Water-absorbent and quick-drying agent, and method for imparting water-absorbent and quick-drying properties

Abstract

[Problem] To provide: a water-absorbing and quick-drying property-imparting agent by which water-absorbing and quick-drying properties can be easily imparted to various materials or articles through a simple process; and a method by which water-absorbing and quick-drying properties can be easily imparted to a predetermined material or article. [Solution] Provided are: a water-absorbing and quick-drying property-imparting agent that contains modified fibroin as an active ingredient; and a method for imparting water-absorbing and quick-drying properties to an article, the method comprising a step for incorporating modified fibroin in an article.

Description

[Technical Field]

[0001] The present invention relates to an agent for imparting quick-drying water absorption properties and a method for imparting quick-drying water absorption properties. [Background technology]

[0002] Generally, clothing such as underwear, innerwear, sportswear, and bedding are required to have moisture-absorbing and quick-drying properties, i.e., to absorb sweat well and dry quickly. For this reason, many underwear and bedding are manufactured using cotton spun yarn, which has relatively high moisture absorption properties.

[0003] However, although cotton has high water absorption, it is difficult to say that it has sufficient quick-drying properties because the absorbed water evaporates slowly. Also, for sportswear and the like, importance is placed on the ability to follow the movements of the human body and stretchability, so polyurethane fibers and polyester fibers are often used, but these synthetic fibers have poor water absorption properties.

[0004] In this context, various techniques have been proposed to improve the moisture-absorbing and quick-drying properties of various fibers, fabrics, etc. For example, Patent Document 1 discloses a moisture-absorbing and quick-drying spun yarn characterized by being composed of a false-twisted spun yarn having a core made of polyester fibers with a modified cross section and a sheath made of staple fibers including cotton fibers, with the core being untwisted and the sheath being in a bound state. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Utility Model Registration No. 3213540 Summary of the Invention [Problem to be solved by the invention]

[0006] For example, in conventional fibers and fabrics, moisture-absorbing and quick-drying properties are achieved by using fibers to which a special structure has been artificially imparted, such as the moisture-absorbing and quick-drying spun yarn described in Patent Document 1. Therefore, the manufacturing process for moisture-absorbing and quick-drying fibers and moisture-absorbing and quick-drying fabrics up to now has inevitably been complicated.

[0007] The present invention aims to provide a water-absorbent, quick-drying agent that can easily impart water-absorbent, quick-drying properties to various materials or articles in a simple process, and a method for easily imparting water-absorbent, quick-drying properties to a predetermined material or article. [Means for solving the problem]

[0008] The present inventors have discovered that modified fibroin has excellent water absorption and quick drying properties, and the present invention is based on this novel finding.

[0009] The present invention relates to, for example, the following inventions. [1] An agent for imparting quick-drying absorbency containing a modified fibroin as an active ingredient. [2] The agent for imparting quick-drying absorbency according to [1], wherein the modified fibroin contains a modified fibroin having an average hydrophobicity index (average HI) of 0 or less. [3] The agent for imparting quick-drying absorbency according to [1] or [2], wherein the modified fibroin contains a modified spider silk fibroin. [4] The agent for imparting quick-drying absorbency according to any of [1] to [3], which is in the form of a fiber. [5] A method for imparting quick-drying absorbency to an article, comprising the step of incorporating modified fibroin into the article. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a water-absorbent, quick-drying agent that can easily impart water-absorbent, quick-drying properties to various materials or articles in a simple process, and a method for easily imparting water-absorbent, quick-drying properties to a specified material or article.

[0011] According to the present invention, for example, quick-drying and moisture-absorbent properties can be imparted to a biodegradable material without impairing its biodegradability by mixing the agent for imparting quick-drying and moisture-absorbent properties of the present invention into the material. Furthermore, according to the present invention, the degree of quick-drying and moisture-absorbent properties of a given product can be adjusted by adjusting the amount of modified fibroin (active ingredient) contained in the product. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram showing an example of a domain sequence of a modified fibroin. [Figure 2] FIG. 1 shows the distribution of z / w (%) values ​​of naturally occurring fibroin. [Figure 3] FIG. 1 shows the distribution of x / y (%) values ​​of naturally occurring fibroin. [Figure 4] FIG. 1 is a schematic diagram showing an example of a domain sequence of a modified fibroin. [Figure 5] FIG. 1 is a schematic diagram showing an example of a domain sequence of a modified fibroin. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0014] [Water-absorbent, quick-drying agent] The water-absorbent, quick-drying agent according to this embodiment contains modified fibroin as an active ingredient. Water-absorbent, quick-drying property refers to the property of absorbing moisture such as sweat and drying quickly. In this specification, moisture may be liquid water or gaseous water. That is, in this specification, water absorption includes moisture absorption. The water-absorbent, quick-drying agent according to this embodiment utilizes the property of modified fibroin, which is excellent in water-absorbent, quick-drying property.

[0015] (Modified fibroin) The modified fibroin according to this embodiment is a fibroin represented by the formula 1: [(A) n Motif-REP] m , or Formula 2: [(A)n Motif-REP] m -(A) n The modified fibroin is a protein containing a domain sequence represented by a 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 not limited to, regions that do not have repeats of the amino acid motif characteristic of fibroin and consist of about 100 amino acid residues.

[0016] As used herein, "modified fibroin" refers to artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be a fibroin whose domain sequence is identical to the amino acid sequence of naturally occurring fibroin. The "naturally occurring fibroin" referred to herein also refers to a fibroin represented by the formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n It is a protein containing a domain sequence represented by a motif.

[0017] A "modified fibroin" may be one that uses the amino acid sequence of naturally occurring fibroin as is, or one that has had its amino acid sequence modified based on the amino acid sequence of naturally occurring fibroin (for example, one that has had its amino acid sequence 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 (for example, one that has the desired amino acid sequence obtained by chemically synthesizing a nucleic acid that encodes a designed amino acid sequence).

[0018] As used herein, the term "domain sequence" refers to a crystalline region specific to fibroin (typically, the amino acid sequence (A) nIt is an amino acid sequence that generates a region (corresponding to a motif) and an amorphous region (typically corresponding to an REP in an amino acid sequence), and is represented by formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n The amino acid sequence represented by the motif (A) n The motif shows an amino acid sequence mainly consisting of alanine residues, and the number of amino acid residues is 2 to 27. (A) n The number of amino acid residues in the motif may be an integer of 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. n The ratio of the number of alanine residues to the total number of amino acid residues in the 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 that the motif is composed of only alanine residues). n At least seven of the motifs may be composed of only 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 of 2 to 300, and may be an integer of 10 to 300. (A) n The motifs may have the same or different amino acid sequences, and the REPs present in multiple instances may have the same or different amino acid sequences.

