Agent for imparting contact cold sensitivity and water absorption and quick drying properties, and method for imparting contact cold sensitivity and water absorption and quick drying properties to an article

Abstract

The purpose of the present invention is to provide a novel agent for imparting cool touch sensation and water-absorbing and quick-drying properties. An agent for imparting cool touch sensation and water-absorbing and quick-drying properties according to the present invention contains, as an active ingredient, a modified fibroin.

Description

Technical Field

[0001] The present invention relates to a contact cold sensitivity and water absorption and quick-drying imparting agent, and a method for imparting contact cold sensitivity and water absorption and quick-drying to an article.

Background Art

[0002] In recent years, high-functional materials having both contact cold sensitivity and water absorption and quick-drying properties have been increasingly used for articles that directly touch the skin, such as summer bedding and clothing. For example, Patent Document 1 discloses a composite spun yarn of triacetate-based staple fiber A having a single-filament fineness of 0.5 to 3.0 dtex and cellulose-based staple fiber B, wherein the mass ratio (A / B) of staple fibers A and B is in the range of 20 / 80 to 80 / 20, and further, the hairiness index of 3 mm or more is 50 pieces / m or less, and the average strength is in the range of 100 CN or more.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] The composite spun yarn disclosed in Patent Document 1 utilizes the advantages of triacetate-based staple fibers. However, synthetic fibers such as triacetate-based staple fibers have a problem of poor biodegradability and causing an environmental load. On the other hand, regarding natural fibers, although silk has relatively high contact cold sensitivity, it cannot fully satisfy the levels required for summer bedding and clothing. Moreover, silk does not have sufficient water absorption and quick-drying properties and cannot be said to have sufficient functions from the viewpoint of preventing stuffiness. Thus, with regard to high-functional materials having both contact cold sensitivity and water absorption and quick-drying properties, it cannot be said that sufficient options have been provided yet.

[0005] An object of the present invention is to provide a novel contact cold sensitivity and water absorption and quick-drying agent. Another object of the present invention is to provide a method for imparting contact cold sensitivity and water absorption and quick-drying property to a predetermined article.

Means for Solving the Problems

[0006] The present inventors have found that modified fibroin has excellent contact cold sensitivity and water absorption and quick-drying properties. The present invention is based on this new finding.

[0007] The present invention relates to, for example, the following inventions. [1] A contact cold sensitivity and water absorption and quick-drying agent containing modified fibroin as an active ingredient. [2] The contact cold sensitivity and water absorption and quick-drying agent according to [1], wherein the modified fibroin contains a modified fibroin having an average value of hydrophobicity index (average HI) exceeding 0. [3] The contact cold sensitivity and water absorption and quick-drying agent according to [1] or [2], wherein the modified fibroin contains modified spider silk fibroin. [4] The contact cold sensitivity and water absorption and quick-drying agent according to any one of [1] to [3], which is in the form of fibers. [5] A method for imparting contact cold sensitivity and water absorption and quick-drying property to an article, the method comprising the step of incorporating modified fibroin into the article.

Effects of the Invention

[0008] According to the present invention, it is possible to provide a novel contact cold sensitivity and water absorption and quick-drying agent. According to the present invention, it is also possible to provide a method for imparting contact cold sensitivity and water absorption and quick-drying property to a predetermined article.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0010] Hereinafter, the mode for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0011] 〔Agent for imparting contact cold sensitivity and water absorption and quick drying property〕 The agent for imparting contact cold sensitivity and water absorption and quick drying property according to the present embodiment contains modified fibroin as an active ingredient.

[0012] Modified fibroin has both contact cold sensitivity (for example, the property of giving a cool feeling when touched) and water absorption and quick drying property (for example, the property of absorbing moisture (for example, sweat) well and drying quickly). The agent for imparting contact cold sensitivity and water absorption and quick drying property according to the present embodiment can utilize these properties of modified fibroin to simultaneously impart contact cold sensitivity and water absorption and quick drying property to a predetermined article.

[0013] (Modified fibroin) The modified fibroin according to the present embodiment has the formula 1: [(A) n Motif-REP] m , or the formula 2: [(A) n Motif-REP] m -(A) nIt is a protein containing a domain sequence represented by a motif. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically regions that do not have repeats of amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

[0014] As used herein, "modified fibroin" means 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 having the same amino acid sequence as that of naturally occurring fibroin. "Naturally occurring fibroin" as used herein also refers to 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.

[0015] "Modified fibroin" may be one that uses the amino acid sequence of naturally occurring fibroin as it is, or one that modifies the amino acid sequence based on the amino acid sequence of naturally occurring fibroin (for example, one that modifies the amino acid sequence by modifying the cloned gene sequence of naturally occurring fibroin), or one that is artificially designed and synthesized without relying on naturally occurring fibroin (for example, one that has a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

[0016] As used herein, "domain sequence" refers to an amino acid sequence that gives rise to a crystal region specific to fibroin (typically corresponding to the (A) n motif of the amino acid sequence.) and an amorphous region (typically corresponding to the REP of the amino acid sequence), and is Formula 1: [(A) n Motif-REP] mor formula 2: [(A) n Motif-REP] m -(A) n means an amino acid sequence represented by the motif. Here, (A) n Motif represents an amino acid sequence mainly composed 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. Also, (A) 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% (which means consisting only of alanine residues). Multiple (A) n Motifs may consist of at least 7 alanine residues only. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may 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. Multiple (A) n Motifs may have the same amino acid sequence as each other, or may have different amino acid sequences. Multiple REPs may have the same amino acid sequence as each other, or may have different amino acid sequences.

[0017] The modified fibroin according to this embodiment can be obtained, for example, by modifying the amino acid sequence corresponding to substituting, deleting, inserting and / or adding one or more amino acid residues to the cloned gene sequence of naturally-derived fibroin. 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 the site-directed mutagenesis method. Specifically, it can be performed according to the methods described in documents such as Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983).

[0018] Naturally occurring fibroin is a protein comprising a domain array represented by formula 1: [(A) n motif-REP] m or formula 2: [(A) n motif-REP] m -(A) n motif, and specifically includes, for example, fibroin produced by insects or spiders.

[0019] Examples of fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Samia cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, and hornet silk protein secreted by the larvae of Vespa simillima xanthoptera.

[0020] More specific examples of fibroin produced by insects include, for example, silkworm fibroin light chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).

[0021] 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 wolf spider, garden spider, black and yellow garden spider, green lynx spider, and marbled orbweaver; spiders belonging to the genus Neoscona such as the Japanese field spider, barn spider, fishing spider, and crab spider; spiders belonging to the genus Pronus such as the false wolf spider; spiders belonging to the genus Cyrtarachne such as the triangular orbweaver and large triangular orbweaver; spiders belonging to the genus Gasteracantha such as the spiny spider and dwarf spiny spider; spiders belonging to the genus Ordgarius such as the bean-shaped orbweaver and armed bean-shaped orbweaver; spiders belonging to the genus Argiope such as the banded garden spider, small banded garden spider, and long-jawed orbweaver; spiders belonging to the genus Arachnura such as the whip spider; spiders belonging to the genus Acusilas such as the jumping spider; spiders belonging to the genus Cytophora such as the gray cross spider, goldenrod spider, and white-banded crab spider; spiders belonging to the genus Poltys such as the funnel-web spider; spiders belonging to the genus Cyclosa such as the trashline orbweaver, four-spotted orbweaver, marbled orbweaver, and black orbweaver; and spiders belonging to the genus Chorizopes such as the Japanese stream spider produce spider silk proteins. Also, spiders belonging to the genus Tetragnatha such as the stretchy spider, long-bodied cellar spider, striped lynx spider, and netted lynx spider; spiders belonging to the genus Leucauge such as the white crab spider, medium-sized white crab spider, and small white crab spider; spiders belonging to the genus Nephila such as the golden orb-weaver and giant golden orb-weaver; spiders belonging to the genus Menosira such as the silver argiope; spiders belonging to the genus Dyschiriognatha such as the dwarf stretchy spider; spiders belonging to the genus Latrodectus such as the black widow, western black widow, northern black widow, and redback spider.Examples include spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as those belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2), ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, Flag, and the like.

