JP7804285B2 - Fiber opening agent - Google Patents

Description

The present invention relates to a fiber-opening agent that is blended into a fiber-reinforced composite material containing a fiber bundle formed by bundling multiple reinforcing fibers and a matrix resin, and that promotes the opening of the fiber bundle.

Fiber-reinforced composite materials, which combine reinforcing fibers with a matrix resin, have traditionally been widely used as high-performance synthetic resin materials. For example, Patent Document 1 describes a fiber-reinforced resin material molded body containing bundles of reinforcing fibers and a matrix resin.

Japanese Patent Application Laid-Open No. 2018-080347

In fiber-reinforced composite materials, if the gaps between fiber bundles are large, resin pools may form in the gaps, resulting in reduced mechanical properties. In response to this, Patent Document 1 proposes a method for producing fiber-reinforced resin materials with minimal variation in fiber content by opening and cutting long fiber bundles.

However, the method described in Patent Document 1 required the fiber bundles to be opened and cut in advance, which resulted in low work efficiency.

The present invention aims to provide a fiber-opening agent that is blended into a fiber-reinforced composite material together with fiber bundles of reinforcing fibers and a matrix resin, and that can promote the opening of fiber bundles of reinforcing fibers during the compounding process.

One aspect of the present invention is a fiber-opening agent that is blended into a fiber-reinforced composite material containing a fiber bundle in which multiple reinforcing fibers are bundled together and a matrix resin to promote the opening of the fiber bundle, the fiber-opening agent containing a structural protein.

In one embodiment, the opening agent may contain the above structural protein as short fibers.

In one aspect, the reinforcing fibers may be short fibers.

In one embodiment, the reinforcing fibers may be carbon fibers.

In one aspect, the structural protein may include modified fibroin.

In one embodiment, the structural protein may include spider silk fibroin.

The present invention provides a fiber-opening agent that is blended into a fiber-reinforced composite material together with fiber bundles of reinforcing fibers and a matrix resin, and that can promote the opening of the fiber bundles of reinforcing fibers during the compounding process.

FIG. 1 is a schematic diagram showing an example of a domain arrangement of spider silk fibroin. FIG. 1 is a schematic diagram showing an example of a domain arrangement of spider silk fibroin. FIG. 1 is a schematic diagram showing an example of a domain arrangement of spider silk fibroin. FIG. 1 is an explanatory diagram illustrating a schematic diagram of an example of a spinning apparatus for producing structural protein fibers. 5(a) is a radar chart showing the distribution of the angles of the carbon fiber bundles relative to a reference line in the fiber reinforced composite material of Example 2-1, in 30° increments. Fig. 5(b) is a radar chart showing the distribution of the angles of the carbon fiber bundles relative to a reference line in the fiber reinforced composite material of Comparative Example 2-1, in 30° increments. 6(a) is a radar chart showing the distribution of the angles of the carbon fiber bundles relative to a reference line in the fiber reinforced composite material of Example 2-2, in 30° increments. Fig. 6(b) is a radar chart showing the distribution of the angles of the carbon fiber bundles relative to a reference line in the fiber reinforced composite material of Comparative Example 2-2, in 30° increments.

The following describes in detail a preferred embodiment of the present invention.

The opening agent of this embodiment is a fiber-reinforced composite material containing a fiber bundle of multiple reinforcing fibers and a matrix resin, and is an opening agent that promotes the opening of the reinforcing fiber bundles. The opening agent of this embodiment contains a structural protein.

Examples of structural proteins include spider silk fibroin, silk fibroin, collagen, resilin, elastin, and keratin, as well as proteins derived from these. Structural proteins may be naturally occurring or artificially produced proteins. Furthermore, the amino acid sequence of the artificial protein may be the same as or different from the amino acid sequence of the naturally occurring protein.

The structural protein is preferably fibroin, more preferably spider silk fibroin.

Naturally occurring spider silk fibroin includes, for example, spider silk fibroin produced by spiders, such as major spindle dragline silk protein, weft silk protein, and small ampullate gland protein. Major spindle dragline silk has repeating regions consisting of crystalline and amorphous regions (also called amorphous regions), giving it both high stress and elasticity. Spider silk weft silk is characterized by having no crystalline regions and repeating regions consisting of amorphous regions. Weft silk has lower stress than major spindle dragline silk, but high elasticity.

Major spindle dragline silk proteins are produced in the major ampullate gland of spiders and are characterized by their excellent toughness. Examples of major spindle dragline silk proteins include the major ampullate gland spidroins MaSp1 and MaSp2 from the orb spider (Nephila clavipes), and ADF3 and ADF4 from the orb spider (Araneus diadematus). ADF3 is one of the two major dragline silk proteins of the orb spider. Spider silk proteins derived from ADF3 are relatively easy to synthesize and have excellent properties in terms of strength, elongation, and toughness.

Weft silk proteins are produced in the flagelliform glands of spiders. Examples of weft silk proteins include flagelliform silk proteins derived from the orb-weaver spider (Nephila clavipes).

Further examples of spider silk fibroin produced by spiders include spiders belonging to the Araneus genus, such as the orb spider, the garden spider, the red orb spider, the green orb spider, and the bean spider; spiders belonging to the Neoscona genus, such as the mountain orb spider, the house spider, the dun orb spider, and the Satsuma spider; spiders belonging to the Pronus genus, such as the little orb spider; spiders belonging to the Cyrtarachne genus, such as the avian orb spider and the large orb spider; spiders belonging to the Gypsy orb spider, such as the spiny orb spider and the stingray spider; spiders belonging to the genus Asteracantha, spiders belonging to the genus Ordgarius such as the orb-weaver spider and the black widow spider, spiders belonging to the genus Argiope such as the orb-weaver spider, spiders belonging to the genus Arachnura such as the brown recluse spider, spiders belonging to the genus Acusilas such as the scraping spider, spiders belonging to the genus Cytophora such as the orb-weaver spider, the orb-weaver spider and the black widow spider, spiders belonging to the genus Poltys such as the house spider spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the Japanese bush spider, the four-headed bush spider, the Japanese bush spider, and the black bush spider, and spiders belonging to the genus Chorizopes, such as the Japanese bush spider; spiders belonging to the genus Tetragnatha, such as the long-legged spider, the long-legged spider, the broad-legged spider, and the scaly bush spider; spiders belonging to the genus Leucauge, such as the large-legged weaver spider, the small-legged weaver spider, and the white-legged weaver spider; and spider silk proteins produced by spiders belonging to the genus Orb-web spider, the orb-web spider, and the giant orb-web spider. spiders belonging to the genus Nephila such as Nephila sp., spiders belonging to the genus Menosira such as Golden Arrow spiders, spiders belonging to the genus Dyschiriognatha such as Small Red-legged Spiders, spiders belonging to the genus Latrodectus such as Black Widow Spiders, Red Back Spiders, Grey Widow Spiders and Three-spotted Widow Spiders, and spiders belonging to the family Tetragnathidae such as Euprosthenops genus.

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

The fibroin may be a modified fibroin. The modified fibroin may be, for example, one whose amino acid sequence has been modified based on the amino acid sequence of naturally occurring fibroin (e.g., one whose amino acid sequence has been modified by modifying the gene sequence of a cloned naturally occurring fibroin), or one that has been artificially designed and synthesized without relying on naturally occurring fibroin (e.g., one having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid that encodes a designed amino acid sequence).

Modified fibroin can be obtained, for example, by modifying the amino acid sequence of a cloned naturally occurring fibroin gene sequence, for example, by substituting, deleting, inserting, and/or adding one or more amino acid residues. 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 site-directed mutagenesis. Specifically, modifications can be performed in accordance with methods described in literature such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

The modified fibroin may be, for example, a fibroin containing a domain sequence represented by formula 1: [(A)n motif-REP]m, or formula 2: [(A)n motif-REP]m-(A)n motif. The modified fibroin may have further amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but are not limited to, regions that do not contain repeats of the amino acid motif characteristic of fibroin and consist of approximately 100 amino acid residues.

As used herein, the term "domain sequence" refers to an amino acid sequence that generates a crystalline region (typically corresponding to the (A)n motif in the amino acid sequence) and an amorphous region (typically corresponding to the REP in the amino acid sequence) that are specific to fibroin, and refers to an amino acid sequence represented by formula 1: [(A)n motif-REP]m or formula 2: [(A)n motif-REP]m-(A)n motif. Here, the (A)n motif represents an amino acid sequence primarily composed of alanine residues, and contains 2 to 27 amino acid residues. The number of amino acid residues in the (A)n motif may be 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. Furthermore, the ratio of the number of alanine residues to the total number of amino acid residues in the (A)n motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning composed only of alanine residues). Of the (A)n motifs present in multiple instances in a domain sequence, at least seven may be composed only of alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may also be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300, and may be an integer from 10 to 300. Multiple (A)n motifs may have the same amino acid sequence as each other or different amino acid sequences. Multiple REPs present may have the same amino acid sequence as each other or different amino acid sequences.