[0019] The modified fibroin according to this embodiment can be obtained, for example, by modifying the amino acid sequence of a cloned gene sequence of naturally occurring fibroin, for example, by substituting, deleting, inserting, and / or adding one or more amino acid residues. The 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, this can be performed in accordance with the methods described in literature, such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology 100, 448 (1983).

[0020] Naturally occurring fibroin has the formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n It is a protein containing a domain sequence represented by a motif, and specific examples include fibroin produced by insects or arachnids.

[0021] Examples of fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, as well as hornet silk proteins excreted by larvae of the Japanese hornet (Vespa simillima xanthoptera).

[0022] A more specific example of fibroin produced by insects is silkworm fibroin L chain (GenBank accession numbers M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

[0023] Examples of fibroin produced by spiders include spider silk proteins produced by spiders belonging to the order Araneae. More specifically, spiders belonging to the genus Araneus, such as the orb spider, the garden spider, the red orb spider, the green orb spider, and the bean spider, spiders belonging to the genus Neoscona, such as the mountain orb spider, the house spider, the dun orb spider, and the Satsuma spider, spiders belonging to the genus Pronus, such as the little orb spider, spiders belonging to the genus Pronus, and the Japanese orb spider, spiders belonging to the genus Arakawa ... Spiders of the genus Cyrtarachne, such as the giant burrowing spider; spiders of the genus Gasteracantha, such as the spiny spider and the stingray spider; spiders of the genus Ordgarius, such as the orb-weaver spider and the dwarf spider; spiders of the genus Argiope, such as the orb-weaver spider, the orb-weaver spider, and the long-jawed orb-weaver spider; spiders belonging to the Arachnura genus such as the brown brook spider; spiders belonging to the Acusilas genus such as the scraping spider; spiders belonging to the Cytophora genus such as the harp spider, the yellow-headed harp spider and the common harp spider; spiders belonging to the Poltys genus such as the house spider; house spiders, four-headed house spiders, and house spiders. Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the Japanese bush spider and the Japanese bush spider, and spiders belonging to the genus Chorizopes, such as the Japanese bush spider, as well as spiders belonging to the genus Tetragnatha, such as the long-legged spider, the long-legged spider, the Japanese bush spider, and the scaly bush spider, and spiders belonging to the genus Argiope, such as the Japanese bush spider Examples of spider silk proteins include those produced by spiders of the genus Leucauge, such as Orb weaver and Orb spider; spiders of the genus Nephila, such as Orb weaver and Orb spider; spiders of the genus Menosira, such as Orb spider; spiders of the genus Dyschiriognatha, such as Orb spider; spiders of the genus Latrodectus, such as the black widow, redback, gray widow, and three-spotted widow; and spiders of the family Tetragnathidae, such as spiders of the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, and Flag.

[0024] 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 ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), and dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank accession numbers 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 clavipes] cruentata] (GenBank accession number ABR37278.1 (amino acid sequence) and the like.

[0025] More specific examples of naturally occurring fibroins include those whose sequence information is registered in NCBI GenBank. For example, these can be confirmed by extracting, from among the sequences registered in NCBI GenBank that contain INV as the division, sequences with spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" as keywords in the definition, sequences with a specific product character string in the CDS, and sequences with a specific character string in the tissue type field in the source.

[0026] The modified fibroin of this embodiment may be modified silk fibroin (a silk protein produced by silkworms with a modified amino acid sequence) or modified spider silk fibroin (a spider silk protein produced by spiders with a modified amino acid sequence).

[0027] Specific examples of modified fibroins include a modified fibroin derived from the major ampullate gland dragline silk protein produced in the spider's major ampullate gland (first modified fibroin), a modified fibroin having a domain sequence with a reduced content of glycine residues (second modified fibroin), (A) n A modified fibroin (third modified fibroin) having a domain sequence with a reduced motif content, a content of glycine residues, and (A) n These include modified fibroins with a reduced motif content (the fourth modified fibroin), modified fibroins with a domain sequence containing areas of locally high hydrophobicity index (the fifth modified fibroin), and modified fibroins with a domain sequence with a reduced content of glutamine residues (the sixth modified fibroin).

[0028] The first modified fibroin is represented by Formula 1: [(A) n Motif-REP] mIn the first modified fibroin, a protein containing a domain sequence represented by (A) n The number of amino acid residues in the motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, even more preferably an integer of 10 to 20, even more preferably an integer of 4 to 16, particularly preferably an integer of 8 to 16, and most preferably an integer of 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. The first modified fibroin is a fibroin represented by formula 1: [(A) n Motif-REP] m The total number of glycine residues, serine residues, and alanine residues contained in the amino acid sequence represented by the formula (I) 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.

[0029] The first modified fibroin has the formula 1: [(A) n Motif-REP] m and the C-terminal sequence is an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 or an amino acid sequence having 90% or more homology to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.

[0030] 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.

[0031] More specific examples of the first modified fibroin include (1-i) a modified fibroin containing the amino acid sequence shown in SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), 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.

[0032] The amino acid sequence shown in SEQ ID NO:4 is the 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 at positions 1 to 13 approximately doubled and mutations performed so that translation terminates at amino acid residue 1154. 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.

[0033] The modified fibroin (1-i) may consist of the amino acid sequence shown in SEQ ID NO:4.

[0034] The second modified fibroin has an amino acid sequence in which the domain sequence has a reduced content of glycine residues compared to naturally-occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence in which, compared to naturally-occurring fibroin, at least one or more glycine residues in REP have been replaced with another amino acid residue.

[0035] The second modified fibroin may have an amino acid sequence in which, compared to naturally occurring fibroin, its domain sequence 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.

[0036] In the second modified fibroin, the proportion of the motif sequences in which the above-mentioned glycine residues have been substituted with other amino acid residues may be 10% or more relative to the total motif sequences.

[0037] The second modified fibroin has the formula 1: [(A) n Motif-REP] m and (A) is located at the most C-terminal side of the domain sequence. n The total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence is z, and the amino acid sequence located most C-terminally from the domain sequence is (A). n When the total number of amino acid residues in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence is represented by w, the z / w ratio may be 30% or more, 40% or more, 50% or more, or 50.9% or more. (A) n The number of alanine residues relative to the total number of amino acid residues in the 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 that it is composed only of alanine residues).

[0038] 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 the same method as the calculation method for the content (z / w) of the amino acid sequence consisting of XGX described below.

