Patent Application
20220411968
The present invention relates to an agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property, and a method of imparting a cool touch feeling property and a water-absorbing and quick-drying property to an article.
In recent years, highly functional materials having both a cool touch feeling property and a water-absorbing and quick-drying property have been used for articles that are brought into direct contact with skin, such as bedding and clothing for summer. For example, Patent Literature 1 discloses a composite spun yarn of a triacetate short fiber A and a cellulose short fiber B having a single yarn fineness of 0.5 to 3.0 dtex, wherein a mass ratio (A/B) of the short fiber A to the short fiber B is in a range of 20/80 to 80/20, a fuzz index of 3 mm or more is 50/m or less, and an average strength is in a range of 100 CN or more.
Patent Literature 1: JP 2015-98662 A
The composite spun yarn disclosed in Patent Literature 1 utilizes advantages of a triacetate short fiber. However, synthetic fibers such as triacetate short fibers are poor in biodegradability and have a problem of causing environmental loads. Meanwhile, with respect to natural fibers, though silk has a relatively high cool touch feeling property, it cannot sufficiently satisfy the level required for bedding and clothing for summer and the like. Further, silk does not have a sufficient water-absorbing and quick-drying property, and also does not have a sufficient function from the viewpoint of prevention of stuffiness and the like. Thus, it cannot be said that sufficient options have yet been provided for highly functional materials having both a cool touch feeling property and a water-absorbing and quick-drying property.
An object of the present invention is to provide an agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property. Another object of the present invention is to provide a method of imparting a cool touch feeling property and a water-absorbing and quick-drying property to a given article.
The present inventors have found that a modified fibroin has an excellent cool touch feeling property and an excellent water-absorbing and quick-drying property. The present invention is based on this novel finding.
The present invention relates to, for example, each of the following inventions.
[1]
An agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property, containing: a modified fibroin as an active ingredient.
[2]
The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to [1], wherein the modified fibroin contains a modified fibroin having an average value of a hydropathy index (average HI) of more than 0.
[3]
The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to [1] or [2], wherein the modified fibroin contains a modified spider silk fibroin.
[4]
The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to any one of [1] to [3], which is in a form of a fiber.
[5]
A method of imparting a cool touch feeling property and a water-absorbing and quick-drying property to an article, the method including the step of: incorporating a modified fibroin into the article.
According to the present invention, a novel agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property can be provided. According to the present invention, a method of imparting a cool touch feeling property and a water-absorbing and quick-drying property to a given article can also be provided.
Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
[Agent for Imparting a Cool Touch Feeling Property and a Water-Absorbing and Quick-Drying Property]
The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present invention contains a modified fibroin as an active ingredient.
Modified fibroins have both a cool touch feeling property (for example, a property of giving a cool feeling at the time of contact) and a water-absorbing and quick-drying property (for example, a property of absorbing moisture (for example, sweat) well and drying quickly.). The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment can simultaneously impart a cool touch feeling property and a water-absorbing and quick-drying property to a given article using these properties of modified fibroins.
(Modified Fibroin)
The modified fibroin according to the present embodiment is a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m−(A)n motif. An amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either or both of the N-terminal side and the C-terminal side of the domain sequence of the modified fibroin. The N-terminal sequence and the C-terminal sequence are not limited thereto, but, typically are regions having no repetitions of amino acid motifs characterized in fibroin, and each consists of amino acids of approximately 100 residues.
The term “modified fibroin” in the present specification refers to an artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin in which a domain sequence is different from an amino acid sequence of a naturally derived fibroin or may be a fibroin in which a domain sequence is the same as an amino acid sequence of a naturally derived fibroin. The term “naturally derived fibroin” as used in the present specification is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m−(A)n motif.
The “modified fibroin” may be a fibroin obtained by using an amino acid sequence of a naturally derived fibroin as it is, a fibroin in which an amino acid sequence is modified based on an amino acid sequence of a naturally derived fibroin (for example, a fibroin in which an amino acid sequence is modified by modifying a cloned gene sequence of a naturally derived fibroin), or a fibroin artificially designed and synthesized independently of a naturally derived fibroin (for example, a fibroin having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding a designed amino acid sequence).