Specific examples of modified fibroins include modified fibroin derived from the major spinal dragline silk protein produced in the major ampullate gland of spiders (first modified fibroin), modified fibroin with a reduced content of glycine residues (second modified fibroin), modified fibroin with a reduced content of (A)n motifs (third modified fibroin), modified fibroin with a reduced content of glycine residues and (A)n motifs (fourth modified fibroin), modified fibroin with a domain sequence including regions with locally high hydrophobicity index (fifth modified fibroin), and modified fibroin with a domain sequence with a reduced content of glutamine residues (sixth modified fibroin).

A modified fibroin (first modified fibroin) derived from the major spinal dragline silk protein produced in the major ampullate gland of spiders includes a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m. In the first modified fibroin, n in formula 1 is preferably an integer from 3 to 20, more preferably an integer from 4 to 20, even more preferably an integer from 8 to 20, even more preferably an integer from 10 to 20, even more preferably an integer from 4 to 16, particularly preferably an integer from 8 to 16, and most preferably an integer from 10 to 16. In the first modified fibroin, the number of amino acid residues constituting REP in formula 1 is preferably 10 to 200, more preferably 10 to 150, even more preferably 20 to 100, and even more preferably 20 to 75. 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 preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more, of the total number of amino acid residues.

The first modified fibroin may be a protein containing units of an amino acid sequence represented by formula 1: [(A)n motif-REP]m, and whose C-terminal sequence is an amino acid sequence shown in any of SEQ ID NOs: 1 to 3, or an amino acid sequence having 90% or more homology to an amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of the C-terminal 50 amino acid residues of the amino acid sequence of ADF3 (GI: 1263287, NCBI), the amino acid sequence shown in SEQ ID NO: 2 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1.

A more specific example of the first modified fibroin is (1-i) a modified fibroin containing the amino acid sequence shown in SEQ ID NO: 4, or (1-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. The sequence identity is preferably 95% or more.

The amino acid sequence shown in SEQ ID NO:4 is an amino acid sequence of ADF3 to which 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 has been added at the N-terminus, with the repeat region from positions 1 to 13 approximately doubled and mutations made so that translation terminates at amino acid residue 1,154. The C-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO:4 is identical to the amino acid sequence shown in SEQ ID NO:3.

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

A modified fibroin with a reduced content of glycine residues (second modified fibroin) has an amino acid sequence in which the domain sequence has a reduced content of glycine residues compared to naturally occurring spider silk fibroin. The second modified fibroin can be said to have an amino acid sequence in which, compared to naturally occurring spider silk fibroin, at least one or more glycine residues in REP have been replaced with another amino acid residue.

The second modified fibroin may have an amino acid sequence whose domain sequence, compared to naturally occurring spider silk fibroin, corresponds to at least one motif sequence selected from GGX and GPGXX (where G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP, in which one glycine residue in at least one or more of the motif sequences has been replaced with another amino acid residue.

The second modified fibroin may have a proportion of motif sequences in which the above-mentioned glycine residues are replaced with other amino acid residues of 10% or more of the total motif sequences.

The second modified fibroin may have an amino acid sequence comprising a domain sequence represented by formula 1: [(A)n motif-REP]m, in which z represents the total number of amino acid residues in an amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the domain sequence excluding the sequence from the C-terminal (A)n motif to the C-terminus of the domain sequence, and w represents the total number of amino acid residues in the domain sequence excluding the sequence from the C-terminal (A)n motif to the C-terminus of the domain sequence, with z/w being 30% or more, 40% or more, 50% or more, or 50.9% or more. The number of alanine residues relative to the total number of amino acid residues in the (A)n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning composed exclusively of alanine residues).

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. The second modified fibroin preferably has a content of the amino acid sequence consisting of GGX in the domain sequence of 30% or less, more preferably 20% or less, even more preferably 10% or less, even more preferably 6% or less, even more preferably 4% or less, and particularly preferably 2% or less. The content of the amino acid sequence consisting of GGX in the domain sequence can be calculated using a method similar to the method for calculating the content (z/w) of the amino acid sequence consisting of XGX below.

The method for calculating z/w will be explained in more detail. First, in spider silk fibroin (modified fibroin) containing a domain sequence represented by Formula 1: [(A)n motif-REP]m, amino acid sequences consisting of XGX are extracted from all REPs contained in the domain sequence, excluding the sequence from the (A)n motif located at the most C-terminal end to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, if 50 amino acid sequences consisting of XGX are extracted (no overlaps), z is 50 x 3 = 150. Furthermore, if there are Xs (middle Xs) contained in two XGXs, such as in the case of an amino acid sequence consisting of XGXGX, the overlaps are deducted from the calculation (5 amino acid residues in the case of XGXGX). w is the total number of amino acid residues contained in the sequence, excluding the sequence from the (A)n motif located at the most C-terminus to the C-terminus of the domain sequence. For example, in the case of the domain sequence shown in Figure 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A)n motif located at the most C-terminal side). Next, z/w (%) can be calculated by dividing z by w.

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, even more preferably 70% or more, and even more preferably 80% or more. There is no particular upper limit to z/w, but it may be, for example, 95% or less.

The second modified fibroin can be obtained, for example, by substituting at least a portion of the base sequence encoding a glycine residue from the cloned gene sequence of naturally occurring spider silk fibroin, thereby modifying it so that it encodes a different amino acid residue. In this case, one glycine residue in the GGX motif or GPGXX motif may be selected as the modified glycine residue, or the substitution may be made so that the z/w ratio is 50.9% or greater. Alternatively, the second modified fibroin can be obtained by, for example, designing an amino acid sequence that satisfies the above-described requirements from the amino acid sequence of naturally occurring spider silk fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification equivalent to substituting a glycine residue in REP with a different amino acid residue from the amino acid sequence of naturally occurring spider silk fibroin, the amino acid sequence may also be modified by substituting, deleting, inserting, and/or adding one or more amino acid residues.

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

More specific examples of the second modified fibroin include (2-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

The modified fibroin (2-i) will now be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by replacing all GGX with GQX in the REP of the amino acid sequence shown in SEQ ID NO: 10, which corresponds to naturally occurring spider silk fibroin. The amino acid sequence shown in SEQ ID NO: 7 is obtained by deleting every third (A)n motif from the N-terminus to the C-terminus of the amino acid sequence shown in SEQ ID NO: 6, and then inserting one [(A)n motif-REP] just before the C-terminus sequence. The amino acid sequence shown in SEQ ID NO: 8 is obtained by inserting two alanine residues into the C-terminus of each (A)n motif of the amino acid sequence shown in SEQ ID NO: 7, substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the N-terminus so that the molecular weight is approximately the same as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a sequence in which a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 11 is repeated four times (however, several amino acid residues on the C-terminal side of the region have been substituted), and a His tag has been added to the C-terminus of the sequence.

The z/w value for the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) is 46.8%. The z/w values for the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. Furthermore, the x/y values at a Giza ratio (described below) of 1:1.8 to 11.3 for 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 are 15.0%, 15.0%, 93.4%, 92.7%, and 89.3%, respectively.

The modified fibroin (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 that has 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 (2-ii) is also a protein that contains a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

The modified fibroin (2-ii) preferably 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 when 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 REP is z and the total number of amino acid residues in REP in the above domain sequence is w, z/w is 50.9% or more.

The second modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows for the isolation, immobilization, detection, visualization, etc. of the modified fibroin.

Examples of tag sequences include affinity tags that utilize specific affinity (binding ability, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). A His tag is a short peptide consisting of approximately 4 to 10 histidine residues, and has the property of specifically binding to metal ions such as nickel, making it useful for isolating modified fibroin by chelating metal chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 12 (an amino acid sequence including a His tag sequence and a hinge sequence).

Tag sequences such as glutathione S-transferase (GST), which specifically binds to glutathione, and maltose binding protein (MBP), which specifically binds to maltose, can also be used.

Furthermore, "epitope tags" that utilize antigen-antibody reactions can also be used. By adding an antigenic peptide (epitope) as a tag sequence, antibodies specific to the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tags, myc tags, and FLAG tags. By using epitope tags, modified fibroin can be easily purified with high specificity.

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

More specific examples of the second modified fibroin containing a tag sequence include (2-iii) a modified fibroin containing an amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.

The amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding the amino acid sequence shown in SEQ ID NO: 12 (including a His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

The modified fibroin (2-iii) may consist of the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin (2-iv) is also a protein comprising a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

The modified fibroin (2-iv) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, and has a z/w ratio of 50.9% or more, where z is 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 REP, and w is the total number of amino acid residues in REP in the domain sequence.