[0022] More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank accession number AAC04504 (amino acid sequence), U37520 (base sequence)), major ampullate spidroin 1 [from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [from Nephila clavata] (GenBank accession number AAL32472 (amino acid sequence), AF441245 (base sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampullate spidroin 2 [from Euprosthenops australis] (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [from Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [from Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [from Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), etc.

[0023] As more specific examples of naturally-derived fibroin, fibroin for which sequence information is registered in NCBI GenBank can be further cited. For example, among the sequences including INV as DIVISION in the sequence information registered in NCBI GenBank, it can be confirmed by extracting the sequences in which spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION, the character string of a specific product from CDS, and the sequences in which a specific character string is described in TISSUE TYPE from SOURCE.

[0024] The modified fibroin according to the present embodiment may be modified silk fibroin (in which the amino acid sequence of the silk protein produced by silkworms is modified), or may be modified spider silk fibroin (in which the amino acid sequence of the spider silk protein produced by spiders is modified).

[0025] As specific examples of the modified fibroin, modified fibroin (first modified fibroin) derived from the major ampullate spidroin protein produced in the major ampullate gland of spiders, modified fibroin (second modified fibroin) having a domain sequence with a reduced glycine residue content, modified fibroin (third modified fibroin) having a domain sequence with a reduced content of (A) motif, modified fibroin (fourth modified fibroin) with a reduced content of glycine residues and (A) motif, modified fibroin (fifth modified fibroin) having a domain sequence including a region with a locally large hydrophobicity index, and modified fibroin (sixth modified fibroin) having a domain sequence with a reduced content of glutamine residues can be cited. n modified fibroin (third modified fibroin) having a domain sequence with a reduced content of (A) motif, modified fibroin (fourth modified fibroin) with a reduced content of glycine residues and (A) motif, modified fibroin (fifth modified fibroin) having a domain sequence including a region with a locally large hydrophobicity index, and modified fibroin (sixth modified fibroin) having a domain sequence with a reduced content of glutamine residues can be cited. n modified fibroin (third modified fibroin) having a domain sequence with a reduced content of (A) motif, modified fibroin (fourth modified fibroin) with a reduced content of glycine residues and (A) motif, modified fibroin (fifth modified fibroin) having a domain sequence including a region with a locally large hydrophobicity index, and modified fibroin (sixth modified fibroin) having a domain sequence with a reduced content of glutamine residues can be cited.

[0026] As the first modified fibroin, Formula 1: [(A) n motif-REP] mExamples include proteins containing a domain array represented by. In the first modified fibroin, (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, still more preferably an integer of 8 to 20, still more preferably an integer of 10 to 20, still 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, in Formula 1, the number of amino acid residues constituting REP is preferably 10 to 200 residues, more preferably 10 to 150 residues, still more preferably 20 to 100 residues, and still more preferably 20 to 75 residues. The first modified fibroin has the 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 is preferably 40% or more, more preferably 60% or more, and still more preferably 70% or more based on the total number of amino acid residues.

[0027] The first modified fibroin has the formula 1: [(A) n Motif-REP] m It may also be a polypeptide containing a unit of the amino acid sequence represented by and having a C-terminal sequence that is the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

[0028] The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of the C-terminal 50 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-terminal 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-terminal of the amino acid sequence shown in SEQ ID NO: 1.

[0029] As a more specific example of the first modified fibroin, there can be mentioned a modified fibroin comprising (1-i) the amino acid sequence represented by 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 represented by SEQ ID NO: 4. The sequence identity is preferably 95% or more.

[0030] The amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of ADF3 with an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, His10 tag, and HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus, where the 1st to 13th repeat regions are increased approximately two-fold and mutated so that translation terminates at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.

[0031] (1-i) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 4.

[0032] The second modified fibroin has an amino acid sequence in which the content of glycine residues in its domain sequence is reduced as compared with that of naturally-derived fibroin. The second modified fibroin can be said to have an amino acid sequence corresponding to the substitution of at least one or a plurality of glycine residues in at least REP with another amino acid residue as compared with naturally-derived fibroin.

[0033] The second modified fibroin may have an amino acid sequence in which, in 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, at least one glycine residue in at least one or a plurality of the motif sequences is substituted with another amino acid residue, as compared with naturally-derived fibroin.

[0034] The proportion of the motif sequence in the second modified fibroin, in which the above-mentioned glycine residue is replaced by another amino acid residue, may be 10% or more based on the total motif sequence.

[0035] The second modified fibroin has the domain sequence represented by Formula 1: [(A) n Motif-REP] m and includes the above domain sequence. When the total number of amino acid residues of 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 (A) n motif located at the most C-terminal side to the C-terminal of the above domain sequence is z, and the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the above domain sequence is w, it may have an amino acid sequence in which z / w is 30% or more, 40% or more, 50% or more, or 50.9% or more. The proportion of alanine residues to the total number of amino acid residues in the (A) n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 100% (which means consisting only of alanine residues).

[0036] Preferably, the second modified fibroin has an increased content ratio of the amino acid sequence consisting of XGX by substituting one glycine residue of the GGX motif with another amino acid residue. Preferably, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is 30% or less, more preferably 20% or less, still more preferably 10% or less, even more preferably 6% or less, still more preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the calculation method of the content ratio (z / w) of the amino acid sequence consisting of the following XGX.

[0037] The method for calculating z / w will be described 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: 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. In addition, for example, in the case of an amino acid sequence consisting of XGXGX, if there are Xs (central Xs) contained in two XGXs, the overlaps are 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-terminus side to the C-terminus of the domain sequence from 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-terminus side). n (The motif is excluded.) Next, z / w (%) can be calculated by dividing z by w.

[0038] Here, the z / w ratio in naturally derived fibroin will be described. First, as described above, fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method exemplified above, and 663 types of fibroin (of which, 415 types were spider-derived fibroin) were extracted. Of all the extracted fibroins, those with the formula 1: [(A) n Using the above-mentioned calculation method, z / w was calculated from the amino acid sequence of naturally-occurring fibroin, which contains 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 is 6% or less. The results are shown in Figure 2. The horizontal axis of Figure 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from Figure 2, z / w in all naturally-occurring fibroins is less than 50.9% (the highest being 50.86%).

[0039] In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, even more preferably 70% or more, and even more preferably 80% or more. There is no particular limitation on the upper limit of z / w, and for example, it may be 95% or less.

[0040] The second modified fibroin can be obtained, for example, by modifying at least a part of the base sequence encoding the glycine residue in the cloned gene sequence of naturally-derived fibroin so as to encode another amino acid residue. At this time, as the glycine residue to be modified, one glycine residue in the GGX motif and the GPGXX motif may be selected, or substitution may be made so that z / w is 50.9% or more. Also, for example, an amino acid sequence satisfying the above-described aspect is designed from the amino acid sequence of naturally-derived fibroin, and it can also be obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to substituting the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally-derived fibroin, an amino acid sequence modification corresponding to substituting, deleting, inserting, and / or adding one or more amino acid residues may be performed.

