Water-absorbing quick-drying agent and method for endowing water-absorbing quick-drying performance

Abstract

Provided are: a water-absorbing quick-drying agent capable of easily imparting water-absorbing quick-drying performance to various materials or articles by a simple step; and a method capable of easily imparting water-absorbing quick-drying performance to a predetermined material or article. A water-absorbing quick-drying agent comprising modified fibroin as an active ingredient; a method for imparting water-absorbing quick-drying properties to an article, said method comprising a step for containing a modified fibroin in the article.

Description

[0001] This application is a divisional application of the following application:

[0002] Invention Title: Water-absorbing quick-drying agent and method for imparting water-absorbing quick-drying properties

[0003] International filing date: June 9, 2020

[0004] International application number: PCT / JP2020 / 022742

[0005] National application number: 202080042228.0 Technical Field

[0006] The invention relates to a water-absorbing quick-drying agent and a method for imparting water-absorbing quick-drying performance. Background Art

[0007] Generally speaking, underwear, sportswear and other clothing as well as beddings require quick-drying, that is, quick-drying performance. Therefore, cotton yarns with good hygroscopicity are mostly used to manufacture underwear and beddings.

[0008] However, although cotton has high water absorption, the absorbed water evaporates slowly, so it is difficult to say that it has sufficient quick-drying performance. In addition, in sportswear, polyurethane fibers and polyester fibers are mostly used because of their emphasis on followability and stretchability to human body movements, but these synthetic fibers have poor water absorption performance.

[0009] Among them, various technologies have been proposed for improving the water absorption and quick-drying performance of various fibers, fabrics, etc. For example, Patent Document 1 discloses a water absorption and quick-drying yarn, characterized in that it has a core portion composed of polyester fibers with a special cross-section and a sheath portion composed of short fibers including cotton fibers, and is composed of a false twisted yarn in which the core portion is in an untwisted state and the sheath portion is in a bundled state.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1 Utility Model Registration No. 3213540 Summary of the invention

[0013] Problems to be solved by the invention

[0014] For example, in conventional fibers and fabrics, water-absorbing and quick-drying properties are achieved by using fibers artificially given special structures, such as the water-absorbing and quick-drying yarn described in Patent Document 1. Therefore, the water-absorbing and quick-drying fibers and water-absorbing and quick-drying fabrics to date cannot avoid the problem that their manufacturing processes become complicated.

[0015] An object of the present invention is to provide a water-absorbing quick-drying agent capable of easily imparting water-absorbing quick-drying properties to various materials or articles in a simple process, and a method for easily imparting water-absorbing quick-drying properties to predetermined materials or articles.

[0016] Methods used to solve problems

[0017] The present inventors have discovered that modified silk fibroin has excellent water absorption and quick-drying properties. The present invention has been completed based on this new finding.

[0018] The present invention relates to, for example, the following inventions.

[0019] [1] A water-absorbing quick-drying agent, comprising modified silk fibroin as an active ingredient.

[0020] [2] The water-absorbing quick-drying agent according to [1], wherein the modified silk fibroin includes a modified silk fibroin having an average hydrophobicity index (average HI) of 0 or less.

[0021] [3] The water-absorbing quick-drying agent according to [1] or [2], wherein the modified silk fibroin comprises modified spider silk fibroin.

[0022] [4] The water-absorbing quick-drying agent according to any one of [1] to [3], which is in the form of fibers.

[0023] [5] A method, specifically a method for imparting water-absorbing quick-drying properties to an article, comprising the step of incorporating modified silk fibroin into the article.

[0024] Effects of the Invention

[0025] According to the present invention, a water-absorbing quick-drying agent capable of easily imparting water-absorbing quick-drying properties to various materials or articles in a simple process and a method for easily imparting water-absorbing quick-drying properties to a predetermined material or article can be provided.

[0026] According to the present invention, for example, by mixing the water-absorbing quick-drying agent of the present invention into a material having biodegradability, water-absorbing quick-drying properties can be imparted without damaging the biodegradability. According to the present invention, the degree of water-absorbing quick-drying properties of an article can also be adjusted by adjusting the amount of modified silk fibroin (active ingredient) contained in a predetermined article. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram showing an example of the domain sequence of modified fibroin.

[0028] Figure 2 This is a graph showing the distribution of z / w (%) values ​​of naturally derived silk fibroin.

[0029] Figure 3 This is a graph showing the distribution of x / y (%) values ​​of naturally derived silk fibroin.

[0030] Figure 4 This is a schematic diagram showing an example of the domain sequence of modified fibroin.

[0031] Figure 5 This is a schematic diagram showing an example of the domain sequence of modified fibroin. DETAILED DESCRIPTION

[0032] Hereinafter, specific embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0033] 〔Water-absorbing and quick-drying agent〕

[0034] The water-absorbing quick-drying agent involved in this embodiment contains modified silk fibroin as an effective ingredient. Water-absorbing quick-drying performance refers to the property of drying quickly while absorbing water such as sweat. In this specification, water can be liquid water or gaseous water. That is, in this specification, water absorption includes moisture absorption. The water-absorbing quick-drying agent involved in this embodiment utilizes the property of modified silk fibroin with excellent water-absorbing quick-drying performance.

[0035] (Remodeled Silk Fibroin)

[0036] The modified silk fibroin involved in this embodiment comprises formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n The modified silk fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically regions that do not have a repeat of the amino acid motif characteristic of silk fibroin and are composed of about 100 amino acid residues, but are not limited thereto.

[0037] In this specification, "modified silk fibroin" refers to artificially produced silk fibroin (artificial silk fibroin). The modified silk fibroin may be a silk fibroin whose domain sequence is different from the amino acid sequence of silk fibroin from natural sources, or a silk fibroin whose amino acid sequence is the same as that of silk fibroin from natural sources. In this specification, "silk fibroin from natural sources" also includes formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A)n The motif represents the domain sequence of a protein.

[0038] "Modified silk fibroin" may be silk fibroin whose amino acid sequence is directly derived from natural sources, or silk fibroin whose amino acid sequence is modified based on the amino acid sequence of natural silk fibroin (for example, silk fibroin obtained by modifying the amino acid sequence of cloned natural silk fibroin gene sequence). In addition, it may be silk fibroin that is artificially designed and synthesized without relying on natural silk fibroin (for example, silk fibroin having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

[0039] In this specification, "domain sequence" refers to a crystal region that is unique to silk fibroin (typically, corresponding to the amino acid sequence (A) n motif) and an amino acid sequence of an amorphous region (typically, corresponding to the amino acid sequence REP), is the formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n The amino acid sequence represented by the motif. Here, (A) n The motif indicates an amino acid sequence mainly composed of alanine residues, with the number of amino acid residues ranging from 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. In addition, as long as (A) n The ratio of the number of alanine residues in the motif to the total number of amino acid residues may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). n At least 7 of the motifs may consist of only alanine residues. REP represents an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. Two or more of (A) n The motifs may be the same amino acid sequence or different amino acid sequences. The multiple REPs may be the same amino acid sequence or different amino acid sequences.

[0040] The modified silk fibroin involved in the present embodiment can be obtained by, for example, modifying the gene sequence of the cloned natural silk fibroin by, for example, replacing, deleting, inserting and / or adding one or more amino acid residues to obtain a modified silk fibroin. The replacement, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art such as site-directed mutagenesis methods. Specifically, it can be performed according to the methods described in the literature such as Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983).

[0041] The natural source of silk fibroin is a protein comprising formula 1: [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m -(A) n Specific examples of proteins having a domain sequence represented by the motif include silk fibroin produced by insects or spiders.

[0042] Examples of silk fibroin produced by insects include silk fibroin produced by silkworms such as the domesticated silkworm (Bombyx mori), the wild silkworm (Bombyx mandarina), the silkworm (Antheraea yamamai), the tussah (Anteraea pernyi), the maple silkworm (Eriogyna pyretorum), the castor silkworm (Pilosamia Cynthia ricini), the catalpa silkworm (Samia cynthia), the camphor silkworm (Caligura japonica), the Indian tussah (Antheraea mylitta), and the amber silkworm (Antheraea assama); and bee silk fibroin produced by the larvae of the bumblebee (Vespasimillima xanthoptera).

[0043] More specific examples of silk fibroin produced by insects include, for example, silkworm silk fibroin L chain (GenBank accession numbers M76430 (base sequence) and AAA27840.1 (amino acid sequence)).

