 and an acceleration voltage of 15 kV. Observed. SEM images of the side and cross sections of the structural protein fibers (spider silk fibroin fibers) obtained in Examples 1 to 6 are shown in FIG. For comparison, side and cross-sectional SEM images of a fiber without particles (Comparative Example 24) are shown in FIG.

As shown in FIGS. 7 and 8, in the recombinant structural protein fibers (Examples 1 to 6) to which the luster-suppressing particles were added obtained in (2), acrylic resin particles, urethane particles, and acrylic urethane composite particles (organic polymer particles) and titanium oxide particles (metal oxide particles) (Comparative Example 25), the particles are dispersed on the fiber surface and inside the fiber without being localized. was confirmed. The holes observed in the fiber cross-section image are the particles dropped from the inside of the fiber during SEM sample preparation (cross-section cutting), or voids (voids) generated at the interface between the particles and the recombinant structural protein during stretching voids).

As described above, it was shown that the structural protein fiber of the present invention can be suitably used as an artificial hair fiber.

DESCRIPTION OF SYMBOLS 1... Extrusion apparatus 2... Undrawn yarn manufacturing apparatus 3... Wet-heat drawing apparatus 4... Drying apparatus 6... Dope liquid 10... Spinning apparatus 20... Solidification liquid tank 21... Drawing bath 36... Raw fiber.

Claims (15)

comprising a recombinant structural protein and gloss-reducing particles incompatible with the recombinant structural protein;
The content of the gloss-suppressing particles is more than 1 part by mass and 12 parts by mass or less with respect to 100 parts by mass of the recombinant structural protein,
A fiber for artificial hair, wherein the luster-suppressing particles are organic polymer particles or metal oxide particles having an average particle size of more than 0.2 μm and not more than 10 μm. 2. The artificial hair fiber according to claim 1, wherein said recombinant structural protein is at least one selected from the group consisting of spider silk fibroin, silk fibroin and keratin. The organic polymer particles are particles made of at least one selected from the group consisting of acrylic resins, urethane resins, acrylic urethane composite resins, polyamide resins and silicone resins, and the metal oxide particles are titanium oxide and silicon dioxide. 3. The artificial hair fiber according to claim 1, which is a particle made of at least one selected from the group consisting of: The artificial hair fiber according to any one of claims 1 to 3, wherein the luster suppressing particles are particles made of at least one selected from the group consisting of acrylic resins, urethane resins and acrylic urethane composite resins. The artificial hair fiber according to any one of claims 1 to 4, wherein said recombinant structural protein is spider silk fibroin. The artificial hair fiber according to any one of claims 1 to 5, wherein said recombinant structural protein is modified spider silk fibroin. The artificial hair fiber according to any one of claims 1 to 6, wherein the luster-suppressing particles have a glass transition temperature of 80°C or less. The artificial hair fiber according to any one of claims 1 to 7, wherein the luster-suppressing particles are urethane resin particles. Artificial hair comprising the fiber for artificial hair according to any one of claims 1 to 8. A method for producing fibers for artificial hair, comprising:
extruding a dispersion comprising a recombinant structural protein, gloss-reducing particles incompatible with the recombinant structural protein, and an organic solvent from a spinneret, and then removing the solvent to form a structural protein fiber;
The content of the gloss-suppressing particles is more than 1 part by mass and not more than 12 parts by mass with respect to 100 parts by mass of the recombinant structural protein, and the gloss-suppressing particles have an average particle diameter of 0.2 μm or more and 10 μm or less. Or metal oxide particles, production method. 11. The method for producing an artificial hair fiber according to claim 10, wherein said recombinant structural protein is at least one selected from the group consisting of spider silk fibroin, silk fibroin and keratin. 12. The method according to claim 10 or 11, wherein the organic polymer particles are particles made of at least one selected from the group consisting of acrylic resins, urethane resins and acrylic urethane composite resins, and the metal oxide particles are titanium oxide particles. A method for producing the described artificial hair fibers. The method for producing an artificial hair fiber according to any one of claims 10 to 12, wherein the recombinant structural protein is spider silk fibroin. The method for producing an artificial hair fiber according to any one of claims 10 to 13, wherein the luster-suppressing particles have a glass transition temperature of 80°C or lower. The method for producing fibers for artificial hair according to any one of claims 10 to 14, wherein the luster-suppressing particles are urethane resin particles.

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