[0041] The above-described another amino acid residue is not particularly limited as long as it is an amino acid residue other than the glycine residue, but hydrophobic amino acid residues such as valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine (M) residue, proline (P) residue, phenylalanine (F) residue, and tryptophan (W) residue, hydrophilic amino acid residues such as glutamine (Q) residue, asparagine (N) residue, serine (S) residue, lysine (K) residue, and glutamic acid (E) residue are preferable, valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine (F) residue, and glutamine (Q) residue are more preferable, and glutamine (Q) residue is still more preferable.

[0042] As a more specific example of the second modified fibroin, there can be mentioned a modified fibroin comprising (2-i) an amino acid sequence represented by 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 represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0043] The modified fibroin of (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 6 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313), which corresponds to naturally-derived fibroin, with GQX. The amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every other (A) n motif from the amino acid sequence represented by SEQ ID NO: 6, from the N-terminal side towards the C-terminal side, and further inserting [(A) n motif-REP] one time in front of the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 8 is obtained by inserting two alanine residues on the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, further substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the C-terminal side so as to have approximately the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence represented by SEQ ID NO: 9 is obtained by adding a predetermined hinge sequence and His tag sequence to the C-terminal of a sequence in which the region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted) is repeated 4 times.

[0044] The value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-derived fibroin) is 46.8%. The values of z / w in the amino acid sequences represented by 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. Also, the values of x / y in the zigzag ratios (described later) of 1:1.8 to 11.3 of the amino acid sequences represented by 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.

[0045] (2-i) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0046] (2-ii) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by the formula 1:[(A) n motif-REP] m Preferably, the above sequence identity is 95% or more.

[0047] (2-ii) When the modified fibroin has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and the total number of amino acid residues of 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 of REP in the above domain sequence is w, it is preferable that z / w is 50.9% or more.

[0048] The second modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. Thereby, isolation, immobilization, detection, visualization, etc. of the modified fibroin become possible.

[0049] As the tag sequence, for example, an affinity tag that utilizes specific affinity (binding property, affinity) with other molecules can be mentioned. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and since it has the property of specifically binding to metal ions such as nickel, it can be used for the isolation of modified fibroin by chelating metal chromatography. As a specific example of the tag sequence, for example, the amino acid sequence represented by SEQ ID NO: 11 (an amino acid sequence containing a His tag sequence and a hinge sequence) can be mentioned.

[0050] Also, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose-binding protein (MBP) that specifically binds to maltose can be used.

[0051] Furthermore, an "epitope tag" that utilizes an antigen-antibody reaction can also be used. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, a myc tag, a FLAG tag, etc. By using the epitope tag, modified fibroin can be easily purified with high specificity.

[0052] Furthermore, those in which the tag sequence can be cleaved with a specific protease can also be used. By subjecting the protein adsorbed via the tag sequence to protease treatment, modified fibroin with the tag sequence cleaved can also be recovered.

[0053] As a more specific example of the modified fibroin containing a tag array, there can be mentioned a modified fibroin containing the amino acid sequence represented by 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 represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0054] The amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are those obtained by adding the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) to the N-terminals of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0055] (2-iii) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0056] (2-iv) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m Preferably, the above sequence identity is 95% or more.

[0057] (2-iv) The modified fibroin has 90% or more sequence identity with the amino acid sequence represented by 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 of 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 of REP in the above domain sequence is w, it is preferable that z / w is 50.9% or more.

[0058] The second modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

[0059] The third modified fibroin has an amino acid sequence in which its domain sequence has a reduced content of (A) motifs as compared with a naturally-derived fibroin. n The domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to the deletion of at least one or more (A) motifs as compared with a naturally-derived fibroin. n

[0060] The third modified fibroin may have an amino acid sequence corresponding to a 10-40% deletion of (A) motifs from a naturally-derived fibroin. n

[0061] The third modified fibroin may have an amino acid sequence in which its domain sequence has a deletion of 1 to 3 (A) motifs per (A) motif from the N-terminal side to the C-terminal side as compared with a naturally-derived fibroin. n per (A) motif n

[0062] The third modified fibroin may have an amino acid sequence in which its domain sequence has a deletion of two consecutive (A) motifs and a deletion of one (A) motif in this order from the N-terminal side to the C-terminal side as compared with a naturally-derived fibroin. n deletion of n

[0063] The third modified fibroin may have an amino acid sequence in which its domain sequence has a deletion of (A) motifs every other one from the N-terminal side to the C-terminal side. n

[0064] The third modified fibroin has the domain array represented by Formula 1: [(A) n Motif-REP] m and, when moving from the N-terminal side to the C-terminal side and sequentially comparing the number of amino acid residues of REP in two adjacent [(A) n Motif-REP] units, and when the number of amino acid residues of REP with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3, the sum of the number of amino acid residues of two adjacent [(A) n Motif-REP] units is defined as x, and the total number of amino acid residues in the domain array is defined as y, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) 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, still more preferably 95% or more, and most preferably 100% (which means consisting only of alanine residues).

[0065] The method for calculating x / y will be described in more detail with reference to FIG. 1. FIG. 1 shows the domain array obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. The domain array has the sequence of (A) n Motif - First REP (50 amino acid residues) - (A) n Motif - Second REP (100 amino acid residues) - (A) n Motif - Third REP (10 amino acid residues) - (A) n Motif - Fourth REP (20 amino acid residues) - (A) n Motif - Fifth REP (30 amino acid residues) - (A) n Motif from the N-terminal side (left side).

[0066] Two adjacent [(A) n Motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side so as not to overlap. At this time, the non-selected [(A) nA [Motif-REP] unit may be present. In FIG. 1, Pattern 1 (comparison between the first REP and the second REP, and comparison between the third REP and the fourth REP), Pattern 2 (comparison between the first REP and the second REP, and comparison between the fourth REP and the fifth REP), Pattern 3 (comparison between the second REP and the third REP, and comparison between the fourth REP and the fifth REP), and Pattern 4 (comparison between the first REP and the second REP) are shown. In addition, there are other selection methods.

[0067] Next, for each pattern, the number of amino acid residues of each REP in two adjacent selected [(A) n Motif-REP] units is compared. The comparison is performed by obtaining the ratio of the number of amino acid residues of the other when the one with fewer amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the second REP is 100 / 50 = 2. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the fifth REP is 30 / 20 = 1.5.

[0068] In FIG. 1, when the one with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is 1.8 to 11.3 for [(A) n The set of [Motif-REP] units is indicated by a solid line. In this specification, this ratio is called the zigzag ratio. When the one with fewer amino acid residues is set to 1 and the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 for [(A) n The set of [Motif-REP] units is indicated by a dashed line.

[0069] In each pattern, the sum of all amino acid residues of two adjacent [(A) n Motif-REP] units indicated by a solid line is added (not only the REP but also the amino acid residues of the (A)n motif). And the added sum Compare the calculated values, and let x be the total value (the maximum value of the total values) of the pattern with the largest total value. In the example shown in FIG. 1, the total value of Pattern 1 is the largest.

[0070] Next, by dividing x by the total number of amino acid residues y in the domain array, x / y (%) can be calculated.

[0071] In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, still 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 limitation on the upper limit of x / y, and it may be, for example, 100% or less. When the Giza ratio is 1:1.9 to 11.3, x / y is preferably 89.6% or more. When the Giza ratio is 1:1.8 to 3.4, x / y is preferably 77.1% or more. When the Giza ratio is 1:1.9 to 8.4, x / y is preferably 75.9% or more. When the Giza ratio is 1:1.9 to 4.1, x / y is preferably 64.2% or more.