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

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

The third modified fibroin may have an amino acid sequence corresponding to a deletion of 10 to 40% of the (A)n motif from naturally occurring spider silk fibroin.

The third modified fibroin may have an amino acid sequence whose domain sequence corresponds to the deletion of one (A)n motif from at least the N-terminus to the C-terminus for every one to three (A)n motifs, compared to naturally occurring spider silk fibroin.

The third modified fibroin may have an amino acid sequence whose domain sequence, compared to naturally occurring spider silk fibroin, corresponds to the deletion of at least two consecutive (A)n motifs from the N-terminus to the C-terminus, and one (A)n motif repeated in this order.

The third modified fibroin may have an amino acid sequence in which its domain sequence is such that at least every third (A)n motif is deleted from the N-terminus to the C-terminus.

The third modified fibroin may have an amino acid sequence comprising a domain sequence represented by formula 1: [(A)n-motif-REP]m, and may have an amino acid sequence in which, when the numbers of amino acid residues in the REP of two adjacent [(A)n-motif-REP] units are compared sequentially from the N-terminus to the C-terminus, the ratio of the number of amino acid residues in the REP of the REP with the fewer amino acid residues is defined as 1, and the maximum sum of the numbers of amino acid residues in two adjacent [(A)n-motif-REP] units such that the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 is defined as x, and the total number of amino acid residues in the domain sequence is defined as y, such that x/y is 20% or more, 30% or more, 40% or more, or 50% or more. The number of alanine residues relative to the total number of amino acid residues in the (A)n-motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning composed exclusively of alanine residues).

The method for calculating x/y will be explained in more detail with reference to Figure 1. Figure 1 shows the domain sequence of spider silk fibroin excluding the N-terminal and C-terminal sequences. The domain sequence has the following sequence from the N-terminus (left side): (A)n motif - first REP (50 amino acid residues) - (A)n motif - second REP (100 amino acid residues) - (A)n motif - third REP (10 amino acid residues) - (A)n motif - fourth REP (20 amino acid residues) - (A)n motif - fifth REP (30 amino acid residues) - (A)n motif.

Two adjacent [(A)n motif-REP] units are selected sequentially from the N-terminus to the C-terminus, with no overlap. At this time, some [(A)n motif-REP] units may not be selected. Figure 1 shows Pattern 1 (comparison of the first REP with the second REP, and the third REP with the fourth REP), Pattern 2 (comparison of the first REP with the second REP, and the fourth REP with the fifth REP), Pattern 3 (comparison of the second REP with the third REP, and the fourth REP with the fifth REP), and Pattern 4 (comparison of the first REP with the second REP). Note that other selection methods also exist.

Next, for each pattern, the number of amino acid residues in each REP in the two selected adjacent [(A)n motif-REP] units is compared. The comparison is performed by determining the ratio of the number of amino acid residues in the one with the fewer amino acid residues, where the number of amino acid residues in the other is set to 1. For example, when comparing the first REP (50 amino acid residues) with the second REP (100 amino acid residues), the ratio of the number of amino acid residues in the second REP is 100/50 = 2 when the number of amino acid residues in the first REP, which has the fewer amino acid residues, is set to 1. Similarly, when comparing the fourth REP (20 amino acid residues) with the fifth REP (30 amino acid residues), the ratio of the number of amino acid residues in the fifth REP is 30/20 = 1.5 when the number of amino acid residues in the fourth REP, which has the fewer amino acid residues, is set to 1.

In Figure 1, pairs of [(A)n motif-REP] units where the ratio of the number of amino acid residues in the other unit to the number of amino acid residues in the unit with the fewer residues is 1 is shown by solid lines. Hereinafter, such ratios will be referred to as Giza ratios. Pairs of [(A)n motif-REP] units where the ratio of the number of amino acid residues in the other unit to the number of amino acid residues in the unit with the fewer residues is 1 is shown by dashed lines.

For each pattern, the total number of amino acid residues in the two adjacent [(A)n motif-REP] units indicated by solid lines is added together (not only the number of REP but also the number of amino acid residues in the (A)n motif). The total sums are then compared, and the total value (maximum total value) of the pattern with the largest total value is designated as x. In the example shown in Figure 1, the total value of pattern 1 is the largest.

Next, 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, even more preferably 70% or more, even more preferably 75% or more, and particularly preferably 80% or more. There is no particular upper limit to x/y, and it may be, for example, 100% or less. When the knurling ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more. When the knurling ratio is 1:1.8 to 3.4, x/y is preferably 77.1% or more. When the knurling ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more. When the knurling 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 seven of the (A)n motifs present in the domain sequence are composed solely of alanine residues, x/y is preferably 46.4% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and particularly preferably 80% or more. There is no particular upper limit to x/y, as long as it is 100% or less.

The third modified fibroin can be obtained, for example, by deleting one or more sequences encoding the (A)n motif from the gene sequence of cloned naturally occurring spider silk fibroin so that x/y is 64.2% or greater. Alternatively, for example, it can be obtained by designing an amino acid sequence corresponding to the deletion of one or more (A)n motifs from the amino acid sequence of naturally occurring spider silk fibroin so that x/y is 64.2% or greater, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the deletion of the (A)n motif from the amino acid sequence of naturally occurring spider silk fibroin, the amino acid sequence may also be modified by substituting, deleting, inserting, and/or adding one or more amino acid residues.

More specific examples of the third modified fibroin include (3-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

The modified fibroin (3-i) will now be described. The amino acid sequence shown in SEQ ID NO: 18 is obtained by deleting every third (A)n motif from the N-terminus to the C-terminus of the amino acid sequence shown in SEQ ID NO: 10, which corresponds to naturally occurring spider silk fibroin, and then inserting one [(A)n motif-REP] just before the C-terminus. The amino acid sequence shown in SEQ ID NO: 7 is obtained by replacing all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 18 with GQX. The amino acid sequence shown in SEQ ID NO: 8 is obtained by inserting two alanine residues into the C-terminus of each (A)n motif of the amino acid sequence shown in SEQ ID NO: 7, substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the N-terminus so that the molecular weight is approximately the same as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a sequence in which a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 11 is repeated four times (however, several amino acid residues on the C-terminal side of the region have been substituted), and a His tag has been added to the C-terminus of the sequence.

The x/y value for the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) at a jaggedness ratio of 1:1.8-11.3 is 15.0%. The x/y values for the amino acid sequence shown in SEQ ID NO: 18 and the amino acid sequence shown in SEQ ID NO: 7 are both 93.4%. The x/y value for the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The x/y value for the amino acid sequence shown in SEQ ID NO: 9 is 89.3%. The z/w values for the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively.

The modified fibroin (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin (3-ii) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin (3-ii) is also a protein that contains a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

The modified fibroin (3-ii) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and when the numbers of amino acid residues in the REP of two adjacent [(A)n motif-REP] units are compared sequentially from the N-terminus to the C-terminus, and the number of amino acid residues in the REP with the fewer amino acid residues is set to 1, the maximum value of the sum of the numbers of amino acid residues in two adjacent [(A)n motif-REP] units in which the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 (Giza ratio of 1:1.8 to 11.3) is defined as x, and y is the total number of amino acid residues in the domain sequence, and x/y is 64.2% or more.

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

More specific examples of the third modified fibroin containing a tag sequence include (3-iii) a modified fibroin containing an amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.

The amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding the amino acid sequence shown in SEQ ID NO: 12 (including a His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

The modified fibroin (3-iii) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin (3-iv) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin (3-iv) is also a protein that contains a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

The modified fibroin (3-iv) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, and when the numbers of amino acid residues in the REP of two adjacent [(A)n motif-REP] units are compared sequentially from the N-terminus to the C-terminus, and the number of amino acid residues in the REP with the fewer amino acid residues is set to 1, the maximum value of the sum of the numbers of amino acid residues in two adjacent [(A)n motif-REP] units such that the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 is defined as x, and the total number of amino acid residues in the domain sequence is y, x/y is 64.2% or more.

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

A modified fibroin (fourth modified fibroin) with a reduced content of glycine residues and (A)n motifs has an amino acid sequence in which the content of (A)n motifs is reduced and the content of glycine residues is reduced compared to naturally occurring spider silk fibroin. The domain sequence of the fourth modified fibroin can be said to have an amino acid sequence in which, compared to naturally occurring spider silk fibroin, at least one or more (A)n motifs are deleted and at least one or more glycine residues in REP are substituted with other amino acid residues. In other words, the fourth modified fibroin is a modified fibroin that combines the characteristics of the modified fibroin (second modified fibroin) with a reduced content of glycine residues and the modified fibroin (third modified fibroin) with a reduced content of (A)n motifs. Specific aspects are as described for the second modified fibroin and the third modified fibroin.