The “domain sequence” as used herein is an amino acid sequence that produces a crystal region (typically, corresponding to the (A)n motif of the amino acid sequence) and an amorphous region (typically, corresponding to the REP of the amino acid sequence) specific to fibroins, and means an amino acid sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m−(A)n motif. Here, the (A)n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues therein is 2 to 27. The number of the amino acid residues in the (A)n 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, the proportion of the number of alanine residues in the total number of amino acid residues in the (A)n motif may be 40% or more, or may also be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that the (A)n motif only consists of alanine residues). At least seven of a plurality of (A)n motifs in the domain sequence may consist of only alanine residues. The REP represents an amino acid sequence consisting of 2 to 200 amino acid residues. The 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. A plurality of (A)n motifs may be the same amino acid sequences or different amino acid sequences. A plurality of REPs may be the same amino acid sequences or different amino acid sequences.
The modified fibroin according to the present embodiment can be obtained by, for example, performing modification of an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues with respect to a cloned gene sequence of a naturally derived fibroin. Substitution, deletion, insertion, and/or addition of the amino acid residues can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, the modification may be performed in accordance with a method described in literatures such as Nucleic Acid Res. 10, 6487 (1982), and Methods in Enzymology, 100, 448 (1983).
The naturally derived fibroin is a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m−(A)n motif, and specific examples thereof include fibroins produced by insects or spiders.
Examples of fibroins produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama and a hornet silk protein secreted by larvae of Vespa simillima xanthoptera.
More specific examples of fibroins produced by insects include the silkworm fibroin L chain (GenBank Accession Nos. M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
Examples of fibroins produced by spiders include spider silk proteins produced by spiders belonging to the order Araneae. More specific examples thereof include spider silk proteins produced by spiders belonging to the genus Araneus, such as Araneus ventricosus, Araneus diadematus, Araneus pinguis, Araneus pentagrammicus, and Araneus nojimai, spiders belonging to the genus Neoscona, such as Neoscona scylla, Neoscona nautica, Neoscona adianta, and Neoscona scylloides, spiders belonging to the genus Pronus, such as Pronous minutus, spiders belonging to the genus Cyrtarachne, such as Cyrtarachne bufo and Cyrtarachne inaequalis, spiders belonging to the genus Gasteracantha, such as Gasteracantha kuhlii and Gasteracantha mammosa, spiders belonging to the genus Ordgarius, such as Ordgarius hobsoni and Ordgarius sexspinosus, spiders belonging to the genus Argiope, such as Argiope amoena, Argiope minuta, and Argiope bruennichi, spiders belonging to the genus Arachnura, such as Arachnura login, spiders belonging to the genus Acusilas, such as Acusilas coccineus, spiders belonging to the genus Cytophora, such as Cyrtophora moluccensis, Cyrtophora exanthematica, and Cyrtophora unicolor, spiders belonging to the genus Poltys, such as Poltys illepidus, spiders belonging to the genus Cyclosa, such as Cyclosa octotuberculata, Cyclosa sedeculata, Cyclosa vallata, and Cyclosa atrata, and spiders belonging to the genus Chorizopes, such as Chorizopes nipponicus, and spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Tetragnatha, such as Tetragnatha praedonia, Tetragnatha maxillosa, Tetragnatha extensa, and Tetragnatha squamata, spiders belonging to the genus Leucauge, such as Leucauge magnifica, Leucauge blanda, and Leucauge subblanda, spiders belonging to the genus Nephila, such as Nephila clavata and Nephila pilipes, spiders belonging to the genus Menosira, such as Menosira ornata, spiders belonging to the genus Dyschiriognatha, such as Dyschiriognatha tenera, spiders belonging to the genus Latrodectus, such as Latrodectus mactans, Latrodectus hasseltii, Latrodectus geometricus, and Latrodectus tredecimguttatus, and spiders belonging to the genus Euprosthenops. Examples of the spider silk proteins include dragline silk proteins such as MaSps (MaSp1 and MaSp2) and ADFs (ADF3 and ADF4), MiSps (MiSp1 and MiSp2), AcSp, PySp, and Flag.
More specific examples of the 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 No. AAL32472 (amino acid sequence), AF441245 (base sequence)), major ampullate spidroin 1 [derived from Euprosthenops australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank Accession No. 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)), and minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank Accession No. ABR37278.1 (amino acid sequence).
More specific examples of the naturally derived fibroin include a fibroin whose sequence information is registered in NCBI GenBank. For example, sequences thereof may be confirmed by extracting sequences in which spidroin, ampullate, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION among sequences containing INV as DIVISION among sequence information registered in NCBI GenBank, sequences in which a specific character string of products is described from CDS, or sequences in which a specific character string is described from SOURCE to TISSUE TYPE.
The modified fibroin according to the present embodiment may be a modified silk fibroin (in which an amino acid sequence of a silk protein produced by silkworm is modified), or may be a modified spider silk fibroin (in which an amino acid sequence of a spider silk protein produced by spiders is modified).