[0072] When the third modified fibroin is a modified fibroin in which at least 7 of the (A) n motifs are composed only of alanine residues, x / y is preferably 46.4% or more, more preferably 50% or more, still 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 limitation on the upper limit of x / y, and it may be 100% or less.

[0073] Here, x / y in naturally occurring fibroin will be described. First, as described above, when confirmed by the method exemplified using fibroin whose amino acid sequence information is registered in NCBI GenBank, 663 types of fibroin (among which 415 types are fibroin derived from spiders) were extracted. Among all the extracted fibroin, Equation 1: [(A)n Motif-REP m From the amino acid sequence of naturally occurring fibroin composed of domain arrays represented by, x / y was calculated by the above-described calculation method. The results when the crimp ratio is 1:1.9 to 4.1 are shown in FIG. 3.

[0074] The horizontal axis in FIG. 3 indicates x / y (%), and the vertical axis indicates frequency. As is clear from FIG. 3, x / y in naturally occurring fibroin is all less than 64.2% (the highest is 64.14%).

[0075] The third modified fibroin can be obtained, for example, from the gene sequence of cloned naturally occurring fibroin by deleting one or more sequences encoding the (A) n motif. Further, for example, from the amino acid sequence of naturally occurring fibroin, one or more (A) n amino acid sequences corresponding to the deletion of the motif so that x / y becomes 64.2% or more can be designed, and the nucleic acid encoding the designed amino acid sequence can also be obtained by chemical synthesis. In any case, in addition to the modification corresponding to the deletion of the (A) n motif from the amino acid sequence of naturally occurring fibroin, modification of the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be further performed.

[0076] More specific examples of the third modified fibroin include (3-i) the amino acid sequence represented by 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) a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0077] (3-i) The modified fibroin will be described. The amino acid sequence represented by SEQ ID NO: 17 is from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin, with every other (A) n motif deleted from the N-terminal side towards the C-terminal side, and further [(A) n motif-REP] inserted once in front of the C-terminal sequence. The amino acid sequences represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 are as described for the second modified fibroin.

[0078] The value of x / y in the ratio of 1:1.8 to 11.3 of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) is 15.0%. The values of x / y in the amino acid sequences represented by SEQ ID NO: 17 and SEQ ID NO: 7 are both 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.1% and 70.0% respectively.

[0079] (3-i) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0080] (3-ii) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m Preferably, the above sequence identity is 95% or more.

[0081] (3-ii) The modified fibroin has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and from the N-terminal side to the C-terminal side, for two adjacent [(A) n motif-REP] units, when comparing the amino acid residue numbers of the REPs of the two adjacent [(A) n motif-REP] units sequentially, and taking the amino acid residue number of the REP with fewer amino acid residues as 1, when the ratio of the amino acid residue number of the other REP is 1.8 to 11.3 (the stagger ratio is 1:1.8 to 11.3), the maximum value of the sum of the amino acid residue numbers of the two adjacent [(A)

[0082] The third modified fibroin may contain the above-described tag sequence at either or both of the N-terminal and C-terminal.

[0083] More specific examples of the modified fibroin containing the tag sequence include the modified fibroin containing the amino acid sequence represented by (3-iii) SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799), or the amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by (3-iv) SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0084] The amino acid sequences represented by 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 represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) to the N-terminals of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0085] (3-iii) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0086] (3-iv) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n Motif-REP] m . Preferably, the above sequence identity is 95% or more.

[0087] (3-iv) The modified fibroin has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and when sequentially comparing the amino acid residue numbers of REP in two adjacent [(A) n Motif-REP] units from the N-terminal side to the C-terminal side, and taking the amino acid residue number of the REP with fewer amino acid residues as 1, when the ratio of the amino acid residue number of the other REP is 1.8 to 11.3, the maximum value of the sum of the amino acid residue numbers of two adjacent [(A) n Motif-REP] units is set as x, and the total amino acid residue number of the domain sequence is set as y, it is preferably that x / y is 64.2% or more.

[0088] The third modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

[0089] The fourth modified fibroin has an amino acid sequence in which the content of the (A) n motif is reduced and the content of glycine residues is reduced, in addition to the domain sequence being compared with the naturally-derived fibroin. The domain sequence of the fourth modified fibroin has at least one or more (A) nIn addition to the motif deletion, it can also be said to have an amino acid sequence corresponding to the substitution of at least one or more glycine residues in the REP with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the second modified fibroin and the third modified fibroin described above. Specific embodiments and the like are as described for the second modified fibroin and the third modified fibroin.

[0090] As a more specific example of the fourth modified fibroin, (4-i) an amino acid sequence represented by 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 with the amino acid sequence represented by 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 the modified fibroin containing the amino acid sequence represented by 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.

[0091] The fifth modified fibroin may have an amino acid sequence in which its domain sequence contains a region with a locally large hydrophobicity index, corresponding to the substitution of one or more amino acid residues in the REP with amino acid residues having a large hydrophobicity index and / or the insertion of one or more amino acid residues with a large hydrophobicity index into the REP, compared to the naturally derived fibroin.

[0092] The region with a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

[0093] The amino acid residues with a large hydrophobicity index described above are more preferably amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A).

[0094] The fifth modified fibroin, compared with naturally-derived fibroin, corresponds to the modification in which one or more amino acid residues in the REP are substituted with amino acid residues having a large hydrophobicity index and / or one or more amino acid residues having a large hydrophobicity index are inserted into the REP. In addition, there may be an amino acid sequence modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues, compared with naturally-derived fibroin.

[0095] The fifth modified f ibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP with hydrophobic amino acid residues (for example, amino acid residues having a positive hydrophobicity index) from the gene sequence of cloned naturally-derived fibroin and / or inserting one or more hydrophobic amino acid residues into the REP. Also, for example, it can be obtained by designing an amino acid sequence corresponding to substitution of one or more hydrophilic amino acid residues in the REP with hydrophobic amino acid residues and / or insertion of one or more hydrophobic amino acid residues into the REP from the amino acid sequence of naturally-derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to substitution of one or more hydrophilic amino acid residues in the REP with hydrophobic amino acid residues and / or insertion of one or more hydrophobic amino acid residues into the REP from the amino acid sequence of naturally-derived fibroin, an amino acid sequence modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues may be further performed.

[0096] The fifth modified fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m and the (A) located on the most C-terminal side nIn all REPs included in the sequence obtained by removing the sequence from the motif to the C-terminus of the above domain sequence from the above domain sequence, when the total number of amino acid residues included in the region where the average value of the hydrophobicity indices of consecutive 4 amino acid residues is 2.6 or more is p, and the most C-terminal (A) n It may have an amino acid sequence in which p / q is 6.2% or more when the total number of amino acid residues included in the sequence obtained by removing the sequence from the motif to the C-terminus of the above domain sequence from the above domain sequence is q.