More specific examples of the fourth modified fibroin include (4-i) modified fibroin containing the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, and (4-ii) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. Specific embodiments of modified fibroin containing the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 are as described above.

A modified fibroin (fifth modified fibroin) having a domain sequence including a region with a high local hydrophobicity index may have an amino acid sequence including a region with a high local hydrophobicity index, which corresponds to one or more amino acid residues in REP being replaced with amino acid residues with a high hydrophobicity index and/or one or more amino acid residues with a high hydrophobicity index being inserted into REP, compared to naturally occurring spider silk fibroin.

It is preferable that the region with a high local hydrophobicity index consists of 2 to 4 consecutive amino acid residues.

It is more preferable that the amino acid residues with a high hydrophobicity index mentioned above are amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A).

The fifth modified fibroin may, in addition to modifications corresponding to the substitution of one or more amino acid residues in REP with amino acid residues having a high hydrophobic index and/or the insertion of one or more amino acid residues having a high hydrophobic index into REP compared to naturally occurring spider silk fibroin, also have modifications in the amino acid sequence corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues compared to naturally occurring spider silk fibroin.

The fifth modified fibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in REP from the gene sequence of cloned naturally occurring spider silk fibroin with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index), and/or by inserting one or more hydrophobic amino acid residues into REP. Alternatively, for example, it can be obtained by designing an amino acid sequence corresponding to the substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and/or the insertion of one or more hydrophobic amino acid residues into REP, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues and/or the insertion of one or more hydrophobic amino acid residues into REP from the amino acid sequence of naturally occurring spider silk fibroin, the amino acid sequence may also be modified by further substituting, deleting, inserting, and/or adding one or more amino acid residues.

The fifth modified fibroin may have an amino acid sequence that includes a domain sequence represented by formula 1: [(A)n motif-REP]m, and in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located most C-terminally to the C-terminus of the domain sequence, p is the total number of amino acid residues contained in a region where the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more, and q is the total number of amino acid residues contained in the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located most C-terminally to the C-terminus of the domain sequence, such that p/q is 6.2% or more.

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

The method for calculating p/q will be explained in more detail. For the calculation, a sequence (hereinafter referred to as "sequence A") is used, which is obtained by excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence represented by Equation 1: [(A)n motif-REP]m. First, the average hydrophobicity index of four consecutive amino acid residues is calculated for all REPs included in sequence A. The average hydrophobicity index is calculated by dividing the sum of the HIs of each amino acid residue included in the four consecutive amino acid residues by 4 (the number of amino acid residues). The average hydrophobicity index is calculated for all four consecutive amino acid residues (each amino acid residue is used to calculate the average one to four times). Next, a region where the average hydrophobicity index of four consecutive amino acid residues is 2.6 or higher is identified. Even if a certain amino acid residue falls into multiple "sequences of four consecutive amino acid residues with an average hydrophobicity index of 2.6 or higher," it is still included as a single amino acid residue in the region. The total number of amino acid residues included in the region is p. The total number of amino acid residues included in sequence A is q.

For example, if 20 "sequences of four consecutive amino acid residues with an average hydrophobicity index of 2.6 or higher" are extracted (with no overlaps), the region where the average hydrophobicity index of the four consecutive amino acid residues is 2.6 or higher will contain 20 consecutive four amino acid residues (with no overlaps), and p is 20 x 4 = 80. Also, if two "sequences of four consecutive amino acid residues with an average hydrophobicity index of 2.6 or higher" overlap by one amino acid residue, the region where the average hydrophobicity index of the four consecutive amino acid residues is 2.6 or higher will contain seven amino acid residues (p = 2 x 4 - 1 = 7, where "-1" is the deduction of the overlap). For example, in the domain sequence shown in Figure 2, there are seven "sequences of four consecutive amino acid residues with an average hydrophobicity index of 2.6 or higher" without overlaps, so p is 7 x 4 = 28. For example, in the case of the domain sequence shown in Figure 2, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (not including the (A)n motif at the end of the C-terminus). Next, p can be divided by q to calculate p/q (%). In the case of Figure 2, the result is 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, even more preferably 20% or more, and even more preferably 30% or more. The upper limit of p/q is not particularly limited, but may be, for example, 45% or less.

The fifth modified fibroin can be obtained, for example, by modifying the amino acid sequence of cloned naturally occurring spider silk fibroin to include a region with a high hydrophobicity index locally, by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in the REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index) and/or by inserting one or more hydrophobic amino acid residues into the REP so that the above-mentioned p/q condition is satisfied. Alternatively, the fifth modified fibroin can be obtained, for example, by designing an amino acid sequence that satisfies the above-mentioned p/q condition from the amino acid sequence of naturally occurring spider silk fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to modifications corresponding to the substitution of one or more amino acid residues in the REP with amino acid residues with a high hydrophobicity index and/or the insertion of one or more amino acid residues with a high hydrophobicity index into the REP, modifications corresponding to the substitution, deletion, insertion, and/or addition of one or more amino acid residues may also be made.

There are no particular restrictions on amino acid residues with a high hydrophobicity index, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are preferred, with valine (V), leucine (L) and isoleucine (I) being more preferred.

Specific examples of the fifth modified fibroin include (5-i) a modified fibroin containing an amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or (5-ii) 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 (5-i) will now be described. The amino acid sequence shown in SEQ ID NO: 22 is obtained by deleting consecutive alanine residues in the (A)n motif of naturally occurring spider silk fibroin so that the number of consecutive alanine residues is five. The amino acid sequence shown in SEQ ID NO: 19 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP in the amino acid sequence shown in SEQ ID NO: 22 at two positions, and deleting some amino acids on the C-terminus so that the molecular weight is approximately the same as that of the amino acid sequence shown in SEQ ID NO: 22. The amino acid sequence shown in SEQ ID NO: 23 is obtained by inserting two alanine residues at the C-terminus of each (A)n motif in the amino acid sequence shown in SEQ ID NO: 22, substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the C-terminus so that the molecular weight is approximately the same as that of the amino acid sequence shown in SEQ ID NO: 22. The amino acid sequence shown in SEQ ID NO: 20 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP into one position in the amino acid sequence shown in SEQ ID NO: 23. The amino acid sequence shown in SEQ ID NO: 21 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP into two positions in the amino acid sequence shown in SEQ ID NO: 23.

The modified fibroin (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 that 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. The modified fibroin (5-ii) is also a protein that contains a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

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

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

More specific examples of the fifth modified fibroin containing a tag sequence include (5-iii) a modified fibroin containing an amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or (5-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

The amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 are obtained by adding the amino acid sequence shown in SEQ ID NO: 12 (including a His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

The modified fibroin (5-iii) may consist of the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

The modified fibroin (5-iv) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. The modified fibroin (5-iv) is also a protein that contains a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

The modified fibroin (5-iv) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, and in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located most C-terminally to the C-terminus of the domain sequence, where p is the total number of amino acid residues contained in a region where the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more, and q is the total number of amino acid residues contained in the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located most C-terminally to the C-terminus of the domain sequence, p/q is 6.2% or more.

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

A modified fibroin (sixth modified fibroin) having a domain sequence with a reduced content of glutamine residues has an amino acid sequence with a reduced content of glutamine residues compared to naturally occurring spider silk fibroin.

It is preferable that the sixth modified fibroin contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

When the sixth modified fibroin contains a GPGXX motif in the REP, the GPGXX motif content is typically 1% or more, may be 5% or more, and preferably 10% or more. There is no particular upper limit to the GPGXX motif content, and it may be 50% or less, or may be 30% or less.

In this specification, the "GPGXX motif content" is a value calculated by the following method.
In spider silk fibroin containing a domain sequence represented by formula 1: [(A)n motif-REP]m or formula 2: [(A)n motif-REP]m-(A)n motif, the GPGXX motif content is calculated as s/t, where s is three times the total number of GPGXX motifs contained in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence (i.e., equivalent to the total number of Gs and Ps in the GPGXX motifs), and t is the total number of amino acid residues in all REPs obtained by excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence and further excluding the (A)n motif.

When calculating the GPGXX motif content, the target is "the sequence excluding the sequence from the C-terminal (A)n motif to the C-terminus of the domain sequence from the domain sequence" because the "sequence from the C-terminal (A)n motif to the C-terminus of the domain sequence" (the sequence corresponding to REP) may contain a sequence that has low correlation with the sequence characteristic of spider silk fibroin, and when m is small (i.e., when the domain sequence is short), this affects the calculation results of the GPGXX motif content, so this influence must be eliminated. Furthermore, 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."