[0039] The calculation method of z / w will be explained in more detail. First, the formula 1: [(A) n Motif-REP] m In a fibroin (modified fibroin or naturally-occurring fibroin) containing a domain sequence represented by the formula (A), the domain sequence is located at the most C-terminal side. n An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, if 50 amino acid sequences consisting of XGX are extracted (no overlaps), z is 50 x 3 = 150. Also, for example, in the case of an amino acid sequence consisting of XGXGX, if two XGX sequences are extracted, If there is an X (middle X) that overlaps, the overlapping X is subtracted from the calculation (in the case of XGXGX, it is 5 amino acid residues). 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-terminal side 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 (the (A)n motif located at the most C-terminal side). n (The motif is excluded.) Next, z / w (%) can be calculated by dividing z by w.

[0040] Here, the z / w ratio in naturally occurring fibroin will be explained. First, as described above, fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, and 663 types of fibroin (of which, 415 types are fibroin derived from spiders) were extracted. Of all the extracted fibroins, those with the formula 1: [(A) nUsing the above-described calculation method, z / w was calculated from the amino acid sequence of naturally occurring fibroin containing a domain sequence represented by the motif [-REP]m and in which the content of the amino acid sequence consisting of GGX in the fibroin was 6% or less. The results are shown in Figure 2. The horizontal axis of Figure 2 represents z / w (%), and the vertical axis represents frequency. As is clear from Figure 2, the z / w of all naturally occurring fibroin was less than 50.9% (the highest was 50.86%).

[0041] 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.

[0042] The second modified fibroin can be obtained, for example, by modifying the gene sequence of a cloned naturally occurring fibroin by substituting at least a portion of the base sequence encoding a glycine residue so that it encodes a different amino acid residue. In this case, the glycine residue to be modified may be one of the glycine residues in the GGX motif and the GPGXX motif, or the glycine residue may be substituted 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 satisfying the above-described aspects from the amino acid sequence of a naturally occurring 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 a naturally occurring fibroin, the amino acid sequence may be further modified by substituting, deleting, inserting, and / or adding one or more amino acid residues.

[0043] The other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but is preferably a hydrophobic amino acid residue such as a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a methionine (M) residue, a proline (P) residue, a phenylalanine (F) residue, or a tryptophan (W) residue, or a hydrophilic amino acid residue such as a glutamine (Q) residue, an asparagine (N) residue, a serine (S) residue, a lysine (K) residue, or a glutamic acid (E) residue, more preferably a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a phenylalanine (F) residue, or a glutamine (Q) residue, and even more preferably a glutamine (Q) residue.

[0044] More specific examples of the second modified fibroin include (2-i) a modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0045] The modified fibroin (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin, in which all GGX in the REP has been replaced with GQX. The amino acid sequence shown in SEQ ID NO: 7 is the amino acid sequence shown in SEQ ID NO: 6, in which every third amino acid (A) has been replaced with GQX from the N-terminus to the C-terminus. n The motif was deleted, and the C-terminal sequence was further shortened to [(A) n The amino acid sequence shown in SEQ ID NO: 8 is a sequence in which one (A) motif [REP] is inserted. nTwo alanine residues were inserted at the C-terminus of the motif, and some glutamine (Q) residues were substituted with serine (S) residues, and some amino acids were deleted at the C-terminus to achieve approximately the same molecular weight as SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 has a predetermined hinge sequence and His tag sequence added to the C-terminus of a sequence in which a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 7 is repeated four times (however, several amino acid residues at the C-terminus of this region have been substituted).

[0046] The z / w value for the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values ​​for the amino acid sequences 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 89.8%, respectively.

[0047] 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.

[0048] The modified fibroin (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin (2-ii) also ... the formula 1: [(A) n Motif-REP] m The above sequence identity is preferably 95% or more.

[0049] 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.

[0050] The second modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows for isolation, immobilization, detection, visualization, etc. of the modified fibroin.

[0051] An example of a tag sequence is an affinity tag that utilizes specific affinity (binding property, 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: 11 (an amino acid sequence including a His tag sequence and a hinge sequence).

[0052] Furthermore, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione, and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

[0053] 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.

[0054] 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 the protease, the modified fibroin from which the tag sequence has been cleaved can be recovered.

[0055] More specific examples of modified fibroins containing tag sequences include (2-iii) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0056] The amino acid sequences shown in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are obtained by adding the amino acid sequence shown in SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0057] The modified fibroin (2-iii) may consist of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0058] The modified fibroin (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin (2-iv) also ...5. The modified fibroin (2- n Motif-REP] m The above sequence identity is preferably 95% or more.

[0059] The modified fibroin (2-iv) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and 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.

[0060] 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 selected depending on the type of host.

[0061] The third modified fibroin has a domain sequence that is different from that of naturally occurring fibroin: (A) n The third modified fibroin has an amino acid sequence with a reduced content of motifs compared to naturally occurring fibroin. The domain sequence of the third modified fibroin has at least one or more (A) n It can be said that it has an amino acid sequence corresponding to the deletion of the motif.

[0062] The third modified fibroin was derived from naturally occurring fibroin (A). n It may also have an amino acid sequence corresponding to a deletion of 10 to 40% of the motif.

[0063] The third modified fibroin has a domain sequence that is different from that of naturally-occurring fibroin in that it has at least one to three (A) n One (A) per motif n The amino acid corresponding to the deletion of the motif It may have an acid sequence.

[0064] The third modified fibroin has a domain sequence that is at least two consecutive domains from the N-terminus to the C-terminus, compared to naturally occurring fibroin (A). n Deletion of motifs and one (A) n The amino acid sequence may be one in which the motif deletions are repeated in this order.

[0065] The third modified fibroin has a domain sequence in which at least every third domain from the N-terminus to the C-terminus is (A) n It may also have an amino acid sequence corresponding to the deletion of the motif.

[0066] The third modified fibroin has the formula 1: [(A) n Motif-REP] m From the N-terminus to the C-terminus, it contains two adjacent domain sequences represented by [(A) n The number of amino acid residues in the REPs of the motif-REP] unit is compared sequentially. When the number of amino acid residues in the REP with the fewer amino acid residues is set to 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3. n When the maximum sum of the numbers of amino acid residues in the motif-REP units is x and the total number of amino acid residues in the domain sequence is y, the amino acid sequence may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) nThe number of alanine residues relative to the total number of amino acid residues in the 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 that it is composed only of alanine residues).

[0067] The method for calculating x / y will be explained in more detail with reference to Figure 1. Figure 1 shows the domain sequence of the modified fibroin excluding the N-terminal sequence and the C-terminal sequence. The domain sequence is as follows from the N-terminal side (left side): (A) n Motif - 1st 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 - 4th REP (20 amino acid residues) - (A) n Motif - 5th REP (30 amino acid residues) - (A) n It has a sequence called a motif.