Specific examples of the modified fibroin include a modified fibroin derived from a major dragline silk protein produced in a major ampullate gland of a spider (first modified fibroin), a modified fibroin containing a domain sequence in which the content of glycine residues is reduced (second modified fibroin), a modified fibroin containing a domain sequence in which the content of an (A)n motif is reduced (third modified fibroin), a modified fibroin in which the content of glycine residues and the content of an (A)n motif are reduced (fourth modified fibroin), a modified fibroin containing a domain sequence including a region locally having a high hydropathy index (fifth modified fibroin), and a modified fibroin containing a domain sequence in which the content of glutamine residues is reduced (sixth modified fibroin).
Examples of the first modified fibroin include a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m. In the first modified fibroin, the number of amino acid residues in the (A)n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, even still more preferably an integer of 10 to 20, still 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 fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, still more preferably 20 to 100 residues, and still even more preferably 20 to 75 residues. In the first modified fibroin, the total number of glycine residues, serine residues, and alanine residues included in the amino acid sequence represented by Formula 1: [(A)n motif-REP]m is preferably 40% or more, more preferably 60% or more, and still more preferably 70% or more, relative to the total number of amino acid residues.
The first modified fibroin may be a polypeptide including an amino acid sequence unit represented by Formula 1: [(A)n motif-REP]m, and including a C-terminal sequence which is an amino acid sequence set forth in any one of SEQ ID NO: 1 to 3 or a C-terminal sequence which is an amino acid sequence having 90% or more homology with the amino acid sequence set forth in any one of SEQ ID NO: 1 to 3.
The amino acid sequence set forth in SEQ ID NO: 1 is identical to an amino acid sequence consisting of 50 amino acid residues of the C-terminus of an amino acid sequence of ADF3 (GI: 1263287, NCBI). The amino acid sequence set forth in SEQ ID NO: 2 is identical to an amino acid sequence set forth in SEQ ID NO: 1 in which 20 amino acid residues have been removed from the C-terminus. The amino acid sequence set forth in SEQ ID NO: 3 is identical to an amino acid sequence set forth in SEQ ID NO: 1 in which 29 amino acid residues have been removed from the C-terminus.
Specific examples of the first modified fibroin include a modified fibroin including (1-i) the amino acid sequence set forth in SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4. The sequence identity is preferably 95% or more.
The amino acid sequence set forth in SEQ ID NO: 4 is obtained by the following mutation: in an amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, a His 10-tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminus, the 1st to 13th repetitive regions are about doubled and the translation ends at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 4 is identical to the amino acid sequence set forth in SEQ ID NO: 3.
The modified fibroin of (1-i) may consist of the amino acid sequence set forth in SEQ ID NO: 4.
The domain sequence of the second modified fibroin has an amino acid sequence in which the content of glycine residues is reduced, as compared with a naturally derived fibroin. It can be said that the second modified fibroin has an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of glycine residues in REP are substituted with another amino acid residue, as compared with a naturally derived fibroin.
The domain sequence of the second modified fibroin may have an amino acid sequence corresponding to an amino acid sequence in which one glycine residue in at least one or the plurality of motif sequences is substituted with another amino acid residue, in at least one motif sequence selected from GGX and GPGXX (where G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP, as compared with a naturally derived fibroin.
In the second modified fibroin, the proportion of the motif sequence in which the glycine residue has been substituted with another amino acid residue may be 10% or more relative to the entire motif sequence.
The second modified fibroin contains a domain sequence represented by Formula 1: [(A)n motif-REP]m, and may have an amino acid sequence in which z/w is 30% or more, 40% or more, 50% or more, or 50.9% or more in a case where the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) included in all REPs in a sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence is defined as z, and the total number of amino acid residues in the sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence is defined as w. The number of alanine residues with respect to the total number of amino acid residues in the (A)n motif is 83% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and even still more preferably 100% (meaning that the (A)n motif consists of only alanine residues).
The second modified fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, still more preferably 10% or less, even still more preferably 6% or less, still further preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the calculation method of the content ratio (z/w) of the amino acid sequence consisting of XGX described below.