[0044] As the silk core protein produced by spiders, for example, spidroin produced by spiders belonging to the order Araneae can be listed. More specifically, spiders belonging to the genus Araneus, such as Araneus fusus, Araneus roxburghii, Araneus nojimai, spiders belonging to the genus Neoscona, such as Araneus spp., Araneus hygrophilus, Araneus shrubbery, and Araneus quasi-Araneus, spiders belonging to the genus Pronus, such as Araneus spp., spiders belonging to the genus Cyrtarachne, such as Araneus spp. and Araneus symmetricus, spiders belonging to the genus Gasteracantha, such as Araneus spp., Araneus spp. and Araneus masculus, spiders belonging to the genus Hesperus, and Araneus spp. Spiders belonging to the genus Ordgarius, such as the stomach spider, the small stomach spider and the horizontal striped spider, such as the spider belonging to the genus Argiope, such as the double-peaked garden spider, the spider belonging to the genus Acusilas, such as the brown hanging leaf spider, the spider belonging to the genus Cytophora, such as the red clouded spider, the flower clouded spider and the full-color clouded spider, the spider belonging to the genus Poltys, such as the ugly cone-headed spider, the eight-headed spider Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the knob spider, the four-protuberant spider, the round-bellied spider and the black-tailed spider, and spiders belonging to the genus Chorizopes, such as the Japanese strong-headed spider, as well as spiders belonging to the genus Tetragnatha, such as the anterior toothed spider, the cone-bellied spider, the straight spider and the scale-patterned spider, spiders belonging to the genus Leucauge, such as the vertical-striped silver-scale spider, the shoulder-spotted silver-scale spider and the small-spotted silver-scale spider, and spiders belonging to the genus Nephila, such as the clavate astilbe and the spotted astilbe. Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as the beautiful wheat spider, spiders belonging to the genus Dyschiriognatha such as the weak thick chelicerae, spiders belonging to the genus Latrodectus such as the red-spotted spider, the geometric spider and the interspotted spider, and spiders belonging to the genus Tetragnathidae such as the genus Euprosthenops. Examples of spider silk proteins include dragline silk proteins such as MaSp (MaSp1 and MaSp2), ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, Flag, etc.

[0045] More specific examples of spider silk proteins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank Accession No. AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence), U37520 (base sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank Accession No. ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata](GenBank Accession Nos. AAL32472 (amino acid sequence), AF441245 (base sequence)), major ampullate spidroin 1[from Euprosthenops australis](GenBank Accession Nos. CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampullate spidroin 2[Euprosthenops australis](GenBank Accession Nos. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1[Nephila clavipes](GenBank Accession No. AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2[Nephila clavipes](GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein[Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), etc.

[0046] As a more specific example of naturally derived silk fibroin, there can be further cited silk fibroin whose sequence information is registered in NCBI GenBank. For example, it can be confirmed by extracting sequences that record spider silk protein, ampulla, silk fibroin, "silk and polypeptide" or "silk and protein" as keywords in the definition (DEFINITION) from sequences containing INV as the classification code (DIVISION) in the sequence information registered in NCBI GenBank, extracting a character string specifying a product from the CDS, and extracting a sequence that records a specified character string in the tissue type (TISSUE TYPE) from the source (SOURCE).

[0047] The modified fibroin involved in this embodiment can be modified silk fibroin (fibroin with modified amino acid sequence of silk protein produced by silkworms) or modified spider silk fibroin (fibroin with modified amino acid sequence of spider silk protein produced by spiders).

[0048] As specific examples of modified silk fibroin, there can be listed modified silk fibroin of large silk tube dragline silk protein produced from the large ampulla gland of spider (first modified silk fibroin), modified silk fibroin having a domain sequence with reduced glycine residue content (second modified silk fibroin), and modified silk fibroin having reduced (A) n The modified silk fibroin with a domain sequence of the motif content (the third modified silk fibroin), the content of glycine residues is reduced and (A) n Motif content modified silk fibroin (the 4th modified silk fibroin), a modified silk fibroin having a domain sequence locally containing a region with a large hydrophobicity index (the 5th modified silk fibroin), and a modified silk fibroin having a domain sequence with reduced content of glutamine residues (the 6th modified silk fibroin).

[0049] The first modified silk fibroin may include a protein comprising the formula 1: [(A) n motif-REP] m In the first modified silk 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, further preferably an integer of 8 to 20, further preferably an integer of 10 to 20, further 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 silk fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, further preferably 20 to 100 residues, and further preferably 20 to 75 residues. In the first modified silk fibroin, Formula 1: [(A) nmotif-REP] m The total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more relative to the total number of amino acid residues.

[0050] The first modified silk fibroin may be a polypeptide comprising formula 1: [(A) n motif-REP] m The amino acid sequence of the unit is shown in SEQ ID NOs: 1 to 3, and the C-terminal sequence is an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3, or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.

[0051] The amino acid sequence shown in sequence number 1 is identical to an amino acid sequence consisting of 50 amino acids at the C-terminus of the amino acid sequence of ADF3 (GI: 1263287, NCBI), the amino acid sequence shown in sequence number 2 is identical to the amino acid sequence after removing 20 residues from the C-terminus of the amino acid sequence shown in sequence number 1, and the amino acid sequence shown in sequence number 3 is identical to the amino acid sequence after removing 29 residues from the C-terminus of the amino acid sequence shown in sequence number 1.

[0052] As a more specific example of the first modified silk fibroin, there can be cited a modified silk fibroin comprising an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in (1-i) sequence number 4 (recombinant spider silk protein ADF3KaiLargeNRSH1) or (1-ii) sequence number 4. The sequence identity is preferably 95% or more.

[0053] The amino acid sequence shown in sequence number 4 is obtained by adding an amino acid sequence (sequence number 5) consisting of a start codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site to the amino acid sequence of ADF3 at the N-terminus, and by mutating the repeat region at positions 1 to 13 to increase by about 2 times and terminating translation at amino acid residue 1154. The amino acid sequence at the C-terminus of the amino acid sequence shown in sequence number 4 is the same as the amino acid sequence shown in sequence number 3.

[0054] The modified silk fibroin of (1-i) can be composed of the amino acid sequence shown in SEQ ID NO:4.

[0055] The second modified silk fibroin has an amino acid sequence in which the content of glycine residues is reduced compared to the silk fibroin from natural sources. The second modified silk fibroin may have an amino acid sequence equivalent to that in which at least one or more glycine residues in REP are replaced with other amino acid residues compared to the silk fibroin from natural sources.

[0056] The domain sequence of the second modified silk fibroin, compared with the silk fibroin of natural origin, may have an amino acid sequence corresponding to a glycine residue in at least one or more of the motif sequences selected from GGX and GPGXX (wherein G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP, in which a glycine residue in the motif sequence is replaced by another amino acid residue.

[0057] In the second modified fibroin, the ratio of the motif sequence in which the glycine residue is substituted with another amino acid residue may be 10% or more relative to the entire motif sequence.

[0058] The second modified silk fibroin comprises formula 1: [(A) n motif-REP] m The domain sequence shown may have the following amino acid sequence: When the amino acid sequence of (A) located closest to the C-terminus is removed from the above domain sequence, n The total number of amino acid residues of the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in all REPs in the sequence obtained by converting the motif to the C-terminus of the above-mentioned domain sequence is set to z, and (A) located most on the C-terminal side is removed from the above-mentioned domain sequence. n When the total number of amino acid residues in the sequence from the motif to the C-terminus of the above domain sequence is w, z / w is 30% or more, 40% or more, 50% or more, or 50.9% or more. (A) n The number of alanine residues in the motif may be 83% or more, preferably 86% or more, more preferably 90% or more, further preferably 95% or more, and even more preferably 100% (meaning consisting of only alanine residues) relative to the total number of amino acid residues.

[0059] The second modified silk fibroin preferably increases the content ratio of the amino acid sequence composed of XGX by replacing one glycine residue in the GGX motif with other amino acid residues. In the second modified silk fibroin, the content ratio of the amino acid sequence composed of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, further preferably 6% or less, further preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence composed 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 composed of XGX described below.

[0060] The calculation method of z / w is further described in detail. First, in the formula 1: [(A) n motif-REP] m In the silk fibroin (modified silk fibroin or naturally derived silk fibroin) having the domain sequence shown, the (A) located closest to the C-terminus is removed from the domain sequence. n The amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence obtained after removing the sequence from the (A)n motif located closest to the C-terminus to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (without repetition), z is 50×3=150. In addition, for example, as in the case of an amino acid sequence consisting of XGXGX, when X (the middle X) contained in 2 XGX exists, the calculation is performed by deducting the repeated part (when it is XGXGX, it is 5 amino acid residues). w is the total number of amino acid residues contained in the sequence obtained after removing the sequence from the (A)n motif located closest to the C-terminus to the C-terminus of the domain sequence from the domain sequence. For example, when it is Figure 1 In the case of the domain sequence shown in FIG. 1 , w is 4+50+4+100+4+10+4+20+4+30=230 (excluding (A) located closest to the C-terminus). n Then, by dividing z by w, z / w (%) can be calculated.

[0061] Here, z / w in natural silk fibroin is described. First, as described above, 663 types of silk fibroin (including 415 types of silk fibroin from spiders) were extracted by confirming the method using the silk fibroin for which amino acid sequence information was registered in NCBI GenBank. n In the amino acid sequence of naturally derived silk fibroin in which the ratio of the amino acid sequence consisting of GGX in silk fibroin is 6% or less, z / w was calculated according to the above calculation method. The result is as follows Figure 2 shown. Figure 2 The horizontal axis represents z / w (%), and the vertical axis represents frequency. Figure 2 It can be seen that the z / w ratio of naturally derived silk fibroin is less than 50.9% (the maximum is 50.86%).

[0062] In the second modified silk fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, further preferably 70% or more, and further preferably 80% or more. The upper limit of z / w is not particularly limited, and may be, for example, 95% or less.