[0068] Two adjacent [(A) n The [(A) motif-REP] units are selected sequentially from the N-terminus to the C-terminus, with no overlaps. n [motif-REP] units may be present. 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 there are other selection methods as well.

[0069] Next, for each pattern, select two adjacent [(A) nThe number of amino acid residues of each REP in the motif-REP unit is compared. The comparison is performed by determining the ratio of the number of amino acid residues of the one with fewer amino acid residues to the number of the other. For example, when comparing a first REP (50 amino acid residues) with a second REP (100 amino acid residues), the ratio of the number of amino acid residues of the second REP is 100 / 50 = 2 when the number of amino acid residues of the first REP, which has fewer amino acid residues, is set to 1. Similarly, when comparing a fourth REP (20 amino acid residues) with a fifth REP (30 amino acid residues), the ratio of the number of amino acid residues of the fifth REP is 30 / 20 = 1.5 when the number of amino acid residues of the fourth REP, which has fewer amino acid residues, is set to 1.

[0070] In Figure 1, when the number of amino acid residues in the one with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues in the other one is 1.8 to 11.3 [(A) n The combination of motif-REP units is shown by a solid line. In this specification, this ratio is called the Giza ratio. When the one with fewer amino acid residues is set to 1, the ratio of the other amino acid residue number is less than 1.8 or more than 11.3 [(A) n The motif-REP] unit pairs are indicated by dashed lines.

[0071] In each pattern, two adjacent [(A) n The numbers of all amino acid residues in the (A)n motif-REP unit are added together (not only the number of REP but also the number of amino acid residues in the (A)n motif). Then, the sums are compared, and the sum of the pattern with the largest sum (maximum sum) is designated as x. In the example shown in Figure 1, the sum of pattern 1 is the largest.

[0072] Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

[0073] 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.

[0074] The third modified fibroin is present multiple times in the domain sequence (A). n In the case of a modified fibroin in which at least seven of the motifs 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.

[0075] Here, we will explain the x / y ratio in naturally occurring fibroin. First, as described above, fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, and 663 types of fibroin (of which, 415 types are fibroin derived from spiders) were extracted. Of all the extracted fibroins, those with the formula 1: [(A) n Motif-REP] m The x / y ratio was calculated by the above-mentioned calculation method from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the following formula: Figure 3 shows the results when the Giza ratio was 1:1.9 to 4.1.

[0076] The horizontal axis of Figure 3 represents x / y (%), and the vertical axis represents frequency. As is clear from Figure 3, the x / y ratio for all naturally occurring fibroins is less than 64.2% (the highest being 64.14%).

[0077] The third modified fibroin is, for example, (A) obtained by cloning the gene sequence of naturally occurring fibroin so that x / y is 64.2% or more. n For example, one or more (A) motifs can be deleted from the amino acid sequence of naturally occurring fibroin so that x / y is 64.2% or more. n Alternatively, the amino acid sequence corresponding to the deletion of the motif may be designed and a nucleic acid encoding the designed amino acid sequence may be chemically synthesized. In either case, the amino acid sequence of naturally occurring fibroin may be derived from (A) n In addition to modifications corresponding to the deletion of a motif, further modifications to the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be made.

[0078] More specific examples of the third modified fibroin include (3-i) a modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0079] The modified fibroin (3-i) will be described. The amino acid sequence shown in SEQ ID NO: 17 is a sequence of amino acids (A) every two amino acids from the N-terminal to the C-terminal of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin. n The motif was deleted, and the C-terminal sequence was further shortened to [(A) nThe amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for the second modified fibroin.

[0080] The x / y value for the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) with 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: 17 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 89.8%. The z / w values ​​for the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively.

[0081] The modified fibroin (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0082] The modified fibroin (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin (3-ii) also ... the formula 1: [(A) n Motif-REP] m The above sequence identity is preferably 95% or more.

[0083] The modified fibroin (3-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and has two adjacent amino acids (A) nThe number of amino acid residues in the REPs of the motif-REP] unit is compared sequentially. When the number of amino acid residues in the REP with the fewer amino acid residues is set to 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 (Giza ratio 1:1.8 to 11.3). Two adjacent [(A) n When the maximum total number of amino acid residues in the motif-REP units is x and the total number of amino acid residues in the domain sequence is y, it is preferable that x / y is 64.2% or more.

[0084] The third modified fibroin may contain the above-mentioned tag sequence at either or both of the N-terminus and C-terminus.

[0085] More specific examples of modified fibroins containing tag sequences include (3-iii) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0086] The amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are obtained by adding the amino acid sequence shown in SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. It is something.

[0087] The modified fibroin (3-iii) may consist of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0088] The modified fibroin (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin (3-iv) also ... the formula 1: [(A)n Motif-REP] m The above sequence identity is preferably 95% or more.

[0089] The modified fibroin (3-iv) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and has two adjacent amino acids (A) n The number of amino acid residues in the REPs of the motif-REP] unit is compared sequentially. When the number of amino acid residues in the REP with the fewer amino acid residues is set to 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3. n When the maximum total number of amino acid residues in the motif-REP units is x and the total number of amino acid residues in the domain sequence is y, it is preferable that x / y is 64.2% or more.

[0090] 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 selected depending on the type of host.

[0091] The fourth modified fibroin has a domain sequence that is different from that of naturally occurring fibroin: (A) n The fourth modified fibroin has an amino acid sequence in which the content of the motif is reduced and the content of glycine residues is reduced. The domain sequence of the fourth modified fibroin has at least one or more (A) n In addition to the deletion of the motif, the fourth modified fibroin has an amino acid sequence in which at least one or more glycine residues in REP have been replaced with other amino acid residues. That is, the fourth modified fibroin is a modified fibroin that combines the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects are as described for the second modified fibroin and the third modified fibroin.

[0092] More specific examples of the fourth modified fibroin include (4-i) modified fibroins comprising the amino acid sequence set forth in SEQ ID NO:7 (Met-PRT410), SEQ ID NO:8 (Met-PRT525), SEQ ID NO:9 (Met-PRT799), SEQ ID NO:13 (PRT410), SEQ ID NO:14 (PRT525), or SEQ ID NO:15 (PRT799), or (4-ii) an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. Specific embodiments of modified fibroins comprising the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15 are as described above.

[0093] The fifth modified fibroin may have an amino acid sequence whose domain sequence includes a region with a localized high hydrophobic index, corresponding to the substitution of one or more amino acid residues in REP with amino acid residues with a high hydrophobic index and / or the insertion of one or more amino acid residues with a high hydrophobic index into REP, compared to naturally occurring fibroin.

[0094] The region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

[0095] The amino acid residue with a high hydrophobicity index is more preferably an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A).