Figure 3 is a schematic diagram showing the domain sequence of spider silk fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to Figure 3. First, in the spider silk fibroin domain sequence shown in Figure 3 (which is of the "[(A)n motif-REP]m-(A)n motif" type), all REPs are contained in "the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence shown as "Region A" in Figure 3). Therefore, the number of GPGXX motifs required to calculate s is 7, and s is 7 x 3 = 21. Similarly, all REPs are contained in "the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence shown as "Region A" in Figure 3). Therefore, the total number of amino acid residues t of all REPs obtained by further excluding the (A)n motif from this 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% in the case of the 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%.

In this specification, the "glutamine residue content" is a value calculated by the following method.
In spider silk fibroin containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif, in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence (the sequence corresponding to "Region A" in Figure 3), the glutamine residue content is calculated as u/t, where u is the total number of glutamine residues contained in that region, and t is the total number of amino acid residues in all REPs excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence. The reason for targeting "the sequence obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence" in calculating the glutamine residue content is the same as that described above.

The sixth modified fibroin may have an amino acid sequence whose domain sequence corresponds to the deletion of one or more glutamine residues in REP or the substitution of other amino acid residues, compared to naturally occurring spider silk fibroin.

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

As shown in Table 1, amino acid residues with a higher hydrophobicity index than glutamine residues include those selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A) are more preferred, and amino acid residues selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) are even more preferred.

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

In this specification, the "hydrophobicity of REP" is a value calculated by the following method.
In spider silk fibroin containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif, in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence (the sequence corresponding to "Region A" in Figure 3), the sum of the hydrophobicity indices of each amino acid residue in that region is v, and the total number of amino acid residues in all REPs excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence and further excluding the (A)n motif is t. The reason for targeting "the sequence obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence" in calculating the hydrophobicity of REP is the same as that described above.

The sixth modified fibroin may have a domain sequence that, compared to naturally occurring spider silk fibroin, is modified by deleting one or more glutamine residues in REP and/or substituting one or more glutamine residues in REP with other amino acid residues, and may also have a further amino acid sequence modification that is equivalent to substituting, deleting, inserting and/or adding one or more amino acid residues.

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

More specific examples of the sixth modified fibroin include (6-i) a modified fibroin comprising the amino acid sequence shown in 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, or (6-ii) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in 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.

We will explain the modified fibroin (6-i).

The amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) is based on the base sequence and amino acid sequence of the naturally occurring fibroin of Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), with amino acid modifications made to improve productivity, such as increasing the number of consecutive alanine residues in the (A)n motif to five. Meanwhile, because no modifications have been made to the glutamine residue (Q) in Met-PRT410, the glutamine residue content is similar to that of naturally occurring fibroin.

The amino acid sequence shown in SEQ ID NO: 27 (M_PRT888) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VLs.

The amino acid sequence shown in SEQ ID NO: 28 (M_PRT965) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with TS and replacing the remaining Qs with A.

The amino acid sequence shown in SEQ ID NO: 29 (M_PRT889) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VL and replacing the remaining Qs with I.

The amino acid sequence shown in SEQ ID NO: 30 (M_PRT916) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VI and replacing the remaining Qs with Ls.

The amino acid sequence shown in SEQ ID NO: 31 (M_PRT918) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VF and replacing the remaining Qs with I.

The amino acid sequence (M_PRT525) shown in SEQ ID NO: 34 is obtained by inserting two alanine residues into the region (A5) of consecutive alanine residues in Met-PRT410 (SEQ ID NO: 7), deleting two domain sequences on the C-terminal side to make the molecular weight approximately the same as that of Met-PRT410, and replacing 13 glutamine residues (Q) with serine residues (S) or proline residues (P).

The amino acid sequence shown in SEQ ID NO: 32 (M_PRT699) is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 34) with VLs.

The amino acid sequence shown in SEQ ID NO: 33 (M_PRT698) is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 34) with VL and replacing the remaining Qs with I.

The amino acid sequence shown in SEQ ID NO: 43 (Met-PRT966) is the amino acid sequence shown in SEQ ID NO: 9 (the amino acid sequence before the amino acid sequence shown in SEQ ID NO: 42 is added to the C-terminus) in which all QQs have been replaced with VFs and the remaining Qs have been replaced with Is.

The amino acid sequences shown in 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 and SEQ ID NO: 43 all have a glutamine residue content of 9% or less (Table 2).

The modified fibroin (6-i) may consist of the amino acid sequence shown in 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.

The modified fibroin (6-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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. The modified fibroin (6-ii) is also 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. The sequence identity is preferably 95% or more.

The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less. Furthermore, 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, which enables the isolation, immobilization, detection, visualization, etc. of the modified fibroin.

More specific examples of the sixth modified fibroin containing a tag sequence include (6-iii) a modified fibroin containing the amino acid sequence shown in 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: 41, or SEQ ID NO: 44, or (6-iv) a modified fibroin containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in 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: 41, or SEQ ID NO: 44.

The amino acid sequences represented by SEQ ID NOs:35, 36, 37, 38, 39, 40, 41, and 44 are the amino acid sequences represented by SEQ ID NOs:27, 28, 29, 30, 31, 32, 33, and 43, respectively, with the amino acid sequence represented by SEQ ID NO:12 (including a His tag sequence and hinge sequence) added to the N-terminus. Because only a tag sequence has been added to the N-terminus, there is no change in the glutamine residue content, and the amino acid sequences represented by SEQ ID NOs:35, 36, 37, 38, 39, 40, 41, and 44 all have a glutamine residue content of 9% or less (Table 3).

The modified fibroin (6-iii) may consist of the amino acid sequence shown in 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: 41 or SEQ ID NO: 44.

The modified fibroin (6-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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: 41, or SEQ ID NO: 44. The modified fibroin (6-iv) is also 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. The sequence identity is preferably 95% or more.

The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less. Furthermore, 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 outside the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

The modified fibroin may be a modified fibroin that combines 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.

The modified fibroin may be hydrophilic or hydrophobic. A hydrophobic modified fibroin is a modified fibroin in which the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin is calculated and then this sum is divided by the total number of amino acid residues (average HI) to obtain a value of 0 or greater. The hydrophobicity index is as shown in Table 1. A hydrophilic modified fibroin is a modified fibroin in which the above average HI is less than 0.

An example of a hydrophobic modified fibroin is the sixth modified fibroin described above. More specific examples of hydrophobic modified fibroins include modified fibroins containing the amino acid sequences shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:43, and the amino acid sequences shown in SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:44.

Examples of hydrophilic modified fibroins include the first, second, third, fourth, and fifth modified fibroins described above. More specific examples of hydrophilic spider silk proteins include modified fibroins containing 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, the amino acid sequence shown in SEQ ID NO:13, SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:15, the amino acid sequence shown in SEQ ID NO:18, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:15, or the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

The above-mentioned modified fibroins can be used alone or in combination of two or more types.

The modified fibroin can be produced, for example, by expressing the nucleic acid using a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence.

There are no particular limitations on the method for producing a nucleic acid encoding a modified fibroin. For example, the nucleic acid can be produced by amplifying and cloning a gene encoding the modified fibroin using techniques such as polymerase chain reaction (PCR), or by chemical synthesis. The method for chemically synthesizing the nucleic acid is also not particularly limited. For example, the gene can be chemically synthesized by linking oligonucleotides automatically synthesized using an AKTA Oligopilot Plus 10/100 (GE Healthcare Japan, Ltd.) or similar equipment using PCR, based on amino acid sequence information for spider silk proteins obtained from the NCBI web database. In this case, to facilitate the purification and/or identification of the modified fibroin, a nucleic acid encoding the modified fibroin may be synthesized that has an amino acid sequence to which an initiation codon and a His10 tag have been added at the N-terminus.

Regulatory sequences are sequences (e.g., promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.) that control the expression of recombinant proteins in hosts and can be selected appropriately depending on the type of host. The promoter may be an inducible promoter that functions in host cells and is capable of inducing the expression of the desired modified fibroin. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be selected appropriately depending on the type of host, such as a plasmid vector, viral vector, cosmid vector, fosmid vector, or artificial chromosome vector. Expression vectors that are capable of autonomous replication in host cells or integration into host chromosomes and contain a promoter in a position that allows transcription of a nucleic acid encoding the modified fibroin are preferably used.

Prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can all be suitably used as hosts.

When using a prokaryote such as a bacterium as a host, the expression vector is preferably a vector that is capable of autonomous replication in the prokaryote and that contains a promoter, a ribosome binding sequence, a nucleic acid encoding the modified fibroin, and a transcription termination sequence. A gene that controls the promoter may also be included.