[0097] For the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. 105-132) is used. Specifically, the hydrophobicity index (hydrophathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

[0098]

Table 1

[0099] The calculation method of p / q will be described in more detail. For the calculation, Formula 1: [(A) n Motif - REP] m From the domain sequence represented by, the most C-terminal (A) nUse the sequence excluding the sequence from the motif to the C-terminus of the domain array (hereinafter referred to as "sequence A"). First, for all REPs contained in sequence A, calculate the average value of the hydrophobicity indices of consecutive 4 amino acid residues. The average value of the hydrophobicity index is obtained by dividing the sum of the HIs of each amino acid residue contained in consecutive 4 amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all consecutive 4 amino acid residues (each amino acid residue is used 1 to 4 times for calculating the average value). Next, identify the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more. Even if a certain amino acid residue belongs to a plurality of "consecutive 4 amino acid residues where the average value of the hydrophobicity index is 2.6 or more", it will be included as 1 amino acid residue in the region. And the total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

[0100] For example, when 20 "consecutive 4 amino acid residues where the average value of the hydrophobicity index is 2.6 or more" are extracted (without duplication), the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more contains 20 consecutive 4 amino acid residues (without duplication), and p is 20×4 = 80. Also, for example, when two "consecutive 4 amino acid residues where the average value of the hydrophobicity index is 2.6 or more" exist with only 1 amino acid residue overlapping, the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more contains 7 amino acid residues (p = 2×4 - 1 = 7. The "-1" is to subtract the overlapping part). For example, in the case of the domain array shown in Figure 4, since there are 7 "consecutive 4 amino acid residues where the average value of the hydrophobicity index is 2.6 or more" without overlap, p is 7×4 = 28. Also, for example, in the case of the domain array shown in Figure 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) n motif that exists at the end of the C-terminus side). Next, by dividing p by q, p / q (%) can be calculated. In the case of Figure 4, it is 28 / 170 = 16.47%.

[0101] In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, still 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, and may be, for example, 45% or less.

[0102] The fifth modified fibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (for example, amino acid residues with a negative hydrophobicity index) in the REP with hydrophobic amino acid residues (for example, amino acid residues with a positive hydrophobicity index) so as to satisfy the above p / q conditions, and / or inserting one or more hydrophobic amino acid residues into the REP, thereby modifying it into an amino acid sequence containing a region with a large hydrophobicity index locally. Also, for example, it can be obtained by designing an amino acid sequence that satisfies the above p / q conditions from the amino acid sequence of naturally-derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to the substitution of one or more amino acid residues in the REP with amino acid residues having a large hydrophobicity index and / or the insertion of one or more amino acid residues having a large hydrophobicity index into the REP compared to naturally-derived fibroin, modifications corresponding to the substitution, deletion, insertion, and / or addition of one or more amino acid residues may also be performed.

[0103] The amino acid residues with a large hydrophobicity index are not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A) are preferred, and valine (V), leucine (L), and isoleucine (I) are more preferred.

[0104] As a more specific example of the fifth modified fibroin, there can be mentioned a modified fibroin comprising (5-i) an amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), or (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0105] The modified fibroin of (5-i) will be described. The amino acid sequence represented by SEQ ID NO: 19 has two amino acid sequences (VLI) each consisting of 3 amino acid residues inserted every other REP, except for the terminal domain sequence at the C-terminal side, with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410), and further some glutamine (Q) residues are substituted with serine (S) residues, and some amino acids at the C-terminal side are deleted. The amino acid sequence represented by SEQ ID NO: 20 has one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP with respect to the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525). The amino acid sequence represented by SEQ ID NO: 21 has two amino acid sequences (VLI) each consisting of 3 amino acid residues inserted every other REP with respect to the amino acid sequence represented by SEQ ID NO: 8.

[0106] The modified fibroin of (5-i) may consist of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0107] The modified fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m . The above sequence identity is preferably 95% or more.

[0108] The modified fibroin of (5-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and the (A) located at the most C-terminal side n In all REPs included in the sequence obtained by removing the sequence from the (A) motif to the C-terminus of the domain sequence from the domain sequence, when the total number of amino acid residues included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is p, and the (A) located at the most C-terminal side n When the total number of amino acid residues included in the sequence obtained by removing the sequence from the (A) motif to the C-terminus of the domain sequence from the domain sequence is q, it is preferable that p / q is 6.2% or more.

[0109] The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.

[0110] More specific examples of the modified fibroin containing a tag sequence include the modified fibroin containing the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665), or SEQ ID NO: 24 (PRT666) in (5-iii), or an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24 in (5-iv).

[0111] The amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 are those obtained by adding the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0112] The modified fibroin of (5-iii) may consist of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0113] (5-iv) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . Preferably, the above sequence identity is 95% or more.

[0114] (5-iv) The modified fibroin has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and in all REPs contained in the sequence obtained by removing the sequence from the (A) n motif at the most C-terminal side to the C-terminal of the domain sequence, when the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif at the most C-terminal side to the C-terminal of the domain sequence is q, preferably p / q is 6.2% or more.

[0115] The fifth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

[0116] The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues as compared with natural fibroin.

[0117] 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 .

[0118] When the sixth modified fibroin contains the GPGXX motif in the REP, the GPGXX motif content rate is usually 1% or more, may be 5% or more, and preferably 10% or more. There is no particular limitation on the upper limit of the GPGXX motif content rate, which may be 50% or less, or may be 30% or less.

[0119] In this specification, the "GPGXX motif content rate" 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 the fibroin (modified fibroin or naturally derived fibroin) containing the domain sequence represented by the motif, for all REPs contained in the sequence obtained by removing the sequence from the most C-terminal (A) n motif to the C-terminus of the domain sequence from the domain sequence, let s be the number obtained by multiplying the total number of GPGXX motifs contained in that region by 3 (i.e., corresponding to the total number of G and P in the GPGXX motif), and when the total number of amino acid residues of all REPs obtained by removing the sequence from the most C-terminal (A) n motif to the C-terminus of the domain sequence from the domain sequence and further removing the (A) n motif is t, the GPGXX motif content rate is calculated as s / t.

[0120] In the calculation of the GPGXX motif content rate, the "sequence obtained by removing the sequence from the most C-terminal (A) n motif to the C-terminus of the domain sequence from the domain sequence" is targeted because the "most C-terminal (A) nThe sequence from the motif to the C-terminus of the domain array (the sequence corresponding to REP) may contain sequences with low correlation to the sequences characteristic of fibroin. When m is small (i.e., when the domain array is short), it affects the calculation result of the GPGXX motif content rate. This is to eliminate this influence. In addition, when the "GPGXX motif" is located at the C-terminus of REP, even if "XX" is, for example, "AA", it is treated as the "GPGXX motif".

[0121] Figure 5 is a schematic diagram showing the domain array of the modified fibroin. The method for calculating the GPGXX motif content rate will be specifically described with reference to Figure 5. First, for the domain array of the modified fibroin shown in Figure 5 (the type of "[(A) n motif - REP] m -(A) n motif"). Since all REPs are included in the sequence obtained by removing the sequence from the most C-terminal (A) n motif to the C-terminus of the domain array from the domain array (the sequence indicated by "Region A" in Figure 5), the number of GPGXX motifs for calculating s is 7, and s is 7×3 = 21. Similarly, since all REPs are included in the sequence obtained by removing the sequence from the most C-terminal (A) n motif to the C-terminus of the domain array from the domain array (the sequence indicated by "Region A" in Figure 5), the total number t of amino acid residues of all REPs after further removing the (A) n motif is 50 + 40 + 10 + 20 + 30 = 150. Next, by dividing s by t, s / t (%) can be calculated. In the case of the modified fibroin in Figure 5, it is 21 / 150 = 14.0%.

[0122] The sixth modified fibroin preferably has a glutamine residue content rate of 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%.