[0096] The fifth modified fibroin may have modifications corresponding to the substitution of one or more amino acid residues in the REP with amino acid residues having a high hydrophobicity index, and / or the insertion of one or more amino acid residues having a high hydrophobicity index into the REP, compared to naturally occurring fibroin, as well as further 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 fibroin.

[0097] 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 a cloned naturally occurring 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, the fifth modified fibroin can be obtained, for example, 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 and / or the insertion of one or more hydrophobic amino acid residues into REP from the amino acid sequence of a naturally occurring fibroin, 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 a naturally occurring fibroin, the amino acid sequence may also be modified by substituting, deleting, inserting, and / or adding one or more amino acid residues.

[0098] The fifth modified fibroin has the formula 1: [(A) n Motif-REP] m and is located at the most C-terminal end (A). n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of amino acid residues contained in the region where the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the region located most C-terminally is defined as (A). nThe domain sequence may have an amino acid sequence in which p / q is 6.2% or more, where q is the total number of amino acid residues contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence.

[0099] The hydrophobicity index of amino acid residues is determined using a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132). Specifically, the hydrophobicity index (hereinafter referred to as "HI") of each amino acid is as shown in Table 1 below.

[0100] [Table 1]

[0101] The calculation method of p / q will be described in more detail. For the calculation, Equation 1: [(A) n Motif-REP] m (A) is located at the C-terminal end of the domain sequence represented by n A sequence excluding the sequence from the motif to the C-terminus of the domain sequence (hereinafter referred to as "sequence A") is used. First, the average hydrophobicity index of four consecutive amino acid residues is calculated for all REPs contained in sequence A. The average hydrophobicity index is calculated by dividing the sum of the HIs of each amino acid residue contained 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 one amino acid residue in the region. The total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.

[0102] For example, if 20 "sequences of four consecutive amino acid residues with an average hydrophobicity index of 2.6 or greater" are extracted (without overlaps), the region with an average hydrophobicity index of 2.6 or greater will contain 20 consecutive four amino acid residues (without overlaps), and p is 20 × 4 = 80. Also, if two "sequences of four consecutive amino acid residues with an average hydrophobicity index of 2.6 or greater" overlap by one amino acid residue, the region with an average hydrophobicity index of 2.6 or greater will contain seven amino acid residues (p = 2 × 4 - 1 = 7, where "-1" is the deduction of the overlap). For example, in the domain sequence shown in Figure 4, there are seven "sequences of four consecutive amino acid residues with an average hydrophobicity index of 2.6 or greater" without overlaps, and p is 7 × 4 = 28. For example, in the case of the domain sequence shown in Figure 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 ((A) at the end of the C-terminal side). n (The motif is not included.) Next, p / q (%) can be calculated by dividing p by q. In the case of Figure 4, it is 28 / 170 = 16.47%.

[0103] 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.

[0104] The fifth modified fibroin can be obtained, for example, by modifying the amino acid sequence of a cloned naturally occurring fibroin to include a region of high hydrophobicity, for example, by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobic index) in the REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobic index) and / or by inserting one or more hydrophobic amino acid residues into the REP so as to satisfy the p / q condition. Alternatively, the fifth modified fibroin can be obtained by, for example, designing an amino acid sequence that satisfies the p / q condition from the amino acid sequence of a naturally occurring fibroin 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 amino acid residues in the REP with amino acid residues with a high hydrophobic index and / or the insertion of one or more amino acid residues with a high hydrophobic 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.

[0105] The amino acid residues with a high hydrophobicity index are not particularly limited, but are preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), and alanine (A), and more preferably valine (V), leucine (L), and isoleucine (I).

[0106] More specific examples of the fifth modified fibroin include (5-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), 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.

[0107] The modified fibroin (5-i) will be explained. The amino acid sequence shown in SEQ ID NO: 19 is a sequence of three amino acid residues in every other REP, except for the C-terminal domain sequence, of the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410). 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 one position in the amino acid sequence shown in SEQ ID NO: 8 (Met-PRT525) at every other REP. 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 two positions in the amino acid sequence shown in SEQ ID NO: 8 at every other REP.

[0108] 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.

[0109] The modified fibroin (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin (5-ii) also ... the formula 1: [(A) n Motif-REP] m The above sequence identity is preferably 95% or more.

[0110] The modified fibroin (5-ii) 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 is located at the most C-terminal side (A) n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of amino acid residues contained in the region where the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the region located most C-terminally is defined as (A). nWhen the total number of amino acid residues contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence is defined as q, p / q is preferably 6.2% or more.

[0111] The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus.

[0112] More specific examples of modified fibroins containing tag sequences include (5-iii) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0113] The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 are obtained by adding the amino acid sequence shown in SEQ ID NO: 11 (including a His tag sequence and a 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.

[0114] The modified fibroin (5-iii) may consist of the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0115] The modified fibroin (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The modified fibroin (5-iv) also contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The modified fibroin (5- n Motif-REP] m The above sequence identity is preferably 95% or more.

[0116] The modified fibroin (5-iv) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24 and is located at the most C-terminal side (A)n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of amino acid residues contained in the region where the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the region located most C-terminally is defined as (A). n When the total number of amino acid residues contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence is defined as q, p / q is preferably 6.2% or more.

[0117] 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 selected depending on the type of host.

[0118] The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues compared to naturally occurring fibroin.

[0119] The sixth modified fibroin preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

[0120] When the sixth modified fibroin contains a GPGXX motif in the REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. There is no upper limit to the GPGXX motif content, and it may be 50% or less, or may be 30% or less.

[0121] As used herein, the "GPGXX motif content" is a value calculated by the following method: Formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n In a fibroin (modified fibroin or naturally-occurring fibroin) containing the domain sequence represented by the motif, (A) is located at the most C-terminal end.n For all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of GPGXX motifs contained in that region is multiplied by three (i.e., the total number of G and P in the GPGXX motif), and the number s is the number located at the most C-terminus (A). n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and then (A) n When the total number of amino acid residues of all REPs excluding the motif is t, the GPGXX motif content is calculated as s / t.

[0122] In calculating the GPGXX motif content, the most C-terminal (A) n The target sequence is the sequence from the motif to the C-terminus of the domain sequence, excluding the sequence from the domain sequence. n The sequence from the 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 fibroin, and when m is small (i.e., when the domain sequence is short), this influence can be affected by the calculation of the GPGXX motif content, so this is to eliminate this influence. Note that when the "GPGXX motif" is located at the C-terminus of REP, even if "XX" is, for example, "AA," it is treated as a "GPGXX motif."