Prokaryotes include microorganisms belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphilum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

When a prokaryote is used as a host, examples of vectors into which nucleic acids encoding modified fibroin can be introduced include pBTrp2 (Boehringer Mannheim), pGEX (Pharmacia), pUC18, pBluescript II, pSupex, pET22b, pCold, pUB110, and pNCO2 (Japanese Patent Application Laid-Open No. 2002-238569).

Eukaryotic hosts include, for example, yeast and filamentous fungi (molds, etc.). Yeasts include, for example, yeasts belonging to the genera Saccharomyces, Pichia, and Schizosaccharomyces. Filamentous fungi include, for example, filamentous fungi belonging to the genera Aspergillus, Penicillium, and Trichoderma.

When a eukaryote is used as the host, examples of vectors for introducing a nucleic acid encoding a modified fibroin include YEp13 (ATCC 37115) and YEp24 (ATCC 37051). Any method for introducing DNA into the host cell can be used to introduce the expression vector into the host cell. Examples include the calcium ion method [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, the spheroplast method, the protoplast method, the lithium acetate method, and the competent method.

In addition to direct expression, nucleic acids can be expressed by a host transformed with an expression vector through secretory production, fusion protein expression, etc., in accordance with the methods described in Molecular Cloning, 2nd Edition.

Modified fibroin can be produced, for example, by culturing a transformed host in a culture medium, producing and accumulating modified fibroin in the culture medium, and then collecting the modified fibroin from the culture medium. The method for culturing a transformed host in a culture medium can be performed according to a method typically used for culturing hosts.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the host and allows efficient cultivation of the host.

Any carbon source that can be assimilated by the host can be used, including, for example, carbohydrates such as glucose, fructose, sucrose, and those containing these, such as molasses, starch, and starch hydrolysates; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.

Nitrogen sources that can be used include, for example, ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria and their digested products.

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.

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

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during cultivation. When culturing microorganisms transformed with an expression vector that uses an inducible promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium when culturing microorganisms transformed with an expression vector that uses the lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing microorganisms transformed with an expression vector that uses the trp promoter.

The modified fibroin produced by the transformed host can be isolated and purified using methods commonly used for isolating and purifying proteins. For example, if the modified fibroin is expressed in a soluble state within the cells, after culturing, the host cells are collected by centrifugation and suspended in an aqueous buffer solution. The host cells are then disrupted using an ultrasonic homogenizer, French press, Manton-Gaulin homogenizer, Dynomill, or the like to obtain a cell-free extract. A purified sample can be obtained from the supernatant obtained by centrifuging the cell-free extract using methods commonly used for isolating and purifying proteins, i.e., solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, or the like, either alone or in combination.

For the above chromatography, column chromatography using Phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), or Sephadex G-150 (Pharmacia Biotech) is preferably used.

Furthermore, if the modified fibroin is expressed by forming an insoluble body within the cells, the host cells can be similarly recovered, disrupted, and centrifuged to recover the insoluble modified fibroin as a precipitate fraction. The recovered insoluble modified fibroin can be solubilized with a protein denaturant. After this procedure, a purified modified fibroin preparation can be obtained using the same isolation and purification method as above.

If the modified fibroin is secreted outside the cells, it can be recovered from the culture supernatant. That is, the culture can be treated by centrifugation or other methods to obtain the culture supernatant, and a purified sample can be obtained from the culture supernatant using the same isolation and purification methods as described above.

In this embodiment, the shape of the structural protein is not particularly limited, but from the viewpoints of excellent dispersibility in the matrix resin and more significantly promoting the opening of fiber bundles of reinforcing fibers, a fibrous or powdery form is preferred. In other words, the opening agent in this embodiment preferably contains structural protein fibers and/or structural protein powder.

The structural protein fibers may be spun from the above-mentioned proteins. Preferably, the structural protein fibers are spider silk fibroin fibers, i.e., spun from polypeptides derived from natural spider silk proteins (artificial spider silk proteins).

Structural protein fibers can be produced by known spinning methods. That is, for example, when producing structural protein fibers, the structural protein is first added to a solvent such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or hexafluoroisopronol (HFIP) along with an inorganic salt as a dissolution promoter, and dissolved to prepare a dope solution. This dope solution can then be spun by a known spinning method such as wet spinning, dry spinning, or dry-wet spinning to obtain the desired structural protein fibers.

Figure 1 is a schematic diagram showing an example of a spinning apparatus for producing structural protein fibers. The spinning apparatus 10 shown in Figure 1 is an example of a spinning apparatus for dry-wet spinning, and has, in order from upstream, an extrusion device 1, a coagulation bath 20, a washing bath 21, and a drying device 4.

The extrusion device 1 has a storage tank 7, in which a dope solution (spinning solution) 6 is stored. A coagulation bath 20 stores a coagulation liquid 11 (e.g., methanol). The dope solution 6 is extruded from a nozzle 9, which is provided with an air gap 19 between the nozzle and the coagulation liquid 11, by a gear pump 8 attached to the lower end of the storage tank 7. The extruded dope solution 6 is supplied into the coagulation liquid 11 through the air gap 19. The solvent is removed from the dope solution 6 in the coagulation liquid 11, causing the protein to coagulate. The coagulated protein is introduced into a washing bath 21, where it is washed with a washing liquid 12 in the washing bath 21. It is then sent to the drying device 4 by a first nip roller 13 and a second nip roller 14 installed in the washing bath 21. At this time, for example, by setting the rotation speed of the second nip roller 14 faster than the rotation speed of the first nip roller 13, structural protein fibers 36 are obtained that are stretched at a ratio corresponding to the rotation speed ratio. The structural protein fibers stretched in the washing solution 12 are dried as they leave the washing bath 21 and pass through the drying device 4, and then are wound up by the winder. In this way, the structural protein fibers are obtained by the spinning device 10 as a wound product 5 that is finally wound up by the winder. Note that 18a to 18g are yarn guides.

The coagulation liquid 11 may be any solution that can be desolvated, such as lower alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, and 2-propanol, as well as acetone. The coagulation liquid 11 may contain water as appropriate. The temperature of the coagulation liquid 11 is preferably 0 to 30°C. The distance the coagulated protein passes through the coagulation liquid 11 (effectively, the distance from thread guide 18a to thread guide 18b) may be long enough to efficiently desolvate the protein, and may be, for example, 200 to 500 mm. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes. Stretching (pre-stretching) may also be performed in the coagulation liquid 11.

The stretching performed in the cleaning bath 21 to obtain structural protein fibers may be so-called wet heat stretching, which is performed in warm water or in a solution of warm water to which an organic solvent or the like has been added. The temperature for this wet heat stretching may be, for example, 50 to 90°C, with 75 to 85°C being preferred. In wet heat stretching, the unstretched yarn (or pre-stretched yarn) can be stretched, for example, 1 to 10 times, with 2 to 8 times being preferred.

The structural protein fibers are preferably short fibers (i.e., fibers with a fiber length of 5 mm or less). The fiber length of the structural protein fibers is preferably 5 mm or less, and more preferably 1 mm or less. Such structural protein fibers have better dispersibility in the matrix resin and more significantly promote the opening of fiber bundles of the reinforcing fibers. Furthermore, there is no particular lower limit to the fiber length of the structural protein fibers, and they may be, for example, 0.1 mm or more, or 0.25 mm or more.

The structural protein fibers may be cut using a fiber cutter to the preferred fiber length described above. In other words, the structural protein fibers may be chopped fibers.

The structural protein powder may be, for example, a structural protein that has been pulverized using a grinder, or may be finely cut using a fiber cutter or the like. From the perspective of easily obtaining a structural protein powder with excellent dispersibility, the structural protein powder may be, for example, a structural protein powder that has been pulverized or cut.

The opening agent of this embodiment may be blended when the reinforcing fiber bundles are combined with the matrix resin. For example, a fiber-reinforced composite material may be produced by mixing the reinforcing fiber bundles, the matrix resin, and the opening agent of this embodiment. Alternatively, a fiber-reinforced composite material may be produced by combining a resin sheet containing the matrix resin and the opening agent with the reinforcing fiber bundles.

The amount of opening agent in this embodiment is preferably blended so as to satisfy the preferred content range in the fiber-reinforced composite material described below.

The opening agent of this embodiment also has the effect of suppressing the orientation of fiber bundles of reinforcing fibers in a fiber-reinforced composite material. That is, the opening agent of this embodiment may be incorporated into a fiber-reinforced composite material containing a fiber bundle formed by bundling multiple reinforcing fibers and a matrix resin, and may function as an orientation suppressor that suppresses the orientation of the fiber bundles.

(fiber reinforced composites)
The fiber reinforced composite material of this embodiment includes fiber bundles of reinforcing fibers, a matrix resin, and the above-mentioned opening agent.