[0123] In this specification, the "glutamine residue content rate" 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 fibroin (modified fibroin or fibroin derived from nature) containing a domain array represented by a motif, the (A) located most C-terminally n In all REPs contained in the sequence obtained by removing the sequence from the (A) motif to the C-terminus of the domain array from the domain array (the sequence corresponding to "Region A" in Fig. 5), when the total number of glutamine residues contained in that region is u, and the (A) located most C-terminally n After removing the sequence from the (A) motif to the C-terminus of the domain array from the domain array, and further removing the (A) n When the total number of amino acid residues of all REPs excluding the motif is t, the glutamine residue content rate is calculated as u / t. In the calculation of the glutamine residue content rate, the reason for targeting "the sequence obtained by removing the sequence from the (A) motif located most C-terminally to the C-terminus of the domain array from the domain array" is the same as the reason described above. n The sixth modified fibroin may have an amino acid sequence corresponding to the deletion of one or more glutamine residues in the REP or the substitution with other amino acid residues as compared with fibroin derived from nature in its domain array.

[0124] The sixth modified fibroin may have an amino acid sequence corresponding to the deletion of one or more glutamine residues in the REP or the substitution with other amino acid residues as compared with fibroin derived from nature in its domain array.

[0125] "Other amino acid residues" may be amino acid residues other than glutamine residues, but are preferably amino acid residues having a larger hydrophobicity index than glutamine residues. The hydrophobicity index of amino acid residues is as shown in Table 1.

[0126] As shown in Table 1, examples of amino acid residues having a higher hydrophobicity index than the glutamine residue include amino acid residues 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.

[0127] For the sixth modified fibroin, the hydrophobicity degree of REP is preferably -0.8 or more, more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular limitation on the upper limit of the hydrophobicity degree of REP, and it may be 1.0 or less, or may be 0.7 or less.

[0128] In this specification, the "hydrophobicity degree 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 derived fibroin) containing a domain sequence represented by a motif, for all REPs contained in the sequence obtained by removing the sequence from the most C-terminal (A) n Motif to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "Region A" in FIG. 5), when the sum of the hydrophobicity indices of each amino acid residue in that region is v, and after removing the sequence from the most C-terminal (A) n Motif to the C-terminal of the domain sequence from the domain sequence, and further (A) nWhen the total number of amino acid residues of all REPs excluding the motif is t, the hydrophobicity degree of the REP is calculated as v / t. In the calculation of the hydrophobicity degree of the REP, the reason for targeting " n the sequence obtained by removing the sequence from the motif to the C-terminal of the domain sequence from the domain sequence" is the same as the reason described above.

[0129] The sixth modified fibroin may have an amino acid sequence modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues in addition to the modification corresponding to deletion of one or more glutamine residues in the REP and / or substitution of one or more glutamine residues in the REP with other amino acid residues, as compared with the domain sequence of the naturally-derived fibroin.

[0130] The sixth modified fibroin can be obtained, for example, by deleting one or more glutamine residues in the REP from the cloned gene sequence of the naturally-derived fibroin and / or substituting one or more glutamine residues in the REP with other amino acid residues. Also, for example, it can be obtained by designing an amino acid sequence corresponding to deletion of one or more glutamine residues in the REP and / or substitution of one or more glutamine residues in the REP with other amino acid residues from the amino acid sequence of the naturally-derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0131] As a more specific example of the sixth modified fibroin, (6-i) a modified fibroin containing the amino acid sequence represented by 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) a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 can be mentioned.

[0132] The modified fibroin of (6-i) will be described. The amino acid sequence represented by SEQ ID NO: 25 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) with VL. The amino acid sequence represented by SEQ ID NO: 26 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with TS and substituting the remaining Q with A. The amino acid sequence represented by SEQ ID NO: 27 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with VL and substituting the remaining Q with I. The amino acid sequence represented by SEQ ID NO: 28 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with VI and substituting the remaining Q with L. The amino acid sequence represented by SEQ ID NO: 29 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with VF and substituting the remaining Q with I.

[0133] The amino acid sequence represented by SEQ ID NO: 30 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence represented by SEQ ID NO: 31 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 8 with VL and substituting the remaining Q with I.

[0134] The amino acid sequence represented by SEQ ID NO: 32 is obtained by replacing all QQ in the sequence of 20 domain sequences existing in the amino acid sequence (Met-PRT410) represented by SEQ ID NO: 7 with VF, and replacing the remaining Q with I, and repeating this region twice.

[0135] The amino acid sequence represented by SEQ ID NO: 41 (Met-PRT917) is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 7 with LI, and replacing the remaining Q with V. The amino acid sequence represented by SEQ ID NO: 42 (Met-PRT1028) is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 7 with IF, and replacing the remaining Q with T.

[0136] 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).

[0137]

Table 2

[0138] (6-i) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42.

[0139] (6-ii) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42. The modified fibroin of (6-ii) also has the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m -(A)n It is a protein containing a domain sequence represented by a motif. The above sequence identity is preferably 95% or more.

[0140] The modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Also, the modified fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.

[0141] The sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, etc. of the modified fibroin.

[0142] More specific examples of the modified fibroin containing a tag sequence include the modified fibroin containing the amino acid sequence represented by 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) in (6-iii), or the modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 in (6-iv).

[0143] The amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 are each the amino acid sequence 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 with the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) added to the N-terminus. Since only the tag sequence is added to the N-terminus, there is no change in the glutamine residue content rate, and the amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 all have a glutamine residue content rate of 9% or less (Table 3).

[0144]

Table 3

[0145] (6-iii) The modified fibroin may consist of the amino acid sequence represented by 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.

[0146] (6-iv) The modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 of (6-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) n Motif-REP] m or the formula 2: [(A) n Motif-REP] m -(A) n Motif. The above sequence identity is preferably 95% or more.

[0147] The modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.

[0148] The sixth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

[0149] The modified fibroin may be a modified fibroin having at least two or more of the characteristics possessed by 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.

[0150] The modified fibroin may be a hydrophilic modified fibroin or a hydrophobic modified fibroin. In the present specification, the "hydrophilic modified fibroin" means a modified fibroin having an average value of hydrophobicity index (average HI) of 0 or less. In the present specification, the "hydrophobic modified fibroin" means a modified fibroin having an average HI exceeding 0. The average HI means a value obtained by obtaining the sum of the hydrophobicity indices (HI) of all amino acid residues constituting the modified fibroin and then dividing the sum by the total number of amino acid residues. The hydrophobicity index is as shown in Table 1. The hydrophilic modified fibroin has sufficient contact cold sensitivity and excellent water absorption and quick drying properties. The hydrophobic modified fibroin has excellent contact cold sensitivity and sufficient water absorption and quick drying properties.

[0151] Examples of the hydrophilic modified fibroin include modified fibroins containing the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0152] Examples of the hydrophobic modified fibroin include modified fibroins containing the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43, and the amino acid sequence represented by 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.

[0153] The modified fibroin according to the present 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 method such as the PCR method. Further, the isolation and purification of the produced modified fibroin can also be carried out by commonly used methods.

[0154] (Contact cold sensitivity and water absorption and quick-drying agent) The contact cold sensitivity and water absorption and quick-drying agent according to the present embodiment may contain one kind of modified fibroin alone, or may contain a combination of two or more kinds of modified fibroins.

[0155] The contact cold sensitivity and water absorption and quick-drying agent according to the present embodiment has a peak heat flow value (q max, W / cm 2 ) evaluated by a contact cold and warmth sensing device of 0.055 W / cm 2 or more, may be 0.060 W / cm 2 or more, and may be 0.065 W / cm2 It may be the above, 0.070 W / cm 2 It may be the above. There is no particular limitation on the upper limit of the peak heat flux value, but it is usually 0.2 W / cm 2 or less. The evaluation of the peak heat flux value can be performed, for example, by the method described in the examples below.

[0156] The contact cold sensitivity and water absorption and quick-drying property imparting agent according to this embodiment may have a water absorption property evaluated according to JIS L 1907 of 60 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, or 5 seconds or less. The evaluation of the water absorption property can be performed, for example, by the method described in the examples below.