[0123] FIG. 5 is a schematic diagram showing the domain sequence of a modified fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. 5. First, the domain sequence of the modified fibroin shown in FIG. 5 ("(A)") n Motif-REP] m -(A) n In the "A" motif type, all REPs are located at the "most C-terminal (A)" n Since the number of GPGXX motifs is 7, s is calculated as 7 × 3 = 21. Similarly, all REPs are located at the C-terminus (A) nThe sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence (the sequence shown as "Region A" in Figure 5) is included in the sequence. n The total number of amino acid residues in all REPs excluding motifs, t, is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t, which is 21 / 150 = 14.0% for the modified fibroin in Figure 5.

[0124] 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%.

[0125] As used herein, the term "glutamine residue content" refers to a value calculated by the following method: Formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n In a fibroin (modified fibroin or naturally-occurring fibroin) containing the domain sequence represented by the motif, (A) is located at the most C-terminal end. n The sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence (the sequence corresponding to "Region A" in Figure 5) from the domain sequence, for all REPs contained in that region, let u be the total number of glutamine residues contained in that region, and let u be the number of glutamine residues located at the most C-terminus (A). n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and then (A) n When the total number of amino acid residues in all REPs excluding the motif is t, the glutamine residue content is calculated as u / t. n The reason for targeting "a sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as that described above.

[0126] The sixth modified fibroin may have an amino acid sequence whose domain sequence corresponds to that of a naturally occurring fibroin in which one or more glutamine residues in the REP have been deleted or substituted with other amino acid residues.

[0127] The "other amino acid residue" may be any amino acid residue other than glutamine, but is preferably an amino acid residue with a higher hydrophobicity index than glutamine. The hydrophobicity indexes of amino acid residues are shown in Table 1.

[0128] 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.

[0129] 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.

[0130] In the present specification, the "hydrophobicity of REP" is a value calculated by the following method: Formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A)n In a fibroin (modified fibroin or naturally-occurring fibroin) containing the domain sequence represented by the motif, (A) is located at the most C-terminal end. n For all REPs contained in the sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence (the sequence corresponding to "Region A" in Figure 5), the sum of the hydrophobicity indexes of each amino acid residue in that region is v, and the residue located most C-terminally (A) n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and then (A) n When the total number of amino acid residues of all REPs excluding motifs is t, The hydrophobicity of REP is calculated as v / t. n The reason for targeting "a sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as that described above.

[0131] The sixth modified fibroin may have a domain sequence that, compared to a naturally occurring fibroin, corresponds to the deletion of one or more glutamine residues in the REP and / or the substitution of one or more glutamine residues in the REP with other amino acid residues, and may also have a modification in the amino acid sequence that corresponds to the substitution, deletion, insertion and / or addition of one or more amino acid residues.

[0132] The sixth modified fibroin can be obtained, for example, by deleting one or more glutamine residues in REP from the gene sequence of a cloned naturally occurring 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 a naturally occurring 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.

[0133] More specific examples of the sixth modified fibroin include (6-i) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or SEQ ID NO: 42 (Met-PRT1028), or (6-ii) modified fibroins containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42.

[0134] The modified fibroin (6-i) will now be described. The amino acid sequence shown in SEQ ID NO: 25 is obtained by substituting all QQs with VL in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410). The amino acid sequence shown in SEQ ID NO: 26 is obtained by substituting all QQs with TS and the remaining Qs with A in the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 27 is obtained by substituting all QQs with VL and the remaining Qs with I in the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 28 is obtained by substituting all QQs with VI and the remaining Qs with L in the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 29 is obtained by substituting all QQs with VF and the remaining Qs with I in the amino acid sequence shown in SEQ ID NO: 7.

[0135] The amino acid sequence shown in SEQ ID NO: 30 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence shown in SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO: 8 with VL and substituting the remaining Qs with I.

[0136] The amino acid sequence shown in SEQ ID NO: 32 is a sequence in which the 20 domain sequence region present in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) is repeated twice, with all QQs replaced with VFs and the remaining Qs replaced with Is.

[0137] The amino acid sequence shown in SEQ ID NO: 41 (Met-PRT917) is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with LI and substituting the remaining Q with V. The amino acid sequence shown in SEQ ID NO: 42 (Met-PRT1028) is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with IF and substituting the remaining Q with T.

[0138] The amino acid sequences represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 all have a glutamine residue content of 9% or less (Table 2).

[0139] [Table 2]

[0140] The modified fibroin (6-i) may consist of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42.

[0141] 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: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42. The modified fibroin (6-ii) also comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42. The modified fibroin (6-ii) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) nIt is a protein containing a domain sequence represented by a motif. The sequence identity is preferably 95% or more.

[0142] The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less, and the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.

[0143] The sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows the modified fibroin to be isolated, immobilized, detected, visualized, and the like.

[0144] More specific examples of modified fibroins containing tag sequences include (6-iii) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028), or (6-iv) modified fibroins containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44.

[0145] The amino acid sequences shown in SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 43, and 44 are obtained by adding the amino acid sequence shown in SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 41, and 42, respectively. 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 shown in SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 43, and 44 all have a glutamine residue content of 9% or less (Table 3).

[0146] [Table 3]

[0147] The modified fibroin (6-iii) may consist of the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 44.

[0148] 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: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 44. The modified fibroin (6-iv) also ... n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n It is a protein containing a domain sequence represented by a motif. The sequence identity is preferably 95% or more.

[0149] The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less, and the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

[0150] 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 selected depending on the type of host.

[0151] 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.

[0152] The modified fibroin is preferably a hydrophilic modified fibroin because it has superior water absorption and quick-drying properties. In this specification, "hydrophilic modified fibroin" refers to a modified fibroin in which the sum of the hydrophobicity indexes (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, resulting in an average HI of 0 or less. The hydrophobicity index is as shown in Table 1. Modified fibroin with an average HI of more than 0 is also sometimes called hydrophobic modified fibroin.

[0153] Examples of hydrophilic modified fibroins 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, and the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0154] 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.

[0155] The modified fibroin according to this embodiment can be produced by a conventional method using a nucleic acid encoding the modified fibroin. The nucleic acid encoding the modified fibroin may be chemically synthesized based on the base sequence information, or may be synthesized using a PCR method or the like. Furthermore, the produced modified fibroin can be isolated and purified by conventional methods.

[0156] (Water-absorbent, quick-drying property imparting agent) The water-absorbent, quick-drying property imparting agent according to this embodiment may contain a single type of modified fibroin, or may contain a combination of two or more types of modified fibroin.

[0157] The water absorbency of the quick-drying agent according to this embodiment may be 60 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, or 5 seconds or less, as evaluated in accordance with JIS L 1907. The water absorbency may be evaluated, for example, by the method described in the examples below.