[0063] For example, the second modified silk fibroin can be obtained by the following modification: from the gene sequence of the cloned natural silk fibroin, at least a part of the base sequence encoding the glycine residue is replaced and made to encode other amino acid residues. At this time, as the modified glycine residue, a glycine residue in the GGX motif and the GPGXX motif can be selected, and it can also be replaced by setting z / w to 50.9% or more. In addition, for example, it can also be obtained by designing an amino acid sequence that meets the above method based on the amino acid sequence of the natural silk fibroin, and by chemically synthesizing the nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification equivalent to replacing the glycine residue in REP with other amino acid residues in the amino acid sequence of the natural silk fibroin, it is also possible to further perform the modification of the amino acid sequence equivalent to the replacement, deletion, insertion and / or addition of one or more amino acid residues.

[0064] The above-mentioned other amino acid residues are not particularly limited as long as they are amino acid residues other than glycine residues, but preferred are hydrophobic amino acid residues such as valine (V) residues, leucine (L) residues, isoleucine (I) residues, methionine (M) residues, proline (P) residues, phenylalanine (F) residues and tryptophan (W) residues, and hydrophilic amino acid residues such as glutamine (Q) residues, asparagine (N) residues, serine (S) residues, lysine (K) residues and glutamic acid (E) residues, more preferred are valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine (F) residues and glutamine (Q) residues, and further preferred is glutamine (Q) residue.

[0065] As a more specific example of the second modified silk fibroin, there can be listed a modified silk fibroin including an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (2-i) sequence number 6 (Met-PRT380), sequence number 7 (Met-PRT410), sequence number 8 (Met-PRT525) or sequence number 9 (Met-PRT799), or (2-ii) sequence number 6, sequence number 7, sequence number 8 or sequence number 9.

[0066] The modified silk fibroin of (2-i) is described. The amino acid sequence represented by sequence number 6 is an amino acid sequence obtained by replacing all GGX in REP of the amino acid sequence represented by sequence number 10 (Met-PRT313) corresponding to the naturally derived silk fibroin with GQX. The amino acid sequence represented by sequence number 7 is obtained by replacing (A) from the N-terminal side to the C-terminal side in the amino acid sequence represented by sequence number 6. n The motif was deleted every 2 sequences, and a [(A) n The amino acid sequence shown in SEQ ID NO: 8 is obtained in each of the amino acid sequences shown in SEQ ID NO: 7 (A) n Two alanine residues are inserted into the C-terminal side of the motif, and further, some glutamine (Q) residues are replaced with serine (S) residues, and some amino acids on the C-terminal side are deleted so that the molecular weight is almost the same as that of sequence number 7. The amino acid sequence shown in sequence number 9 is a sequence in which a region of 20 domain sequences present in the amino acid sequence shown in sequence number 7 (wherein several amino acid residues on the C-terminal side of the region are replaced) is repeated 4 times, and a predetermined hinge sequence and His tag sequence are added to the C-terminus of the sequence.

[0067] The z / w value in the amino acid sequence represented by sequence number 10 (corresponding to naturally derived silk fibroin) is 46.8%. The z / w values ​​in the amino acid sequence represented by sequence number 6, the amino acid sequence represented by sequence number 7, the amino acid sequence represented by sequence number 8, and the amino acid sequence represented by sequence number 9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. In addition, the x / y values ​​of the amino acid sequences represented by sequence numbers 10, 6, 7, 8, and 9, with a sawtooth ratio (described below) of 1:1.8 to 11.3, are 15.0%, 15.0%, 93.4%, 92.7%, and 89.8%, respectively.

[0068] The modified silk fibroin of (2-i) can be composed of the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0069] The modified silk fibroin of (2-ii) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified silk fibroin of (2-ii) also comprises formula 1: [(A) n motif-REP] m The above sequence identity is preferably 95% or more.

[0070] The modified silk fibroin (2-ii) has a sequence identity of more than 90% with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and when the total number of amino acid residues of the amino acid sequence composed of XGX (wherein X represents an amino acid residue other than glycine) contained in REP is set to z and the total number of amino acid residues of REP in the above-mentioned domain sequence is set to w, z / w is preferably more than 50.9%.

[0071] The second modified silk fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. Thus, separation, fixation, detection, and visualization of the modified silk fibroin can be achieved.

[0072] As a tag sequence, for example, an affinity tag that utilizes specific affinity (binding property, affinity) with other molecules can be listed. As a specific example of an affinity tag, a histidine tag (His tag) can be listed. The His tag is a short peptide composed of about 4 to 10 histidine residues arranged in an array, and has the property of specifically binding to metal ions such as nickel, and can therefore be used for the separation of modified silk fibroin by chelating metal chromatography. As a specific example of a tag sequence, for example, the amino acid sequence shown in sequence number 11 (an amino acid sequence comprising a His tag sequence and a hinge sequence) can be listed.

[0073] Alternatively, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose may be used.

[0074] Furthermore, an "epitope tag" that utilizes an antigen-antibody reaction can also be used. By adding a peptide (epitope) for displaying antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tags, myc tags, FLAG tags, and the like. By utilizing epitope tags, modified silk fibroin can be easily purified with high specificity.

[0075] Furthermore, a tag obtained by removing a tag sequence using a specific protease may be used. The modified silk fibroin after the removal of the tag sequence may be recovered by treating the protein adsorbed via the tag sequence with a protease.

[0076] As more specific examples of modified silk fibroin containing a tag sequence, there can be listed modified silk fibroin including an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (2-iii) sequence number 12 (PRT380), sequence number 13 (PRT410), sequence number 14 (PRT525) or sequence number 15 (PRT799), or (2-iv) sequence number 12, sequence number 13, sequence number 14 or sequence number 15.

[0077] The amino acid sequences shown in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding the amino acid sequence shown in SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0078] The modified silk fibroin of (2-iii) can be composed of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0079] The modified silk fibroin of (2-iv) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified silk fibroin of (2-iv) also comprises formula 1: [(A) n motif-REP] m The above sequence identity is preferably 95% or more.

[0080] The modified silk fibroin (2-iv) has a sequence identity of more than 90% with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and when the total number of amino acid residues of the amino acid sequence composed of XGX (wherein X represents an amino acid residue other than glycine) contained in REP is set to z and the total number of amino acid residues of REP in the above-mentioned domain sequence is set to w, z / w is preferably more than 50.9%.

[0081] The second modified silk fibroin may contain a secretion signal for releasing the protein produced by the recombinant protein production system to the outside of the host. The sequence of the secretion signal may be appropriately set according to the type of the host.

[0082] The third modified silk fibroin has a domain sequence compared to the silk fibroin from natural sources (A)n The amino acid sequence of the modified silk fibroin can have a structure equivalent to the deletion of at least one or more (A) compared to the silk fibroin from natural sources. n The amino acid sequence of the motif.

[0083] The third modified silk fibroin may have (A) equivalent to 10 to 40% missing from the natural silk fibroin. n The amino acid sequence of the motif.

[0084] The third domain sequence of the modified silk fibroin may have at least 1 to 3 (A) equivalent to each from the N-terminal side to the C-terminal side compared to the naturally derived silk fibroin. n One missing (A) in the motif n The amino acid sequence of the motif.

[0085] The domain sequence of the third modified silk fibroin may have at least two consecutive (A) repeating in sequence from the N-terminal side to the C-terminal side, compared with the natural silk fibroin. n The deletion of the motif and one (A) n The amino acid sequence of the missing motif.

[0086] The domain sequence of the third modified silk fibroin may have a deletion (A) at least every 2 occurrences from the N-terminal side to the C-terminal side. n The amino acid sequence of the motif.

[0087] The third modified silk fibroin comprises formula 1: [(A) n motif-REP] m The domain sequence shown may have the following amino acid sequence: two adjacent amino acids [(A) n The number of amino acid residues of the REP in the motif-REP] unit is set to 1, and the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 for the two adjacent [(A) n When the maximum value of the total value obtained by adding the number of amino acid residues of the motif-REP] unit is x and the total number of amino acid residues of the domain sequence is y, x / y is 20% or more, 30% or more, 40% or more or 50% or more. (A) n The number of alanine residues in the motif may be 83% or more, preferably 86% or more, more preferably 90% or more, further preferably 95% or more, and even more preferably 100% (meaning consisting of only alanine residues) relative to the total number of amino acid residues.

[0088] Reference Figure 1The calculation method of x / y will be described in further detail. Figure 1 The domain sequence after removing the N-terminal sequence and the C-terminal sequence from the modified silk fibroin is shown. The domain sequence has (A) from the N-terminal side (left side) n Motif-1st REP (50 amino acid residues)-(A) n Motif-2nd REP (100 amino acid residues)-(A) n Motif-3rd REP (10 amino acid residues)-(A) n Motif-4th REP (20 amino acid residues)-(A) n Motif-5th REP (30 amino acid residues)-(A) n The sequence of the motif.

[0089] Select two adjacent ones from the N-terminal side to the C-terminal side in a non-repeating manner [(A) n Motif-REP] unit. In this case, there may also be unselected [(A) n motif-REP] unit. Figure 1 Mode 1 (comparison between the 1st REP and the 2nd REP, and between the 3rd REP and the 4th REP), Mode 2 (comparison between the 1st REP and the 2nd REP, and between the 4th REP and the 5th REP), Mode 3 (comparison between the 2nd REP and the 3rd REP, and between the 4th REP and the 5th REP), and Mode 4 (comparison between the 1st REP and the 2nd REP) are shown. In addition, there are other selection methods.