[0157] The contact cold sensitivity and water absorption and quick-drying property imparting agent according to this embodiment may have a quick-drying property evaluated by measuring the diffusible residual moisture rate of 100 minutes or less, 90 minutes or less, 80 minutes or less, or 70 minutes or less. The diffusible residual moisture rate (%) is a value calculated by the following formula. Diffusible residual moisture rate (%) = weight of water at each time (g) / weight of water at the start of measurement (g) × 100 The quick-drying property means the time required for the diffusible residual moisture rate to reach 10% or less. The evaluation of the quick-drying property can be performed, for example, by the method described in the examples below.

[0158] The contact cold sensitivity and water absorption and quick-drying property imparting agent according to this embodiment may further contain other additives (components other than the active ingredient) according to its form, use, etc. Examples of the additives include plasticizers, leveling agents, cross-linking agents, crystal nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers, and synthetic resins. The content of the additives can be set within a range that does not impair the effects of the present invention.

[0159] The contact cold-sensitivity and water-absorbing and quick-drying agent according to this embodiment may be in any form, such as powdery, pasty, or liquid (e.g., suspension, solution). The contact cold-sensitivity and water-absorbing and quick-drying agent according to this embodiment may also be in the form of, for example, fibers, films, gels, porous bodies, particles, etc. The form of the contact cold-sensitivity and water-absorbing and quick-drying agent according to this embodiment may be appropriately set according to the type of the article to which the contact cold-sensitivity and water-absorbing and quick-drying properties are to be imparted and the use of the article.

[0160] Since the contact cold-sensitivity and water-absorbing and quick-drying agent according to this embodiment contains modified fibroin as a main component, it can be formed into any of the above-mentioned forms. The molded body may be one formed by molding modified fibroin itself, or may be one formed by combining modified fibroin with other materials.

[0161] When preparing the contact cold-sensitivity and water-absorbing and quick-drying agent according to this embodiment in a powdery 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 necessary.

[0162] When preparing the contact cold-sensitivity and water-absorbing and quick-drying agent according to this embodiment in a liquid form (e.g., solution), for example, the protein obtained by the above-described method for producing modified fibroin may be dissolved in a solvent capable of dissolving modified fibroin to form a liquid (modified fibroin solution). The modified fibroin solution may contain other additives as necessary. Examples of the solvent 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.

[0163] When preparing the contact cold-sensitivity and water-absorbing and quick-drying agent according to this embodiment in the form of fibers, for example, the above-described modified fibroin solution may be used as a dope solution and spun by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning to obtain fibers (modified fibroin fibers). The form of the fibers may be single yarn, or may be composite yarns such as blended yarns, mixed fiber yarns, mixed woven yarns, interwoven yarns, twisted yarns, and covering yarns, or may be non-woven fabrics or the like.

[0164] The modified fibroin fibers may be short fibers or long fibers. Further, the modified fibroin fibers may be alone or may be combined with other fibers. That is, a single yarn composed only of modified fibroin fibers or a composite yarn formed by combining modified fibroin fibers and other fibers may be used alone or in combination. The above single yarn and the above composite yarn may be spun yarns obtained by twisting short fibers, or may be filament yarns obtained by twisting or not twisting long fibers. Further, the modified fibroin fibers, whether short fibers or long fibers, can be used as fibers without being processed into yarns, 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, hemp, animal hair fibers, and silk. When used in combination with other fibers, based on the total amount of the fibers, the content of the modified fibroin fibers is preferably 20% by mass or more, more preferably 30% by mass or more, still more preferably 40% by mass or more, and even more preferably 50% by mass or more.

[0165] When preparing the contact cold-sensitivity and water-absorbing and quick-drying agent according to this embodiment in the form of films, gels, porous bodies, particles, etc., for example, it can be produced according to the methods described in JP-A-2009-505668, JP-A-2009-505668, Patent No. 5678283, Patent No. 4638735, etc.

[0166] 〔Method for imparting contact cold-sensitivity and water-absorbing and quick-drying property to an article〕 The method for imparting contact cold sensitivity and rapid water absorption and drying properties to an article according to this embodiment includes a step of incorporating modified fibroin into the article. Since the modified fibroin according to the present invention has both contact cold sensitivity and rapid water absorption and drying properties, by incorporating it into an article, contact cold sensitivity and rapid water absorption and drying properties can be imparted to the article.

[0167] The article is not particularly limited as long as it is to be imparted with contact cold sensitivity and rapid water absorption and drying properties. Specifically, examples include fibers, fabrics, knitted fabrics, non-woven fabrics, cotton, sponges, films, resins, composite materials (regardless of the form of the contact cold sensitivity and rapid water absorption and drying agent according to this embodiment), and various articles produced using them.

[0168] In the step of incorporating modified fibroin, it may be a step of incorporating the contact cold sensitivity and rapid water absorption and drying agent according to the present invention described above to thereby incorporate modified fibroin. The method for incorporating modified fibroin is not particularly limited, and it may be a method of mixing modified fibroin into a material (raw material), or it may be a method of forming an article by combining the contact cold sensitivity and rapid water absorption and drying agent prepared in the form of the above-described molded body with another material (such as a molded body). Further, it may be a method of forming an article (molded body) by molding modified fibroin itself (which may contain other additives as necessary).

[0169] The content of the modified fibroin in the article is preferably 20% by mass or more, more preferably 30% by mass or more, still more preferably 40% by mass or more, and even more preferably 50% by mass or more, based on the total amount of the article. The upper limit of the content of the modified fibroin may be 100% by mass or 90% by mass or less, based on the total amount of the article. By adjusting the content of the modified fibroin in the article, the contact cold sensitivity and the water absorption and quick-drying properties of the article can be controlled. That is, since the modified fibroin according to the present invention has both contact cold sensitivity and water absorption and quick-drying properties, the contact cold sensitivity and the water absorption and quick-drying properties of the article can be made higher as the content of the modified fibroin in the article is increased.

[0170] The present invention described above can also be regarded as the use of modified fibroin for imparting contact cold sensitivity and water absorption and quick-drying properties to an article, which comprises a step of incorporating modified fibroin into the article.

Examples

[0171] Hereinafter, the present invention will be described more specifically based on examples and the like. However, the present invention is not limited to the following examples.

[0172] 〔Test Example 1: Production of Modified Fibroin Fibers〕 (1) Preparation of Expression Vector Modified fibroin (PRT966) having the amino acid sequence shown in SEQ ID NO: 40 and modified fibroin (PRT799) having the amino acid sequence shown in SEQ ID NO: 15 were designed. Nucleic acids encoding the designed modified fibroin were synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Then, after the nucleic acid was excised by restriction enzyme treatment with NdeI and EcoRI, it was recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

[0173] (2) Expression of Protein The obtained expression vector was used to transform Escherichia coli BLR(DE3). The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture broth was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that the OD 600 was 0.005. The temperature of the culture broth was maintained at 30 °C, and flask culture was carried out until the OD 600 reached 5 (about 15 hours) to obtain a seed culture broth.

[0174]

Table 4

[0175] The seed culture broth was added to a jar fermenter supplemented with 500 mL of production medium (Table 5) so that the OD 600 was 0.05. The temperature of the culture broth was maintained at 37 °C and cultured under constant control at pH 6.9. Also, the dissolved oxygen concentration in the culture broth 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 / L glucose, 120 g / L Yeast Extract) was added at a rate of 1 mL / min. The temperature of the culture broth was maintained at 37 °C and cultured under constant control at pH 6.9. Also, the dissolved oxygen concentration in the culture broth was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture broth to a final concentration of 1 mM to induce the expression of modified fibroin. 20 hours after the addition of IPTG, the culture broth was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture broth before and after the addition of IPTG, and the expression of the target modified fibroin was confirmed by the appearance of a band of the target modified fibroin size depending on the addition of IPTG.