In this embodiment, the matrix resin may be any known resin used in fiber-reinforced composites, without any particular limitation. Examples of matrix resins include vinyl ester resin, unsaturated polyester, phenolic resin, melamine resin, epoxy resin, polyamide resin, methyl methacrylate, polypropylene, nylon, polyethylene, polystyrene, polyacetal, polycarbonate, ABS, AES, PET, PBT, PPS, LCP, PEEK, polyurethane, urea resin, and silicone resin. The matrix resin may be a thermoplastic resin or a thermosetting resin.

The matrix resin content is not particularly limited, but may be, for example, 35% by mass or more, and preferably 45% by mass or more. The matrix resin content may be, for example, 65% by mass or less, and preferably 55% by mass or less.

In this embodiment, the reinforcing fibers can be any known reinforcing fibers used in fiber-reinforced composites, without any particular limitations. Suitable reinforcing fibers include, for example, carbon fiber, boron fiber, aramid fiber, polyethylene fiber, and Zylon (registered trademark) fiber. Other reinforcing fibers that can be used include synthetic fibers such as polyamide fiber and polyimide fiber; metal fibers such as alumina fiber, titanium fiber, steel fiber, and copper fiber; natural fibers derived from plants such as bamboo fiber, kenaf fiber, and bagasse fiber; and inorganic fibers such as glass fiber and basalt fiber.

The fiber length of the reinforcing fibers is not particularly limited, and they may be so-called long fibers (e.g., fibers with a fiber length of more than 50 mm), short fibers (e.g., fibers with a fiber length of 50 mm or less), or continuous fibers.

In a preferred embodiment, the reinforcing fibers may be carbon fibers. In such fiber-reinforced composites, the opening agent has a more pronounced effect of promoting the opening of fiber bundles. Short carbon fibers with a fiber length of 50 mm or less are more preferred.

The reinforcing fiber content is not particularly limited, but may be, for example, 25% by mass or more, and preferably 35% by mass or more. The reinforcing fiber content may be, for example, 55% by mass or less, and preferably 45% by mass or less.

The amount of the opening agent is not particularly limited, but may be, for example, 0.5% by mass or more, and preferably 1% by mass or more. The amount of the opening agent may be, for example, 10% by mass or less, and preferably 5% by mass or less.

The fiber-reinforced composite may further contain other components in addition to those listed above. Examples of other components include fillers, curing agents, low-profile agents, and internal mold release agents.

Examples of fillers include calcium carbonate, aluminum hydride, barium sulfate, clay, and mica. The filler content is not particularly limited and may be, for example, 0.5% by mass or more, preferably 1% by mass or more, or 50% by mass or less, preferably 20% by mass or less.

The manufacturing method of fiber-reinforced composite materials is not particularly limited. Examples of methods that can be used to combine matrix resin and reinforcing fibers include hand layup, spray-up, SMC press, which compresses a sheet-like mixture of reinforcing fibers and resin components in a mold, RTM, which injects resin into a mold filled with fibers similar to injection molding, autoclave molding, and de-autoclave molding. Other known methods that can be used include thermoplastic resin injection, BMC (bulk molding compound) injection of thermosetting resin, GMT (glass-mat reinforced thermoplastics) press, which impregnates a mat of fibers with a thermoplastic resin, papermaking, and needle punching.

The timing of adding the opening agent is not particularly limited; for example, it may be added in advance to the resin component in the above-mentioned compounding method, or it may be added at the same time as compounding.

Below, the first and second embodiments are described as specific examples of methods for producing fiber-reinforced composite materials.

The manufacturing method according to the first aspect includes a step of mixing reinforcing fibers with a resin composition containing a resin component and an opening agent. The method for mixing the resin composition and reinforcing fibers is not particularly limited, and may be, for example, an extruder, a kneader, or the like.

The resin component in the first aspect is a component that forms the matrix resin of the fiber-reinforced composite material and may be, for example, a thermoplastic resin or a thermosetting resin.

When the resin component is a thermosetting resin, the resin composition may further contain a curing agent, and the above-mentioned manufacturing method may further include a step of curing the resin composition. The curing agent and curing method are not particularly limited, and known curing agents and curing methods can be applied depending on the type of thermosetting resin, etc.

The manufacturing method according to the second aspect includes a sheeting process in which a resin composition containing a resin component and a fiber-opening agent is flattened into a sheet to form a resin sheet, and a compounding process in which reinforcing fibers are placed on the resin sheet to compound the resin sheet and the reinforcing fibers.

In the second aspect, the resin component is a component that forms the matrix resin of the fiber-reinforced composite and may be, for example, a thermoplastic resin or a thermosetting resin.

When the resin component is a thermosetting resin, the resin composition may further contain a curing agent, and the above-mentioned manufacturing method may further include a step of curing the resin composition. The curing agent and curing method are not particularly limited, and known curing agents and curing methods can be applied depending on the type of thermosetting resin, etc.

In the second aspect, the method for molding the resin composition is not particularly limited, and examples include methods such as compression molding.

In the second aspect, the method for combining the resin sheet and reinforcing fibers is not particularly limited, and examples include a method of scattering reinforcing fibers on a resin sheet.

The uses of the fiber-reinforced composite material according to this embodiment are not particularly limited. For example, the fiber-reinforced composite material can be suitably used for components such as frames, inner panels, and outer panels of structures such as automobiles, railway vehicles, ships, aircraft, rockets, artificial satellites, and robots.

Fiber-reinforced composites can be used in automobile brake discs, wheels, fuel tanks, hoods, roofs, side doors, back doors, luggage inner and outer panels, exterior parts, structural parts, undercarriage parts, engine-related parts, fuel cell and electric vehicle parts, and other parts. Exterior parts include bumpers, rocker moldings, pillars, and roof moldings. Structural parts include bumper reinforcements, rear floor pans, crash boxes, dash panels, rear partitions, seatback frames, and braces. Undercarriage parts include lower absorbers and undercovers. Engine-related parts include engine covers, engine undercovers, cylinder head covers, oil pans, radiator supports, and timing belt and chain covers. Fuel cell and electric vehicle parts include stack frames, stack end plates, fuel cell cells, inverter covers, inverter cases, rotor motors, stator motors, and reactors. Other parts include brackets, anchors, pedals, sheet metal parts, etc.

Fiber-reinforced composites can also be suitably used for aircraft nose landing gear doors, main landing gear doors, floor beams, engine covers, ailerons, wing-body fairings, horizontal stabilizers, vertical stabilizers, rudders, elevators, etc.

Fiber-reinforced composites can also be used effectively in applications such as ship masts, drone frames, suitcases, cargo containers, housings for electronic and electrical devices (e.g., personal computers, displays, projectors, cameras, mobile phones, smartphones, tablets, etc.), fishing equipment (e.g., fishing rods and reels), golf club shafts, golf club heads, rackets (e.g., for tennis, badminton, squash, and table tennis), automobile frames, front forks, rims, baseball bats, skis, hockey sticks, ski poles, kendo bamboo swords, Japanese bows and arrows, Western-style bows, table tennis tables, billiard cues, gateball sticks, construction and civil engineering materials, wind power generation blades (wind turbines), flywheels, pipes, parabolic antennas, prosthetic limbs, wheelchairs, beds, portable ramps, crutches, and artificial bones.

The above describes a preferred embodiment of the present invention, but the present invention is not limited to the above embodiment.

For example, as described above, the opening agent of the above embodiment also has the effect of suppressing the orientation of reinforcing fiber bundles in a fiber-reinforced composite material. Therefore, one aspect of the present invention can be described as an orientation suppressor that is blended into a fiber-reinforced composite material containing a fiber bundle formed by bundling multiple reinforcing fibers and a matrix resin, and suppresses the orientation of the reinforcing fiber bundles.

The present invention will be described in more detail below based on examples. However, the present invention is not limited to the following examples.

(Example 1-1)
[Protein production]
(1) Preparation of Expression Vector A modified fibroin (spider silk fibroin, hereinafter also referred to as "PRT799") having SEQ ID NO: 15 was designed based on the base sequence and amino acid sequence of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415) as a protein contained in the spinning dope. Note that the amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence in which amino acid residues have been substituted, inserted, and deleted with the aim of improving productivity compared to the amino acid sequence of fibroin derived from Nephila clavipes, and further has the amino acid sequence represented by SEQ ID NO: 12 (tag sequence and hinge sequence) added to the N-terminus.

Next, a nucleic acid encoding the modified fibroin PRT799, which has the designed amino acid sequence shown in SEQ ID NO: 15, was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. The nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised using restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+), respectively, to obtain expression vectors.