[0090] Next, for each pattern, compare the selected two adjacent [(A) n The number of amino acid residues of each REP in the [motif-REP] unit. Comparison is performed by finding the ratio of the number of amino acid residues of the other party when the one with relatively fewer amino acid residues is set to 1. For example, when comparing the 1st REP (50 amino acid residues) and the 2nd REP (100 amino acid residues), when the 1st REP with relatively fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the 2nd REP is 100 / 50=2. Similarly, when comparing the 4th REP (20 amino acid residues) and the 5th REP (30 amino acid residues), when the 4th REP with relatively fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the 5th REP is 30 / 20=1.5.

[0091] Figure 1 In the figure, the solid line indicates that when the one with the relatively smaller number of 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 [(A) nIn this specification, this ratio is referred to as the sawtooth ratio. The dashed line indicates that when the number of amino acid residues of the side with relatively fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other side is less than 1.8 or greater than 11.3 [(A) n motif-REP] unit.

[0092] In each mode, two adjacent solid lines [(A) n The number of all amino acid residues in the [motif-REP] unit is added (not only REP, but also the number of amino acid residues in the (A)n motif). Then, the added totals are compared, and the total value of the pattern with the largest total value (the maximum total value) is set to x. Figure 1 In the example shown, the total value of mode 1 is the largest.

[0093] Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

[0094] In the third modified silk fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% ​​or more, further preferably 70% or more, further preferably 75% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited, for example, it can be 100% or less. When the sawtooth ratio is 1:1.9 to 11.3, x / y is preferably 89.6% or more, when the sawtooth ratio is 1:1.8 to 3.4, x / y is preferably 77.1% or more, when the sawtooth ratio is 1:1.9 to 8.4, x / y is preferably 75.9% or more, and when the sawtooth ratio is 1:1.9 to 4.1, x / y is preferably 64.2% or more.

[0095] When the third modified silk fibroin has multiple (A) in the domain sequence n When at least 7 of the motifs of the modified silk fibroin are composed only of alanine residues, x / y is preferably 46.4% or more, more preferably 50% or more, further preferably 55% or more, further preferably 60% or more, further preferably 70% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited, and it can be 100% or less.

[0096] Here, x / y in natural silk fibroin is described. First, as described above, 663 types of silk fibroin (including 415 types of silk fibroin derived from spiders) were extracted by confirming the method using the silk fibroin for which amino acid sequence information is registered in NCBI GenBank. n motif-REP] mIn the amino acid sequence of naturally derived fibroin composed of the indicated domain sequence, x / y was calculated according to the above-mentioned calculation method. Figure 3 The results are shown when the sawtooth ratio is 1:1.9 to 4.1.

[0097] Figure 3 The horizontal axis represents x / y (%), and the vertical axis represents frequency. Figure 3 It is found that the x / y ratio in the naturally derived silk fibroin is less than 64.2% (the maximum is 64.14%).

[0098] For example, the gene sequence encoding (A) can be cloned from a naturally derived silk fibroin gene sequence so that x / y is 64.2% or more. n In addition, for example, one or more of the amino acid sequences of the silk fibroin from natural sources may be designed so that x / y is set to 64.2% or more. n The amino acid sequence in which the motif is deleted is obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, except for the deletion of (A) in the amino acid sequence of the silk fibroin from natural sources n In addition to modifications corresponding to the motif, further modifications of the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed.

[0099] As a more specific example of the third modified silk fibroin, there can be listed a modified silk fibroin including an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (3-i) sequence number 17 (Met-PRT399), sequence number 7 (Met-PRT410), sequence number 8 (Met-PRT525) or sequence number 9 (Met-PRT799), or (3-ii) sequence number 17, sequence number 7, sequence number 8 or sequence number 9.

[0100] The modified silk fibroin of (3-i) is described. The amino acid sequence represented by SEQ ID NO: 17 is obtained by modifying (A) from the N-terminal side to the C-terminal side in the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to the naturally derived silk fibroin. n The motif was deleted every 2 sequences, and a [(A) n The amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified silk fibroin.

[0101] The amino acid sequence shown in sequence number 10 (equivalent to naturally derived silk fibroin) has a sawtooth ratio of 1:1.8 to 11.3, and the x / y value is 15.0%. The x / y values ​​in the amino acid sequence shown in sequence number 17 and the amino acid sequence shown in sequence number 7 are both 93.4%. The x / y value in the amino acid sequence shown in sequence number 8 is 92.7%. The x / y value in the amino acid sequence shown in sequence number 9 is 89.8%. The z / w values ​​in the amino acid sequences shown in sequence number 10, sequence number 17, sequence number 7, sequence number 8, and sequence number 9 are 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively.

[0102] The modified silk fibroin of (3-i) can be composed of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0103] The modified silk fibroin of (3-ii) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified silk fibroin of (3-ii) also comprises formula 1: [(A) n motif-REP] m The above sequence identity is preferably 95% or more.

[0104] The modified silk fibroin of (3-ii) has a sequence identity of more than 90% with the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and two adjacent amino acids [(A) n The number of amino acid residues of the REP in the motif-REP] unit is set to 1, and the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 (the sawtooth ratio is 1:1.8 to 11.3). n When the maximum value of the total value obtained by adding the number of amino acid residues in the motif-REP] unit is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

[0105] The third modified silk fibroin may contain the tag sequence at either or both of the N-terminus and the C-terminus.

[0106] As more specific examples of modified silk fibroin containing a tag sequence, there can be listed modified silk fibroin including an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (3-iii) sequence number 18 (PRT399), sequence number 13 (PRT410), sequence number 14 (PRT525) or sequence number 15 (PRT799), or (3-iv) sequence number 18, sequence number 13, sequence number 14 or sequence number 15.

[0107] The amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding the amino acid sequence shown in SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0108] The modified silk fibroin of (3-iii) can be composed of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0109] The modified silk fibroin of (3-iv) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified silk fibroin of (3-iv) also comprises formula 1: [(A) n motif-REP] m The above sequence identity is preferably 95% or more.

[0110] The modified silk fibroin of (3-iv) has a sequence identity of more than 90% with the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and two adjacent [(A) n The number of amino acid residues of the REP in the motif-REP] unit is set to 1, and the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 for the two adjacent [(A) n When the maximum value of the total value obtained by adding the number of amino acid residues in the motif-REP] unit is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

[0111] The third modified silk fibroin may contain a secretion signal for releasing the protein produced by the recombinant protein production system to the outside of the host. The sequence of the secretion signal may be appropriately set according to the type of the host.

[0112] The fourth modified silk fibroin has a structural domain sequence that is different from the natural silk fibroin, in addition to reducing (A) nThe silk fibroin of the invention also has an amino acid sequence with a reduced content of glycine residues in addition to the motif. The structural domain sequence of the fourth modified silk fibroin is compared with the silk fibroin from natural sources, in addition to at least one or more (A) n In addition to the motif deletion, it can also have an amino acid sequence corresponding to at least one or more glycine residues in REP replaced by other amino acid residues. That is, the fourth modified silk fibroin is a modified silk fibroin having the characteristics of the second modified silk fibroin and the third modified silk fibroin mentioned above. The specific implementation method is as described in the second modified silk fibroin and the third modified silk fibroin.

[0113] As a more specific example of the fourth modified silk fibroin, there can be cited a modified silk fibroin comprising an amino acid sequence having a sequence identity of 90% or more to (4-i) the amino acid sequence of 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) the amino acid sequence of 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 silk fibroin comprising the amino acid sequence of 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.

[0114] The domain sequence of the 5th modified silk fibroin, compared with the silk fibroin from natural sources, may have an amino acid sequence equivalent to one or more amino acid residues in REP being replaced with amino acid residues with a large hydrophobicity index and / or one or more amino acid residues with a large hydrophobicity index being inserted into REP, and locally containing a region with a large hydrophobicity index.

[0115] The region having a large local hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

[0116] The amino acid residue having a large hydrophobicity index is more preferably an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A).

[0117] The 5th modified silk fibroin, compared with the silk fibroin from natural sources, is equivalent to replacing one or more amino acid residues in REP with amino acid residues with a large hydrophobicity index and / or inserting one or more amino acid residues with a large hydrophobicity index into REP. In addition, compared with the silk fibroin from natural sources, it can also undergo a modification of the amino acid sequence equivalent to replacing, deleting, inserting and / or adding one or more amino acid residues.

[0118] For example, the fifth modified silk fibroin can be obtained by replacing one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobic index) in REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobic index) from the gene sequence of the cloned natural silk fibroin, and / or inserting one or more hydrophobic amino acid residues into REP. In addition, for example, an amino acid sequence equivalent to replacing one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues and / or inserting one or more hydrophobic amino acid residues into REP from the amino acid sequence of the natural silk fibroin can be designed, and the nucleic acid encoding the designed amino acid sequence can be obtained by chemically synthesizing. In any case, in addition to the modification equivalent to replacing one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues and / or inserting one or more hydrophobic amino acid residues into REP from the amino acid sequence of the natural silk fibroin, the modification of the amino acid sequence equivalent to the replacement, deletion, insertion and / or addition of one or more amino acid residues can also be performed.