[0178] (3) Protein purification The cells collected 2 hours after adding IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF, and the cells were disrupted using a high-pressure homogenizer (manufactured by GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity was achieved. 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, stirred with a stirrer at 60 °C for 30 minutes, and dissolved. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36 / 32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, and the moisture was removed using a freeze dryer, and modified fibroin (PRT966 and PRT799) was obtained by recovering the freeze-dried powder.

[0179] PRT966 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] (4) Production of modified fibroin fibers Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent, and the freeze-dried powder of modified fibroin was added thereto to a concentration of 24% by mass, and dissolved using a shaker for 3 hours. Then, insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning dope).

[0181] The prepared spinning dope was filtered through a metal filter with an aperture of 5 μm at 60°C, then left standing in a 30 mL stainless steel syringe to defoam, and then extruded from a solid nozzle with a needle diameter of 0.2 mm into a 100% methanol coagulation bath. The extrusion temperature was 60°C. After coagulation, the obtained raw yarn was wound up and naturally dried to obtain modified fibroin fibers (raw material fibers).

[0182] [Test Example 2: Evaluation of water absorption and quick drying property] (1) Production of knitted fabric Using each of the raw material fibers prepared in Test Example 1, a knitted fabric was produced by flat knitting using a flat knitting machine. For comparison, commercially available silk fibers, cotton fibers, and polyester fibers were prepared as raw material fibers.

[0183] For the knitted fabric using PRT966 fibers as the raw material fiber, the fineness was 1 / 30 Nm (wool count single yarn) and the gauge number was 18. For the knitted fabric using PRT799 fibers as the raw material fiber, the fineness was 1 / 30 Nm (wool count single yarn) and the gauge number was 16. For the knitted fabrics using other raw material fibers, the fineness and gauge number were adjusted so as to have almost the same cover factor as the knitted fabrics using PRT966 fibers and PRT799 fibers. Specifically, it was as follows. Silk: fineness 2 / 60 Nm (double yarn), gauge number 14; Cotton: fineness 2 / 34 Nm (double yarn), gauge number 14; Polyester: fineness 1 / 60 Nm (single yarn), gauge number 14

[0184] (2) Evaluation of water absorption The water absorption was evaluated by a test according to JIS L 1907 (Test method for water absorption of textile products - Drop method). Specifically, under standard environment (temperature 20 ± 2°C / humidity 65 ± 4%RH), water was dropped one by one from a burette onto the surface of the knitted fabric prepared above, and the time until the dropped water droplet was absorbed by the knitted fabric (specular reflection disappeared) was measured (the maximum measurement time was 60 seconds). The results are shown in Table 6.

[0185] (3) Evaluation of quick drying property The quick-drying property was evaluated by measuring the diffusible residual moisture content. Specifically, 0.6 mL of tap water was dropped onto the back side of the knitted fabric under standard environmental conditions (temperature: 20 ± 2°C / humidity: 65 ± 4% RH), and the weight of the water was determined by measuring the weight of the knitted fabric every certain period of time (every 5 minutes). The diffusible residual moisture content was calculated using the following formula. Diffusible residual moisture content (%) = weight of water at each time (g) / weight of water at the start of measurement (g) × 100 The measurement was carried out until the diffusible residual moisture content reached 10% or less (i.e., the weight of water became 10% of 0.6 mL = 60 μL (60 mg)), and the time required for the diffusible residual moisture content to reach 10% was determined. The results are shown in Table 6.

[0186]

Table 6

[0187] It can be understood that the modified fibroins (PRT799 and PRT966) are excellent in water absorption and quick-drying properties. In particular, it can be understood that the hydrophilic modified fibroin (PRT799) is excellent in all aspects of water absorption and quick-drying properties.

[0188] 〔Test Example 3: Evaluation of contact cold sensitivity〕 (1) Production of knitted fabric Using each raw material fiber prepared in Test Example 1, a knitted fabric was produced by flat knitting using a flat knitting machine. For comparison, commercially available wool fibers, cashmere fibers, silk fibers, cotton fibers, rayon fibers, and polyester fibers were prepared as raw material fibers.

[0189] For the knitted fabric using PRT966 fiber as the raw material fiber, the fineness was 1 / 30 Nm (wool count single yarn) and the gauge number was 18. For the knitted fabric using PRT799 fiber as the raw material fiber, the fineness was 1 / 30 Nm (wool count single yarn) and the gauge number was 16. For the knitted fabrics using other raw material fibers, the fineness and gauge number were adjusted so as to have almost the same cover factor as the knitted fabrics using PRT966 fiber and PRT799 fiber. Specifically, it was as follows. Wool Fiber fineness: 2 / 30 Nm (double yarn), Gauge number: 14 Cashmere Fiber fineness: 1 / 32 Nm (single yarn), Gauge number: 14 Silk Fiber fineness: 2 / 60 Nm (double yarn), Gauge number: 14 Cotton Fiber fineness: 2 / 34 Nm (double yarn), Gauge number: 14 Rayon Fiber fineness: 1 / 38 Nm (single yarn), Gauge number: 14 Polyester Fiber fineness: 1 / 60 Nm (single yarn), Gauge number: 14

[0190] (2) Evaluation of contact cold sensitivity Cut into 10 cm × 10 cm to obtain test pieces (samples), and left in the test environment (temperature 20 ± 2°C, relative humidity 65 ± 4%) for 4 hours or more. Then, using a contact cold and warm sensing device (KES-F7 Thermo Lab II type, manufactured by Kato Tech Co., Ltd.) set at 30°C, after overlapping the sensor on the hot plate to make the sensor temperature constant, the sensor was brought into contact with the back surface of the test piece, and under the condition of a temperature difference (ΔT) of 10°C between the test piece and the sensor, the peak value of the heat flux (q max, W / cm 2 ) of each test piece was measured. The larger the peak value of the heat flux, the more heat is transferred from the contacting object (such as the skin) to the test piece, which means it has the property of high contact cold sensitivity and can be evaluated as such. The results are shown in Table 7.

[0191]

Table 7

[0192] It can be understood that the modified fibroin (PRT966 and PRT799. In particular, PRT966) is excellent in contact cold sensitivity.

Claims

Claim 1 containing modified fibroin as an active ingredient, wherein the modified fibroin has an amino acid sequence with a reduced glycine residue content in its domain sequence as compared to naturally-derived fibroin, and is a contact cold-sensitivity and water-absorbing and quick-drying property-imparting agent. Claim 2 The contact cold-sensitivity and water-absorbing and quick-drying property-imparting agent according to claim 1, wherein the modified fibroin includes a modified fibroin having an average value of hydrophobicity index (average HI) exceeding 0. Claim 3 The contact cold-sensitivity and water-absorbing and quick-drying property-imparting agent according to claim 1 or 2, wherein the modified fibroin includes modified spider silk fibroin. Claim 4 The contact cold-sensitivity and water-absorbing and quick-drying property-imparting agent according to any one of claims 1 to 3, which is in the form of fibers. Claim 5 A method for imparting contact cold-sensitivity and water-absorbing and quick-drying properties to an article, comprising the step of incorporating modified fibroin into the article, wherein the modified fibroin has an amino acid sequence with a reduced glycine residue content in its domain sequence as compared to naturally-derived fibroin.

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