(2) Protein Expression Escherichia coli BLR(DE3) was transformed with the expression vector obtained in (1). The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that the OD600 became 0.005. The culture solution temperature was maintained at 30°C, and flask culture was continued until the OD600 reached 5 (approximately 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) so that the OD600 reached 0.05. The culture temperature was maintained at 37°C, and the pH was controlled to a constant 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g/L glucose, 120 g/L yeast extract) was added at a rate of 1 mL/min. The culture temperature was maintained at 37°C, and the pH was controlled at a constant 6.9. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours. 1 M isopropyl-β-thiogalactopyranoside (IPTG) was then added to the culture medium to a final concentration of 1 mM to induce expression of the modified fibroin. Twenty hours after IPTG addition, the culture medium was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture medium before and after IPTG addition. Expression of the desired modified fibroin protein was confirmed by the appearance of a band of the desired spider silk fibroin size, which was dependent on IPTG addition.

(3) Protein Purification The bacterial cells harvested 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed bacterial cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF, and the cells were disrupted using a high-pressure homogenizer (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 highly purified. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg/mL, and dissolved by stirring with a stirrer at 60°C for 30 minutes. After dissolution, the solution was dialyzed against water using a dialysis tube (Cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.) The white aggregated protein obtained after dialysis was recovered by centrifugation, and the water was removed using a freeze-dryer. The freeze-dried powder was recovered to obtain the desired modified fibroin protein (PRT799).

[Production of fiber-opening agent]
The above-mentioned PRT799 was added to dimethyl sulfoxide (DMSO) to a concentration of 24% by mass, and then LiCl was added as a dissolution promoter to a concentration of 4.0% by mass. The mixture was then dissolved using a shaker for 3 hours. Dust and bubbles were then removed to obtain a dope solution. The solution viscosity of the dope solution was 5000 cP (centipoise) at 90°C.

The obtained dope solution was used in a spinning apparatus 10 shown in Fig. 4 to carry out a known dry-wet spinning process to obtain a spider silk fibroin monofilament. The dry-wet spinning process was carried out under the following conditions:
Extrusion nozzle diameter: 0.1 mm
Extrusion rate: 327.6 ml/h
Temperature of coagulation liquid (methanol): 2°C
Winding speed: 99.5 m/min
Stretching ratio: 4.52 times Drying temperature: 80℃
Air gap length: 5 mm

48 spider silk fibroin monofilaments were bundled together and cut to an average length of 0.5 mm using a fiber cutting machine to obtain short fiber-forming opening agent (A-1).

[Production of fiber-reinforced composite materials]
A resin composition was obtained by kneading vinyl ester resin (Neopol 8025 manufactured by Japan U-Pica Co., Ltd.), calcium carbonate (Whiten SB Blue manufactured by Bihoku Funka Kogyo Co., Ltd.), the above-mentioned fiber-opening agent (A-1), and other additives in a mixer. The resulting resin composition was flattened into a sheet, and carbon fiber (TC36P 12K manufactured by Taiwan Plastics Co., Ltd., fiber bundle diameter 7 μm, cut into 1-inch (approximately 25 mm) pieces) was sprinkled on top. A sheet of the above-mentioned resin composition was then placed on top of the sheet to obtain a laminate sandwiching the carbon fiber between the resin sheets. The mass ratio of vinyl ester resin, calcium carbonate, carbon fiber, fiber-opening agent, and additives in the laminate was 47.8:4.8:40.3:2.4:4.7. The laminate was compression molded using a mold heated to 140°C to obtain a 300 mm x 300 mm x 2 mm flat plate-shaped molded product (fiber-reinforced composite material).

The resulting molded body was cut into 100 x 25 mm pieces to prepare test pieces for bending tests, and the bending properties of the test pieces were measured using a bending tester (Shimadzu Corporation, AG-20kNX). The test speed was 5 mm/min, and the distance between supports was 80 mm. The results are shown in Table 6.

Next, 21 locations of the carbon fiber bundles that could be observed from the surface of the resulting molded body were randomly selected, and the fiber bundle diameter was measured using a microscope with measuring functions (KEYENCE Corporation, VHX-5000). The average of the measurements from the 21 locations was 9.63 mm. The degree of opening from the fiber bundle diameter (7 μm) before compounding was 137.6%.

(Comparative Example 1-1)
A flat-plate molded body (fiber-reinforced composite material) was obtained in the same manner as in Example 1-1, except that no opening agent was added to the resin composition and the mass ratio of vinyl ester resin, calcium carbonate, carbon fiber, and additives in the laminate was 48.8:4.9:40.4:5.9. The bending properties and carbon fiber bundles of the obtained molded body were measured in the same manner as in Example 1-1. The results are shown in Table 6.

(Example 2-1)
In the same manner as in Example 1-1, a 300 mm x 300 mm x 2 mm plate-shaped molded product (fiber reinforced composite material) was obtained.
The 300 mm × 300 mm surface of the obtained molded body was divided into 12 × 10 sections, and images of three randomly selected sections (1 section: 25 mm × 30 mm) were taken with a microscope (KEYENCE Corporation, VHX-5000). The orientation of the carbon fiber bundles was evaluated from the images. Specifically, the horizontal line was used as the reference line, and the angle between the reference line and the fiber bundles visible in the image was measured using image processing software (Mitani Corporation, WinROOF2015). The distribution of the fiber bundle orientation (angle) relative to the reference line was displayed in a radar chart in 30° increments. The results are shown in Figure 5(a).

(Comparative Example 2-1)
A 300 mm × 300 mm × 2 mm plate-shaped molded body (fiber-reinforced composite material) was obtained in the same manner as in Comparative Example 1-1. The orientation of the carbon fiber bundles of the obtained molded body was evaluated in the same manner as in Example 2-1. The results are shown in Figure 5(b).

(Example 2-2)
[Protein production]
A modified fibroin (spider silk fibroin, hereinafter also referred to as "PRT966") having the amino acid sequence shown in SEQ ID NO: 44 was designed, and a nucleic acid encoding the modified fibroin PRT966 having the designed amino acid sequence shown in SEQ ID NO: 44 was synthesized. Using this nucleic acid, a modified fibroin (PRT966) was obtained in the same manner as in "Protein Production" in Example 1-1.

[Production of fiber-opening agent]
A short fiber opening agent (A-2) having an average length of 0.5 mm was obtained in the same manner as in Example 1-1, except that the above-mentioned PRT966 was used instead of PRT799.

[Production of fiber-reinforced composite materials]
A flat-plate-shaped molded body (fiber-reinforced composite material) was obtained in the same manner as in Example 2-1, except that TC35 48K carbon fiber (manufactured by Taiwan Plastics Corporation) was used, the opening agent (A-2) was used instead of the opening agent (A-1), and the amount of the opening agent was adjusted so that the mass ratio of vinyl ester resin, calcium carbonate, carbon fiber, opening agent, and additives was 47.5:4.7:40.4:1.2:6.2. The fiber bundle orientation of the obtained molded body was determined in the same manner as in Example 2-1. The results are shown in Figure 6(a).

(Comparative Example 2-2)
A flat-plate molded body (fiber-reinforced composite material) was obtained in the same manner as in Example 2-1, except that no opening agent was added to the resin composition and the mass ratio of vinyl ester resin, calcium carbonate, carbon fiber, and additives in the laminate was 48.1:4.8:40.9:6.2. The orientation of the fiber bundles in the obtained molded body was determined in the same manner as in Example 2-1. The results are shown in Figure 6(b).

Comparing Example 2-1 with Comparative Example 2-1, and comparing Example 2-2 with Comparative Example 2-2, it was confirmed that the Examples had a more random orientation than the Comparative Examples, and that the opening agent could also be used as an orientation inhibitor.

Claims (6)

A fiber-opening agent that includes a fiber bundle in which a plurality of reinforcing fibers are bundled and a matrix resin, the fiber-opening agent being blended into a fiber-reinforced composite material in which the content of the matrix resin is 55% by mass or less and the content of the reinforcing fibers is 35% by mass or more , and that promotes opening of the fiber bundles,
A fiber opening agent containing structural proteins as short fibers . A fiber-opening agent that is blended into a fiber-reinforced composite material containing a fiber bundle in which a plurality of reinforcing fibers are bundled and a matrix resin, and that promotes opening of the fiber bundle,
containing structural proteins as short fibers,
The opening agent has a value of 0 or more when the sum of the hydrophobicity index (HI) of all amino acid residues constituting the structural protein is calculated and the sum is divided by the total number of amino acid residues. The opening agent according to claim 1 or 2, wherein the reinforcing fibers are short fibers. The opening agent according to any one of claims 1 to 3, wherein the reinforcing fibers are carbon fibers. The fiber-opening agent according to any one of claims 1 to 4, wherein the structural protein comprises modified fibroin. The fiber-opening agent according to any one of claims 1 to 5, wherein the structural protein includes spider silk fibroin.