[0119] The fifth modified silk fibroin comprises formula 1: [(A) n motif-REP] m The domain sequence shown in the figure may have the following amino acid sequence: (A) located at the most C-terminal side is removed from the above domain sequence n Of all REPs contained in a sequence obtained by converting the motif to the C-terminus of the above domain sequence, the total number of amino acid residues contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and (A) located closest to the C-terminus is removed from the above domain sequence. n When the total number of amino acid residues contained in the sequence from the motif to the C-terminus of the above domain sequence is q, p / q is 6.2% or more.

[0120] Regarding 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.M01.Bio1., 157, pp.105-132) is used. Specifically, the hydrophobicity index (hydrophilic index, hereinafter also referred to as "HI") of each amino acid is shown in the following Table 1.

[0121] [Table 1]

[0122] Amino Acids HI Amino Acids HI Isoleucine (Ile) 4.5 Tryptophan (Trp) -0.9 Valine (Val) 4.2 Tyrosine (Tyr) -1.3 Leucine (Leu) 3.8 Proline (Pro) -1.6 Phenylalanine (Phe) 2.8 Histidine (His) -3.2 Cysteine ​​(Cys) 2.5 Asparagine (Asn) -3.5 Methionine (Met) 1.9 Aspartic acid (Asp) -3.5 Alanine (Ala) 1.8 Glutamine (Gln) -3.5 Glycine (Gly) -0.4 Glutamate (Glu) -3.5 Threonine (Thr) -0.7 Lysine (Lys) -3.9 Serine (Ser) -0.8 Arginine (Arg) -4.5

[0123] The calculation method of p / q is further described in detail. In the calculation, the formula 1 is used: [(A) n motif-REP] m The domain sequence shown is excluding the domain located closest to the C-terminus (A) n The sequence obtained after the sequence from the motif to the C-terminus of the domain sequence (hereinafter referred to as "sequence A"). First, among all REPs contained in sequence A, the average value of the hydrophobicity index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the sum of the HI of each amino acid residue contained in the 4 consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all 4 consecutive amino acid residues (each amino acid residue is used for the calculation of 1 to 4 average values). Next, determine the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is greater than 2.6. Even if a certain amino acid residue belongs to multiple "4 consecutive amino acid residues with an average value of the hydrophobicity index of more than 2.6", it will be included in the region as an amino acid residue. Moreover, the total number of amino acid residues contained in the region is p. In addition, the total number of amino acid residues contained in sequence A is q.

[0124] For example, when 20 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" are extracted (without duplication), the region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more contains 20 consecutive 4 amino acid residues (without duplication), and p is 20×4=80. In addition, for example, in the case where only one amino acid residue is repeated in 2 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", the region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more contains 7 amino acid residues (p=2×4-1=7. "-1" is the subtraction of the repeated part). For example, in Figure 4 In the case of the domain sequence shown in FIG. , there are 7 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" without duplication, so p is 7×4=28. Figure 4 In the case of the domain sequence shown in FIG. 1 , q is 4+50+4+40+4+10+4+20+4+30=170 (excluding the last (A) at the C-terminal end). n Then, by dividing p by q, p / q (%) can be calculated. Figure 4 In this case, 28 / 170=16.47%.

[0125] In the fifth modified silk fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, further preferably 20% or more, further preferably 30% or more. The upper limit of p / q is not particularly limited, and may be, for example, 45% or less.

[0126] For example, for the amino acid sequence of the cloned natural source silk fibroin, by replacing one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobic index) in REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobic index), and / or inserting one or more hydrophobic amino acid residues in REP so that it satisfies the above-mentioned p / q condition, and by transforming it into an amino acid sequence that locally contains a region with a large hydrophobic index, the fifth modified silk fibroin is obtained. In addition, for example, it can also be obtained by designing an amino acid sequence that satisfies the above-mentioned p / q condition based on the amino acid sequence of the natural source silk fibroin, and by chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, compared with the natural source silk fibroin, in addition to the transformation equivalent to replacing one or more amino acid residues in REP with amino acid residues with a large hydrophobic index, and / or inserting one or more amino acid residues with a large hydrophobic index in REP, it can also be further transformed by replacing, deleting, inserting and / or adding one or more amino acid residues.

[0127] The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.

[0128] As a more specific example of the fifth modified silk fibroin, there can be listed a modified silk fibroin including an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (5-i) sequence number 19 (Met-PRT720), sequence number 20 (Met-PRT665) or sequence number 21 (Met-PRT666), or (5-ii) sequence number 19, sequence number 20 or sequence number 21.

[0129] The modified silk fibroin of (5-i) is described. The amino acid sequence shown in sequence number 19 is an amino acid sequence (VLI) consisting of three amino acid residues inserted at two locations every other REP, in addition to the terminal domain sequence on the C-terminal side, relative to the amino acid sequence shown in sequence number 7 (Met-PRT410), and further a part of the glutamine (Q) residues are replaced with serine (S) residues, and a part of the amino acids on the C-terminal side are deleted. The amino acid sequence shown in sequence number 20 is an amino acid sequence (VLI) consisting of three amino acid residues inserted at one location every other REP, relative to the amino acid sequence shown in sequence number 8 (Met-PRT525). The amino acid sequence shown in sequence number 21 is an amino acid sequence (VLI) consisting of three amino acid residues inserted at two locations every other REP, relative to the amino acid sequence shown in sequence number 8.

[0130] The modified silk fibroin of (5-i) can be composed of the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0131] The modified silk fibroin of (5-ii) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified silk fibroin of (5-ii) also comprises formula 1: [(A) n motif-REP] m The above sequence identity is preferably 95% or more.

[0132] The modified silk fibroin (5-ii) has a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and preferably, the amino acid sequence of (A) located closest to the C-terminus is removed from the domain sequence. n Of all REPs contained in the sequence obtained by converting the motif to the C-terminus of the domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the residues (A) located closest to the C-terminus are removed from the domain sequence. n When the total number of amino acid residues contained in the sequence from the motif to the C-terminus of the domain sequence is q, p / q is 6.2% or more.

[0133] The fifth modified silk fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.

[0134] As more specific examples of modified silk fibroin containing a tag sequence, there can be listed modified silk fibroin including an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (5-iii) sequence number 22 (PRT720), sequence number 23 (PRT665) or sequence number 24 (PRT666), or (5-iv) sequence number 22, sequence number 23 or sequence number 24.

[0135] The amino acid sequences shown in SEQ ID NOs: 22, 23 and 24 are obtained by adding the amino acid sequence shown in SEQ ID NOs: 11 (including a His tag sequence and a hinge sequence) to the N-termini of the amino acid sequences shown in SEQ ID NOs: 19, 20 and 21, respectively.

[0136] The modified silk fibroin of (5-iii) can be composed of the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0137] The modified silk fibroin of (5-iv) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. The modified silk fibroin of (5-iv) also comprises formula 1: [(A) n motif-REP] m The above sequence identity is preferably 95% or more.

[0138] The modified silk fibroin (5-iv) has a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and preferably, the amino acid sequence of (A) located closest to the C-terminus is removed from the domain sequence. n Of all REPs contained in the sequence obtained by converting the motif to the C-terminus of the domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is defined as p, and the residues (A) located closest to the C-terminus are removed from the domain sequence. n When the total number of amino acid residues contained in the sequence from the motif to the C-terminus of the domain sequence is q, p / q is 6.2% or more.

[0139] The fifth modified silk fibroin may contain a secretion signal for releasing the protein produced by the recombinant protein production system to the outside of the host. The sequence of the secretion signal may be appropriately set according to the type of the host.

[0140] The sixth modified silk fibroin has an amino acid sequence with a reduced content of glutamine residues compared to silk fibroin from natural sources.

[0141] The sixth modified silk fibroin preferably contains at least one motif selected from the group consisting of a GGX motif and a GPGXX motif in the amino acid sequence of REP.

[0142] When the sixth modified silk fibroin includes a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited and may be 50% or less, or 30% or less.

[0143] In this specification, the "GPGXX motif content rate" is a value calculated by the following method.

[0144] In the inclusion formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n In the silk fibroin (modified silk fibroin or naturally derived silk fibroin) having the domain sequence shown in the motif, the (A) located at the most C-terminal side is removed from the domain sequence. n Among all REPs contained in the sequence obtained from the sequence from the GPGXX motif to the C-terminus of the domain sequence, the number obtained by multiplying the total number of GPGXX motifs contained in the region by 3 (i.e., the total number of G and P in the GPGXX motif) is set as s, and the (A) located closest to the C-terminus is removed from the domain sequence. n The sequence from the motif to the C-terminus of the domain sequence and excluding (A) n When the total number of amino acid residues of all REPs obtained after the motif is set to t, the GPGXX motif content ratio is calculated as s / t.

[0145] In the calculation of the GPGXX motif content, the motif (A) located closest to the C-terminus was removed from the domain sequence. n The purpose of taking the sequence obtained from the motif to the C-terminus of the domain sequence as the target is to exclude the following influence: "(A) located at the most C-terminal side n The sequence from the motif to the C-terminus of the domain sequence (the sequence equivalent to REP) sometimes contains a sequence with low correlation with the characteristic sequence of fibroin, which will affect the calculation result of the GPGXX motif content when m is small (that is, when the domain sequence is short). In addition, when the "GPGXX motif" is located at the C-terminus of REP, it is treated as the "GPGXX motif" even if "XX" is "AA", for example.