[0158] The water-absorbing and quick-drying agent according to this embodiment is evaluated by measuring the diffusible residual moisture content. The quick-drying property may be 100 minutes or less, 90 minutes or less, 80 minutes or less, or 70 minutes or less. The diffusible residual moisture content (%) is a value calculated by the following formula: Diffusible residual moisture content (%) = Weight of water (g) at each time / Weight of water (g) at the start of measurement × 100. The quick-drying property refers to the time required for the diffusible residual moisture content to reach 10% or less. The quick-drying property can be evaluated, for example, by the method described in the Examples below.

[0159] The water-absorbing and quick-drying property-imparting agent according to the present embodiment may further contain other additives (components other than the active ingredient) depending on the form, application, etc. Examples of additives include plasticizers, leveling agents, crosslinking agents, crystal nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers, and synthetic resins. The content of the additives may be 50 parts by mass or less relative to 100 parts by mass of the total amount of the water-absorbing and quick-drying property-imparting agent.

[0160] The water-absorbent, quick-drying property imparting agent according to this embodiment may be in any form, for example, a powder, a paste, or a liquid (for example, a suspension or a solution). The water-absorbent, quick-drying property imparting agent according to this embodiment may also be in the form of, for example, fibers, a film, a gel, a porous body, particles, or the like. The form of the water-absorbent, quick-drying property imparting agent according to this embodiment may be appropriately determined depending on the object to which the water-absorbent, quick-drying property is to be imparted (the article to which the water-absorbent, quick-drying property is to be imparted) and its intended use.

[0161] The water-absorbing and quick-drying property imparting agent according to this embodiment contains modified fibroin as a main component, and therefore can be molded into any of the above-mentioned shapes. The molded article may be molded from the modified fibroin itself, or may be molded from a combination of the modified fibroin and other materials.

[0162] When preparing the water-absorbing and quick-drying property-imparting agent according to the present embodiment in powder form, for example, the protein obtained by the above-described method for producing modified fibroin may be dried to form a powder. The protein powder may contain other additives as needed.

[0163] When preparing the water-absorbing and quick-drying property-imparting agent according to this embodiment in a liquid form (e.g., a solution), for example, the protein obtained by the above-described method for producing modified fibroin may be dissolved in a solvent capable of dissolving the modified fibroin to form a liquid (modified fibroin solution). The modified fibroin solution may contain other additives as needed. Examples of solvents capable of dissolving modified fibroin include dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP). An inorganic salt may be added to the solvent as a dissolution promoter.

[0164] When the water-absorbing and quick-drying property-imparting agent according to this embodiment is prepared in the form of a fiber, for example, the modified fibroin solution described above may be used as a dope solution and spun into a fiber (modified fibroin fiber) by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning. The form of the fiber may be a single yarn, or a composite yarn such as a blended yarn, a mixed yarn, a mixed woven yarn, a union yarn, a ply-twisted yarn, or a covered yarn, or may be a nonwoven fabric, etc.

[0165] The modified fibroin fiber may be either a staple fiber or a filament. The modified fibroin fiber may be the modified fibroin fiber alone or may be combined with other fibers. That is, a single yarn consisting of the modified fibroin fiber alone and a composite yarn consisting of the modified fibroin fiber combined with other fibers may be used alone or in combination. The single yarn and the composite yarn may be a spun yarn in which staple fibers are twisted together, or a filament yarn in which filaments are twisted or not twisted together. The modified fibroin fiber, whether a staple fiber or a filament fiber, may be used as is, without being processed into a yarn, either alone or in combination with other fibers. Examples of other fibers include synthetic fibers such as nylon and polyester, regenerated fibers such as cupra and rayon, and natural fibers such as cotton and hemp. When used in combination with other fibers, the content of the modified fibroin fiber is preferably 20% by mass or more, more preferably 30% by mass or more, even more preferably 40% by mass or more, and even more preferably 50% by mass or more, based on the total amount of fibers.

[0166] When the water-absorbing and quick-drying property-imparting agent according to the present embodiment is prepared in the form of a film, a gel, a porous body, particles, or the like, it can be produced in accordance with the methods described in, for example, JP-A-2009-505668, JP-A-2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735, and the like.

[0167] [Method for imparting moisture-absorbing and quick-drying properties to an article] The method for imparting moisture-absorbing and quick-drying properties to an article according to this embodiment comprises the step of incorporating a modified fibroin into the article. The modified fibroin according to the present invention has excellent moisture-absorbing and quick-drying properties, and therefore, by incorporating the modified fibroin into an article, moisture-absorbing and quick-drying properties can be imparted to the article.

[0168] The article is not particularly limited as long as it is one to which moisture-absorbing and quick-drying properties should be imparted, and specific examples thereof include fibers, woven fabrics, knitted fabrics, nonwoven fabrics, cotton, sponges, films, resins, composite materials (regardless of the form of the moisture-absorbing and quick-drying properties imparting agent according to the present embodiment), and various articles made using them.

[0169] The step of incorporating the modified fibroin may be a step of incorporating the modified fibroin by incorporating the water-absorbent, quick-drying property-imparting agent according to the present invention described above. The method of incorporating the modified fibroin is not particularly limited, and may involve mixing the modified fibroin with the material (raw material), or may involve combining the water-absorbent, quick-drying property-imparting agent prepared in the form of the above-described molded body with other materials (molded body, etc.) to form an article. Alternatively, the modified fibroin (which may contain other additives as necessary) itself may be molded to form an article (molded body).

[0170] The content of the modified fibroin in the article is preferably 20% by mass or more, more preferably 30% by mass or more, even more preferably 40% by mass or more, and even more preferably 50% by mass or more, based on the total weight of the article. The upper limit of the modified fibroin content may be 100% by mass or 90% by mass or less, based on the total weight of the article. The water absorption and quick-drying properties of the article can be controlled by adjusting the content of the modified fibroin in the article. In other words, because the modified fibroin according to the present invention has excellent water absorption and quick-drying properties, the water absorption and quick-drying properties of the article can be improved as the content of the modified fibroin in the article increases. [Example]

[0171] The present invention will be described in more detail below with reference to examples, etc. However, the present invention is not limited to the following examples.

[0172] [Production of modified fibroin] (1) Preparation of expression vectors A modified fibroin (PRT918) having the amino acid sequence shown in SEQ ID NO: 37 and a modified fibroin (PRT799) having the amino acid sequence shown in SEQ ID NO: 15 were designed. Nucleic acids encoding the designed modified fibroins were synthesized. An NdeI site was added to the 5' end of the nucleic acid and an EcoRI site downstream of the termination codon. This nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

[0173] (2) Protein Expression Escherichia coli BLR(DE3) was transformed with the obtained expression vector. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture was added to 100 mL of seed culture medium (Table 4) containing ampicillin at OD 200. 600 The culture temperature was kept at 30°C, and the OD 600 The flask culture was continued until the fertilization rate reached 5 (about 15 hours), and a seed culture solution was obtained.