[0146] Figure 5 Schematic diagram showing the domain sequence of modified silk fibroin. Figure 5 The calculation method of GPGXX motif content rate is described in detail. Figure 5 The domain sequence of the modified silk fibroin shown ("[(A) n motif-REP] m -(A) n motif" type), all REPs are included in the "removal from the domain sequence from the (A) located at the most C-terminal side" n The sequence obtained by the sequence from the motif to the C-terminus of the domain sequence" ( Figure 5 Therefore, the number of GPGXX motifs used to calculate s is 7, and s is 7×3=21. Similarly, since all REPs are included in the "region A" located closest to the C-terminal side of the domain sequence, n The sequence obtained by the sequence from the motif to the C-terminus of the domain sequence" ( Figure 5 Therefore, (A) is further removed from the sequence. n The total number of amino acid residues of all REPs obtained after the motif is 50+40+10+20+30=150. Next, by dividing s by t, s / t (%) can be calculated. Figure 5 In the case of modified silk fibroin, it is 21 / 150=14.0%.

[0147] In the sixth modified silk fibroin, the glutamine residue content is preferably 9% or less, more preferably 7% or less, further preferably 4% or less, and particularly preferably 0%.

[0148] In the present specification, the "glutamine residue content" is a value calculated by the following method.

[0149] In the inclusion formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n In the silk fibroin (modified silk fibroin or naturally derived silk fibroin) having the domain sequence shown in the motif, the (A) located at the most C-terminal side is removed from the domain sequence. n The sequence obtained by the sequence from the motif to the C-terminus of the domain sequence (equivalent to Figure 5 Among all REPs contained in the "region A" of the domain sequence, when the total number of glutamine residues contained in the region is set to u, and the (A) located closest to the C-terminus is removed from the domain sequence n The sequence from the motif to the C-terminus of the domain sequence and further removed (A) nWhen the total number of amino acid residues of all REPs obtained after the motif is set as t, the glutamine residue content is calculated as u / t. In the calculation of the glutamine residue content, "removing the (A) located at the most C-terminal side from the domain sequence" is used. n The reason why the "sequence obtained from the sequence from the motif to the C-terminus of the domain sequence" is the object is the same as the above reason.

[0150] The domain sequence of the sixth modified silk fibroin may have an amino acid sequence corresponding to a deletion or substitution of one or more glutamine residues in REP with other amino acid residues, compared with the silk fibroin derived from natural sources.

[0151] The "other amino acid residues" may be any amino acid residue other than the glutamine residue, and are preferably amino acid residues having a hydrophobicity index greater than that of the glutamine residue. The hydrophobicity indexes of the amino acid residues are shown in Table 1.

[0152] As shown in Table 1, examples of amino acid residues having a hydrophobicity index greater than that of glutamine residues 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 them, more preferred are amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), and alanine (A), and even more preferred are amino acid residues selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F).

[0153] The hydrophobicity of REP in the sixth modified silk fibroin is preferably -0.8 or higher, more preferably -0.7 or higher, further preferably 0 or higher, further preferably 0.3 or higher, and particularly preferably 0.4 or higher. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or lower, or 0.7 or lower.

[0154] In this specification, "the hydrophobicity degree of REP" is a value calculated by the following method.

[0155] In the inclusion formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n In the silk fibroin (modified silk fibroin or naturally derived silk fibroin) having the domain sequence shown in the motif, the (A) located at the most C-terminal side is removed from the domain sequence.n The sequence obtained by the sequence from the motif to the C-terminus of the domain sequence (equivalent to Figure 5 Among all REPs contained in the "region A" of the domain sequence, when the sum of the hydrophobicity indexes of the amino acid residues in the region is set to v, and the residue (A) located closest to the C-terminus is removed from the domain sequence. n The sequence from the motif to the C-terminus of the domain sequence and further removed (A) n When the total number of amino acid residues of all REPs obtained after the motif is set as t, the hydrophobicity of REP is calculated as v / t. In the calculation of the hydrophobicity of REP, "removing the (A) located closest to the C-terminus from the domain sequence" is used. n The reason why the "sequence obtained from the sequence from the motif to the C-terminus of the domain sequence" is the object is the same as the above reason.

[0156] The domain sequence of the sixth modified silk fibroin, compared with the silk fibroin of natural origin, is equivalent to the deletion of one or more glutamine residues in REP and / or the replacement of one or more glutamine residues in REP with other amino acid residues, and can also be further modified by replacing, deleting, inserting and / or adding one or more amino acid residues.

[0157] For example, the sixth modified silk fibroin can be obtained by deleting one or more glutamine residues in REP and / or replacing one or more glutamine residues in REP with other amino acid residues in the gene sequence of the cloned natural silk fibroin. In addition, for example, an amino acid sequence equivalent to the amino acid sequence of the natural silk fibroin in which one or more glutamine residues in REP are deleted and / or replaced with other amino acid residues can be designed, and the nucleic acid encoding the designed amino acid sequence can be obtained by chemically synthesizing it.

[0158] As more specific examples of the sixth modified silk fibroin, there are listed sequence numbers (6-i) 25 (Met-PRT888), 26 (Met-PRT965), 27 (Met-PRT889), 28 (Met-PRT916), 29 (Met-PRT918), 30 (Met-PRT699), 31 (Met-PRT698), 32 (Met -PRT966), sequence number 41 (Met-PRT917) or sequence number 42 (Met-PRT1028), or a modified silk fibroin having an amino acid sequence shown in (6-ii) sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 41 or sequence number 42 with a sequence identity of more than 90%.

[0159] The modified silk fibroin of (6-i) is described. The amino acid sequence represented by sequence number 25 is an amino acid sequence obtained by replacing all QQ in the amino acid sequence (Met-PRT410) represented by sequence number 7 with VL. The amino acid sequence represented by sequence number 26 is an amino acid sequence obtained by replacing all QQ in the amino acid sequence represented by sequence number 7 with TS, and replacing the remaining Q with A. The amino acid sequence represented by sequence number 27 is an amino acid sequence obtained by replacing all QQ in the amino acid sequence represented by sequence number 7 with VL, and replacing the remaining Q with I. The amino acid sequence represented by sequence number 28 is an amino acid sequence obtained by replacing all QQ in the amino acid sequence represented by sequence number 7 with VI, and replacing the remaining Q with L. The amino acid sequence represented by sequence number 29 is an amino acid sequence obtained by replacing all QQ in the amino acid sequence represented by sequence number 7 with VF, and replacing the remaining Q with I.

[0160] The amino acid sequence represented by SEQ ID NO: 30 is an amino acid sequence in which all QQ in the amino acid sequence (Met-PRT525) represented by SEQ ID NO: 8 are replaced with VL. The amino acid sequence represented by SEQ ID NO: 31 is an amino acid sequence in which all QQ in the amino acid sequence represented by SEQ ID NO: 8 are replaced with VL, and the remaining Q is replaced with I.

[0161] The amino acid sequence shown in SEQ ID NO: 32 is an amino acid sequence obtained by repeating the region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) twice, in which all QQ in the sequence are replaced with VF, and the remaining Q is replaced with I.

[0162] The amino acid sequence represented by SEQ ID NO: 41 (Met-PRT917) is an amino acid sequence in which all QQ in the amino acid sequence represented by SEQ ID NO: 7 are replaced by LI, and the remaining Q is replaced by V. The amino acid sequence represented by SEQ ID NO: 42 (Met-PRT1028) is an amino acid sequence in which all QQ in the amino acid sequence represented by SEQ ID NO: 7 are replaced by IF, and the remaining Q is replaced by T.

[0163] The glutamine residue contents of the amino acid sequences shown in SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 41 and 42 are all less than 9% (Table 2).

[0164] [Table 2]

[0165] Modified silk fibroin Glutamine residue content GPGXX motif content REP hydrophobicity Met-PRT410 (serial number 7) 17.7％ 27.9％ -1.52 Met-PRT888 (serial number 25) 6.3％ 27.9％ -0.07 Met-PRT965 (serial number 26) 0.0％ 27.9％ -0.65 Met-PRT889 (serial number 27) 0.0％ 27.9％ 0.35 Met-PRT916 (Serial No. 28) 0.0％ 27.9％ 0.47 Met-PRT918 (serial number 29) 0.0％ 27.9％ 0.45 Met-PRT699 (serial number 30) 3.6％ 26.4％ -0.78 Met-PRT698 (Serial No. 31) 0.0％ 26.4％ -0.03 Met-PRT966 (Serial No. 32) 0.0％ 28.0％ 0.35 Met-PRT917 (Serial No. 41) 0.0％ 27.9％ 0.46 Met-PRT1028 (Serial No. 42) 0.0％ 28.1％ 0.05

[0166] The modified silk fibroin of (6-i) can be composed of the amino acid sequence shown in sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 41 or sequence number 42.

[0167] The modified silk fibroin of (6-ii) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42. The modified silk fibroin of (6-ii) also comprises Formula 1: [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m -(A) n The protein having the domain sequence shown in the motif. The sequence identity is preferably 95% or more.

[0168] The modified silk fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. The modified silk fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.

[0169] The sixth modified silk fibroin may include a tag sequence at either or both of the N-terminus and the C-terminus, thereby enabling separation, fixation, detection, and visualization of the modified silk fibroin.

[0170] As more specific examples of modified silk fibroin containing a tag sequence, there can be listed modified silk fibroin including the amino acid sequence shown in (6-iii) sequence number 33 (PRT888), sequence number 34 (PRT965), sequence number 35 (PRT889), sequence number 36 (PRT916), sequence number 37 (PRT918), sequence number 38 (PRT699), sequence number 39 (PRT698), sequence number 40 (PRT966), sequence number 43 (PRT917) or sequence number 44 (PRT1028), or modified silk fibroin including an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in (6-iv) sequence number 33, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 43 or sequence number 44.

[0171] The amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 have the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, and SEQ ID NO: 42 added to the N-terminus of the amino acid sequences shown in SEQ ID NO: 11 (including the His tag sequence and the hinge sequence). Since only the tag sequence was added to the N-terminus, the glutamine residue content did not change, and the glutamine residue content of the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 was 9% or less (Table 3).

[0172] [Table 3]

[0173] Modified silk fibroin Glutamine residue content GPGXX motif content REP hydrophobicity PRT888 (Serial No. 33) 6.3％ 27.9％ -0.07 PRT965 (serial number 34) 0.0％ 27.9％ -0.65 PRT889 (serial number 35) 0.0％ 27.9％ 0.35 PRT916 (serial number 36) 0.0％ 27.9％ 0.47 PRT918 (serial number 37) 0.0％ 27.9％ 0.45 PRT699 (serial number 38) 3.6％ 26.4％ -0.78 PRT698 (serial number 39) 0.0％ 26.4％ -0.03 PRT966 (serial number 40) 0.0％ 28.0％ 0.35 PRT917 (serial number 43) 0.0％ 27.9％ 0.46 PRT1028 (serial number 44) 0.0％ 28.1％ 0.05

[0174] The modified silk fibroin of (6-iii) can be composed of the amino acid sequence shown in sequence number 33, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 43 or sequence number 44.

[0175] The modified silk fibroin of (6-iv) comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. The modified silk fibroin of (6-iv) also comprises Formula 1: [(A) n motif-REP] m , or formula 2: [(A)n motif-REP] m -(A) n The protein having the domain sequence shown in the motif. The sequence identity is preferably 95% or more.

[0176] The modified silk fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. The modified silk fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.

[0177] The sixth modified silk fibroin may contain a secretion signal for releasing the protein produced by the recombinant protein production system to the outside of the host. The sequence of the secretion signal may be appropriately set according to the type of the host.

[0178] The modified silk fibroin may be a modified silk fibroin having at least two or more characteristics among the characteristics of the first modified silk fibroin, the second modified silk fibroin, the third modified silk fibroin, the fourth modified silk fibroin, the fifth modified silk fibroin, and the sixth modified silk fibroin.

[0179] As modified silk fibroin, hydrophilic modified silk fibroin is preferred due to its superior water absorption and quick-drying properties. In the present specification, "hydrophilic modified silk fibroin" refers to a modified silk fibroin in which the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified silk fibroin is obtained, and then the sum is divided by the number of all amino acid residues to obtain a value (average HI) of less than 0. The hydrophobicity index is shown in Table 1. In addition, modified silk fibroin with an average HI greater than 0 is sometimes referred to as hydrophobic modified silk fibroin.

[0180] As hydrophilic modified silk fibroin, for example, there can be listed modified silk fibroin including the amino acid sequence shown in sequence number 4, the amino acid sequence shown in sequence number 6, sequence number 7, sequence number 8 or sequence number 9, the amino acid sequence shown in sequence number 13, sequence number 11, sequence number 14 or sequence number 15, the amino acid sequence shown in sequence number 18, sequence number 7, sequence number 8 or sequence number 9, the amino acid sequence shown in sequence number 17, sequence number 11, sequence number 14 or sequence number 15, and the amino acid sequence shown in sequence number 19, sequence number 20 or sequence number 21.

[0181] As hydrophobic modified silk fibroin, for example, there can be listed modified silk fibroin comprising the amino acid sequence shown in sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 33 or sequence number 43, and the amino acid sequence shown in sequence number 35, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 41 or sequence number 44.

[0182] The modified silk fibroin involved in this embodiment can be manufactured by conventional methods using a nucleic acid encoding the modified silk fibroin. The nucleic acid encoding the modified silk fibroin can be chemically synthesized based on the base sequence information, or can be synthesized using the PCR method, etc. In addition, the separation and purification of the manufactured modified silk fibroin can also be carried out by conventional methods.

[0183] (Water-absorbing and quick-drying agent)

[0184] The water-absorbing quick-drying agent according to the present embodiment may contain only one type of modified silk fibroin, or may contain two or more types of modified silk fibroin in combination.

[0185] The water-absorbing quick-drying agent according to this embodiment may have a water absorption performance of 60 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, or 5 seconds or less, as evaluated according to JIS L 1907. The water absorption performance may be evaluated, for example, by the method described in the Examples described below.

[0186] The quick-drying performance of the water-absorbing quick-drying agent according to the present embodiment can be evaluated by measuring the diffusible residual moisture content in a time period of 100 minutes or less, 90 minutes or less, 80 minutes or less, or 70 minutes or less. The diffusible residual moisture content (%) is a value calculated by the following formula.

[0187] Diffusible residual moisture content (%) = weight of water at each time (g) / weight of water at the start of measurement (g) × 100

[0188] The quick-drying performance refers to the time required for the diffusible residual moisture content to reach 10% or less. The quick-drying performance can be evaluated by the method described in the Examples described below, for example.

[0189] The water-absorbing quick-drying agent involved in the present embodiment may also contain other additives (ingredients other than the active ingredient) according to its form, purpose, etc. As additives, for example, plasticizers, leveling agents, crosslinking agents, crystal nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers and synthetic resins may be listed. Relative to the total amount of 100 parts by mass of the water-absorbing quick-drying agent, the content of the additive may be less than 50 parts by mass.

[0190] The water-absorbing quick-drying agent involved in this embodiment can be in any form of powder, paste, liquid (such as suspension, solution), etc. The water-absorbing quick-drying agent involved in this embodiment can also be in the form of fiber, film, gel, porous body, granule, etc. The form of the water-absorbing quick-drying agent involved in this embodiment can be appropriately set according to the object to which the water-absorbing quick-drying performance is given (the article to which the water-absorbing quick-drying performance is given) and its use.

[0191] The quick-drying water-absorbing agent according to the present embodiment contains modified silk fibroin as a main component, and thus can be formed into any of the above-mentioned forms. The formed body can be a formed body formed of modified silk fibroin itself, or a formed body formed of a combination of modified silk fibroin and other materials.

[0192] When the water-absorbing quick-drying agent according to the present embodiment is prepared in a powdered form, for example, the protein obtained by the above-mentioned method for producing modified silk fibroin may be dried and powdered. The protein powder may contain other additives as required.

[0193] In the case of preparing the water-absorbing quick-drying agent involved in the present embodiment in a liquid (e.g., solution) form, for example, a solvent that can dissolve the modified silk fibroin can be used to dissolve the protein obtained by the manufacturing method of the above-mentioned modified silk fibroin and make it into a liquid (modified silk fibroin solution). In the modified silk fibroin solution, other additives can also be included as needed. As solvents that can dissolve the modified silk fibroin, for example, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid and hexafluoroisopropanol (HFIP) can be listed. In the solvent, an inorganic salt can also be added as a dissolution promoter.

[0194] When the water-absorbing quick-drying agent of the present embodiment is prepared in the form of fibers, for example, the modified silk fibroin solution can be used as a spinning solution, and the fibers (modified silk fibroin fibers) can be spun by a known spinning method such as wet spinning, dry spinning, dry-wet spinning or melt spinning. As the fiber form, it can be a single yarn, or a composite yarn such as a blended yarn, a mixed fiber yarn, a blended knitted yarn, a cross-twisted yarn, a double-twisted yarn, and a core-spun yarn, or a non-woven fabric.

[0195] The modified silk fibroin fiber can be a short fiber or a long fiber. In addition, the modified silk fibroin fiber can be used alone or in combination with other fibers. That is, the single yarn consisting only of the modified silk fibroin fiber and the composite yarn formed by combining the modified silk fibroin fiber with other fibers can be used alone or in combination. The above-mentioned single yarn and the above-mentioned composite yarn can be a spun yarn formed by twisting the short fiber, or a long yarn formed by twisting or not twisting the long fiber. In addition, the modified silk fibroin fiber can be a short fiber or a long fiber, and can also be used alone or in combination with other fibers without being processed into a spun yarn. As other fibers, for example, synthetic fibers such as nylon and polyester, regenerated fibers such as cuprammonium fibers and rayon, natural fibers such as cotton and flax can be cited. When used in combination with other fibers, based on the total amount of fiber, the content of the modified silk fibroin fiber is preferably 20% by mass or more, more preferably 30% by mass or more, further preferably 40% by mass or more, and further more preferably 50% by mass or more.