[0174] [Table 4]

[0175] The seed culture solution was added to a jar fermenter containing 500 mL of production medium (Table 5) and measured at OD 600 The culture temperature was maintained at 37°C and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration.

[0176] [Table 5]

[0177] Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g glucose / L, 120 g yeast extract / L) was added at a rate of 1 mL / min. The culture temperature was maintained at 37°C, and the pH was controlled at 6.9. The culture was continued for 20 hours, maintaining the dissolved oxygen concentration in the culture at 20% of the dissolved oxygen saturation concentration. 1 M isopropyl-β-thiogalactopyranoside (IPTG) was then added to the culture to a final concentration of 1 mM to induce the expression of the modified fibroin. 20 hours after IPTG addition, the culture was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the cultures before and after IPTG addition. Expression of the desired modified fibroin was confirmed by the appearance of a band of the desired size, which was dependent on the addition of IPTG.

[0178] (3) Protein Purification. The cells were harvested 2 hours after the addition of IPTG and washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF, and 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 at 60°C for 30 minutes. After dissolution, the cells were dialyzed against water using a dialysis tube (Cellulose tube 36 / 32, Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation, and the water was removed using a freeze-dryer. The freeze-dried powder was collected to obtain modified fibroins (PRT918 and PRT799).

[0179] PRT918 is a hydrophobic modified fibroin with an average HI greater than 0. PRT799 is a hydrophilic modified fibroin with an average HI less than or equal to 0.

[0180] [Production of protein fibers] Dimethyl sulfoxide (DMSO) with LiCl dissolved to a concentration of 4.0% by mass was prepared as a solvent, and the freeze-dried powder of modified fibroin was added to the solvent to a concentration of 24% by mass and dissolved for 3 hours using a shaker. After that, insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning dope).

[0181] The prepared spinning solution was filtered at 60°C through a metal filter with 5 μm openings, then allowed to stand in a 30 mL stainless steel syringe to degas, and then discharged through a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The discharge temperature was 60°C. After coagulation, the obtained raw yarn was wound and air-dried to obtain modified fibroin fiber (raw fiber).

[0182] For comparison, commercially available silk fibers, cotton fibers, and polyester fibers were prepared as raw fibers.

[0183] [Manufacturing of knitted fabrics] Each raw fiber was used to manufacture knitted fabrics by flat knitting using a flat knitting machine. The knitted fabric using PRT918 fiber as the raw fiber had a thickness of 1 / 30N (single wool count) and a gauge of 18. The knitted fabric using PRT799 fiber as the raw fiber had a thickness of 1 / 30N (single wool count) and a gauge of 16. The knitted fabrics using other raw fibers had almost the same cover factor as the knitted fabrics using PRT918 fiber and PRT799 fiber. The thickness and gauge were adjusted to match the target. Specifically, they are as follows: Silk: Thickness: 2 / 60N (single yarn), Gauge: 14 Cotton: Thickness: 2 / 34N (double yarn), Gauge: 14 Polyester: Thickness: 1 / 60N (single yarn), Gauge: 14

[0184] [Evaluation of Water Absorbency and Quick-Drying Properties] (Evaluation of Water Absorbency) Water absorbency was evaluated by a test in accordance with JIS L 1907 (Testing Method for Water Absorbency of Textile Products, Drop Method). Specifically, under standard conditions (temperature 20±2°C / humidity 65±4%RH), one drop of water was dropped from a burette onto the surface of the knitted fabric prepared above, and the time until the dropped water was absorbed by the knitted fabric (the specular reflection disappeared) was measured (maximum measurement time 60 seconds). The results are shown in Table 6.

[0185] (Evaluation of Quick-Drying Property) Quick-drying property was evaluated by measuring the diffusible residual moisture content. Specifically, under standard conditions (temperature 20±2°C / humidity 65±4% RH), 0.6 mL of tap water was dropped onto the backside of the knitted fabric, and the weight of the water was measured at regular intervals (every 5 minutes). The diffusible residual moisture content was calculated using the following formula: Diffusible residual moisture content (%) = weight of water (g) at each time point / weight of water (g) at the start of measurement × 100. Measurements were continued until the diffusible residual moisture content was 10% or less (i.e., 10% of the weight of water = 60 μL (60 mg)), and the time required for the diffusible residual moisture content to reach 10% was calculated. The results are shown in Table 6.

[0186] [Table 6]

[0187] It can be seen that the modified fibroins (PRT799 and PRT918) are excellent in water absorption and quick-drying properties. In particular, it can be seen that the hydrophilic modified fibroin (PRT799) is excellent in both water absorption and quick-drying properties.

Claims

1. It contains modified fibroin as an active ingredient, The modified fibroin has the formula 1: [(A) ｎ motif-REP] ｍ , or Formula 2: [(A) ｎ motif-REP] ｍ -(A) ｎ A water-absorbing and quick-drying agent comprising a domain sequence represented by a motif, the domain sequence being different from the amino acid sequence of naturally occurring fibroin. [In Formula 1 and Formula 2, (A) ｎ The motif has 2 to 27 amino acid residues, and (A) ｎ The ratio of the number of alanine residues to the total number of amino acid residues in the motif is 40% or more, REP represents an amino acid sequence consisting of 2 to 200 amino acid residues, m represents an integer of 2 to 300, and a plurality of m exists (A). ｎ The motifs may have the same or different amino acid sequences, and multiple REPs may have the same or different amino acid sequences.

2. The water-absorbing and quick-drying agent according to claim 1, wherein the modified fibroin comprises a modified fibroin having an average hydrophobicity index (average HI) of 0 or less.

3. The water-absorbing and quick-drying property imparting agent according to claim 1 or 2, which is in the form of fibers.

4. A method for imparting moisture-wicking and quick-drying properties to an article, comprising: Including a modified fibroin in the article, The modified fibroin has the formula 1: [(A) ｎ motif-REP] ｍ , or Formula 2: [(A) ｎ motif-REP] ｍ -(A) ｎ The method comprises a domain sequence represented by a motif, and the domain sequence is different from the amino acid sequence of naturally occurring fibroin. [In Formula 1 and Formula 2, (A) ｎ The motif has 2 to 27 amino acid residues, and (A) ｎ The ratio of the number of alanine residues to the total number of amino acid residues in the motif is 40% or more, REP represents an amino acid sequence consisting of 2 to 200 amino acid residues, m represents an integer of 2 to 300, and a plurality of m exists (A). ｎ The motifs may have the same or different amino acid sequences, and multiple REPs may have the same or different amino acid sequences.

Images