[0196] When the water-absorbing quick-drying agent according to the present embodiment is prepared in the form of a film, gel, porous body, granules, etc., it can be produced according to the methods described in, for example, Japanese Patent Application Publication No. 2009-505668, Japanese Patent Application Publication No. 5678283, Japanese Patent Application No. 4638735, etc.

[0197] [Method for imparting water-absorbing and quick-drying properties to articles]

[0198] The method of imparting quick-drying water absorption to an article according to this embodiment comprises the step of including the modified silk fibroin in the article. The modified silk fibroin according to the present invention has excellent quick-drying water absorption, so by including it in an article, the article can be endowed with quick-drying water absorption.

[0199] The article is not particularly limited as long as it is an article that should be endowed with water-absorbing and quick-drying properties. Specifically, fibers, woven fabrics, knitted fabrics, nonwoven fabrics, cotton, sponges, films, resins, composite materials (regardless of the form of the water-absorbing and quick-drying agent involved in this embodiment), and various articles made from them can be cited.

[0200] In the process of including modified silk fibroin, it is also possible to include the process of modified silk fibroin by including the water-absorbing quick-drying agent involved in the present invention. There is no particular limitation as a method of including modified silk fibroin, and it can be a method of mixing modified silk fibroin in a material (raw material), or it can be a method of combining the water-absorbing quick-drying agent prepared in the form of the above-mentioned molded body with other materials (molded bodies, etc.) to form an article. In addition, it can also be a method of forming an article (molded body) by molding the modified silk fibroin (which can also contain other additives as needed) itself.

[0201] Based on the total amount of the article, the content of the modified silk fibroin in the article is preferably 20% by mass or more, more preferably 30% by mass or more, further preferably 40% by mass or more, and further more preferably 50% by mass or more. Based on the total amount of the article, the upper limit of the content of the modified silk fibroin can be 100% by mass, or it can be 90% by mass or less. By adjusting the content of the modified silk fibroin in the article, the water absorption and quick-drying performance of the article can be controlled. That is, since the modified silk fibroin involved in the present invention has excellent water absorption and quick-drying performance, the water absorption and quick-drying performance of the article can be further improved as the content of the modified silk fibroin in the article is increased.

[0202] Example

[0203] Hereinafter, the present invention will be described in more detail based on Examples etc. However, the present invention is not limited to the following Examples.

[0204] [Manufacturing of modified silk fibroin]

[0205] (1) Preparation of expression vector

[0206] A modified silk fibroin (PRT918) having the amino acid sequence shown in sequence number 37 and a modified silk fibroin (PRT799) having the amino acid sequence shown in sequence number 15 were designed. A nucleic acid encoding the designed modified silk fibroin was synthesized. In the nucleic acid, an NdeI site was attached to the 5' end and an EcoRI site was attached downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was treated with restriction enzymes using NdeI and EcoRI and excised, and then recombined into a protein expression vector pET-22b (+) to obtain an expression vector.

[0207] (2) Protein expression

[0208] The obtained expression vector was used to transform E. coli BLR (DE3). The transformed E. coli was cultured in 2 mL LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin to a concentration of OD 0. 600The culture temperature was maintained at 30°C and flask culture was performed until the OD 600 When the temperature reaches 5 (about 15 hours), a seed culture solution is obtained.

[0209] [Table 4]

[0210] Seed culture medium

[0211]

[0212] The seed culture was added to a fermenter containing 500 mL of production medium (Table 5) to an OD of 600 The culture medium temperature was maintained at 37°C and the pH was controlled to be constant at 6.9. In addition, the dissolved oxygen concentration in the culture medium was maintained at 20% of the saturated dissolved oxygen concentration.

[0213] [Table 5]

[0214] Production medium

[0215]

[0216] After the glucose in the production medium is completely consumed, the feed solution (glucose 455g / 1L, yeast extract 120g / 1L) is immediately added at a rate of 1mL / min. The culture solution temperature is maintained at 37°C, and the pH is constantly controlled at 6.9 for cultivation. In addition, the dissolved oxygen concentration in the culture solution is maintained at 20% of the saturated dissolved oxygen concentration and cultured for 20 hours. Then, 1M isopropyl-β-thiogalactopyranoside (IPTG) is added to the culture solution to make its final concentration 1mM, and the induced modified silk fibroin is expressed. After 20 hours after the addition of IPTG, the culture solution is centrifuged and the bacteria are recovered. SDS-PAGE is performed using bacteria prepared from the culture solution before and after the addition of IPTG, and the expression of the target modified silk fibroin is confirmed based on the appearance of bands of the size of the target modified silk fibroin that depends on the addition of IPTG.

[0217] (3) Protein purification

[0218] The cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer containing about 1 mM PMSF.

[0219] In Tris-HCl buffer (pH7.4), the cells were broken using a high-pressure homogenizer (manufactured by GEA Niro Soavi). The broken cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20mM Tris-HCl buffer (pH7.4) until it reached a high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10mM sodium dihydrogen phosphate, 20mM NaCl, 1mM Tris-HCl, pH7.0) at a concentration of 100mg / mL, and stirred with a stirrer at 60°C for 30 minutes to dissolve it. After dissolution, dialyzed with water using a dialysis tube (cellulose tube 36 / 32 manufactured by Sanko Junyaku Co., Ltd.). The white coagulated protein obtained after dialysis was recovered by centrifugation, the water was removed by a freeze dryer, and the modified silk fibroin (PRT918 and PRT799) was obtained by recovering the freeze-dried powder.

[0220] PRT918 is a hydrophobic modified silk fibroin with an average HI exceeding 0. PRT799 is a hydrophilic modified silk fibroin with an average HI below 0.

[0221] 〔Production of protein fibers〕

[0222] Dimethyl sulfoxide (DMSO) in which LiCl was dissolved was prepared as a solvent to a concentration of 4.0% by mass, to which freeze-dried powder of modified silk fibroin was added to a concentration of 24% by mass, and dissolved using an oscillator for 3 hours. Then, insoluble matter and bubbles were removed to obtain a modified silk fibroin solution (spinning stock solution).

[0223] The prepared spinning solution was filtered at 60°C using a metal filter with a mesh size of 5 μm, then placed in a 30 mL stainless steel syringe and allowed to stand for degassing, and then ejected from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The ejection temperature was 60°C. After coagulation, the obtained raw silk was wound up and dried naturally to obtain a modified silk core protein fiber (raw material fiber).

[0224] For comparison, commercially available silk fibers, cotton fibers, and polyester fibers were prepared as raw material fibers.

[0225] 〔Manufacture of knitted fabrics〕

[0226] Knitted fabrics are manufactured by weaving with a weft knitting machine using various raw materials. The knitted fabric using PRT918 fiber as the raw material fiber has a thickness of 1 / 30N (single yarn count) and a needle gauge of 18. The knitted fabric using PRT799 fiber as the raw material fiber has a thickness of 1 / 30N (single yarn count) and a needle gauge of 16. The thickness and needle gauge of the knitted fabric using other raw materials are adjusted so that they have approximately the same cover factor as the knitted fabric using PRT918 fiber and PRT799 fiber. Specifically, it is as follows.

[0227] Silk thickness: 2 / 60N (monofilament), needle length: 14

[0228] Cotton thickness: 2 / 34N (double wire), needle length: 14

[0229] Polyester thickness: 1 / 60N (monofilament), needle gauge: 14

[0230] [Evaluation of water absorption and quick-drying performance]

[0231] (Water absorption performance evaluation)

[0232] The water absorption performance was evaluated by a test based on JIS L 1907 (test method for water absorption performance of fiber products - drip method). Specifically, under a standard environment (temperature 20±2°C / humidity 65±4%RH), one drop of water was dripped from a dropper onto the surface of the knitted fabric prepared above, and the time until the dripped water drop was absorbed by the knitted fabric (mirror reflection disappeared) was measured (the maximum measurement time was 60 seconds). The results are shown in Table 6.

[0233] (Quick-drying performance evaluation)

[0234] The quick-drying performance is evaluated by measuring the diffusive residual moisture rate. Specifically, in a standard environment (temperature 20±2°C / humidity 65±4%RH), 0.6mL of tap water is dripped onto the inner side of the knitted fabric, and the weight of the knitted fabric is measured every certain period of time (every 5 minutes). The weight of the water is obtained and the diffusive residual moisture rate is calculated by the following formula.

[0235] Diffusible residual moisture content (%) = weight of water at each time (g) / weight of water at the start of measurement (g) × 100

[0236] The measurement was performed until the diffusible residual water content became 10% or less (ie, 10% of the weight of 0.6 mL of water = 60 μL (60 mg)), and the time required until the diffusible residual water content became 10% was calculated.

[0237] [Table 6]

[0238] Water absorption (seconds) Quick-drying (points) Modified Silk Fibroin (PRT918) >60 54 Modified Silk Fibroin (PRT799) 3 66 silk >60 134.3 cotton 1 125.7 Polyester >60 300.8

[0239] It can be understood that the modified silk fibroin (PRT799 and PRT918) has excellent water absorption and quick-drying properties. In particular, it can be understood that the hydrophilic modified silk fibroin (PRT799) has excellent water absorption and quick-drying properties.

Claims

1. A water-absorbing quick-drying agent, wherein: It contains modified silk fibroin as an active ingredient.

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