The method of calculating z/w will be described in more detail. First, the amino acid sequence consisting of XGX is extracted from all REPs included in a sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence in the fibroin containing a domain sequence represented by Formula 1: [(A)n motif-REP]m (modified fibroin or naturally derived fibroin). The total number of amino acid residues constituting XGX is z. For example, in a case where 50 amino acid sequences consisting of XGX are extracted (there is no overlap), z is 50×3=150. Also, for example, in a case where X (central X) included in two XGXs exists as in a case of the amino acid sequence consisting of XGXGX, z is calculated by subtracting the overlapping portion (in a case of XGXGX, it is 5 amino acid residues). w is the total number of amino acid residues included in a sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in a case of the domain sequence shown in
Here, z/w in a naturally derived fibroin will be described. First, as described above, 663 types of fibroins (415 types of fibroins derived from spiders among them) were extracted by confirming fibroins with amino acid sequence information registered in NCBI GenBank by an exemplified method. The values of z/w were calculated by the calculation method described above, from amino acid sequences of naturally derived fibroins which include a domain sequence represented by Formula 1: [(A)n motif-REP]m and in which the content ratio of the amino acid sequence consisting of GGX in the fibroin is 6% or less, among all the extracted fibroins. The results are shown in
In the second modified fibroin, z/w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, even still more preferably 70% or more, and still further preferably 80% or more. The upper limit of z/w is not particularly limited, but may be 95% or less, for example.
The second modified fibroin can be obtained by, for example, substituting and modifying at least a part of a base sequence encoding a glycine residue from a cloned gene sequence of a naturally derived fibroin so as to encode another amino acid residue. In this case, one glycine residue in a GGX motif or a GPGXX motif may be selected as the glycine residue to be modified, and substitution may be performed so that z/w is 50.9% or more. In addition, the second modified fibroin can also be obtained by, for example, designing an amino acid sequence satisfying each of the above aspects from the amino acid sequence of a naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to substitution of a glycine residue in REP with another amino acid residue from the amino acid sequence of a naturally derived fibroin, modification of the amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may be performed.
The above-described another amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but it is preferably a hydrophobic amino acid residue such as a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a methionine (M) residue, a proline (P) residue, a phenylalanine (F) residue, or a tryptophan (W) residue, or a hydrophilic amino acid residue such as a glutamine (Q) residue, an asparagine (N) residue, a serine (S) residue, a lysine (K) residue, or a glutamic acid (E) residue, more preferably a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a phenylalanine (F) residue, or a glutamine (Q) residue, and still more preferably a glutamine (Q) residue.
More specific examples of the second modified fibroin include a modified fibroin including (2-i) the amino acid sequence set forth in SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met-PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (2-i) will be described. The amino acid sequence set forth in SEQ ID NO: 6 is obtained by substituting all GGXs with GQX in REP of the amino acid sequence set forth in SEQ ID NO: 10 (Met-PRT313) corresponding to a naturally derived fibroin. The amino acid sequence set forth in SEQ ID NO: 7 is obtained by deleting every other two (A)n motifs from the N-terminal side to the C-terminal side from the amino acid sequence set forth in SEQ ID NO: 6 and further inserting one [(A)nmotif-REP] before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 8 is obtained by inserting two alanine residues on the C-terminal side of each (A)n motif of the amino acid sequence set forth in SEQ ID NO: 7 and further substituting a part of glutamine (Q) residues with a serine (S) residue to delete a part of amino acids on the C-terminal side so as to be almost the same as the molecular weight of SEQ ID NO: 7. The amino acid sequence set forth in SEQ ID NO: 9 is obtained by adding a predetermined hinge sequence and a His tag sequence to the C-terminus of a sequence obtained by repeating a region of 20 domain sequences (where several amino acid residues on the C-terminal side of the region are substituted) present in the amino acid sequence set forth in SEQ ID NO: 7 four times.
The value of z/w in the amino acid sequence set forth in SEQ ID NO: 10 (corresponding to naturally derived fibroin) is 46.8%. The values of z/w in the amino acid sequence set forth in SEQ ID NO: 6, the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, and the amino acid sequence set forth in SEQ ID NO: 9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. In addition, the values of x/y in the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 at a Giza ratio (described below) of 1:1.8 to 11.3 are 15.0%, 15.0%, 93.4%, 92.7%, and 89.8%, respectively.
The modified fibroin of (2-i) may consist of the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (2-ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.
It is preferable that the modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and z/w is 50.9% or more in a case where the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) included in REP is defined as z, and the total number of amino acid residues of REP in the domain sequence is defined as w.
The second modified fibroin may have a tag sequence at either or both of the N-terminus and the C-terminus. This enables the modified fibroin to be isolated, immobilized, detected, and visualized.
The tag sequence may be, for example, an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. Specific examples of the affinity tag include a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged and has a property of specifically binding to a metal ion such as nickel. Thus, the His tag can be used for isolation of a modified fibroin by chelating metal chromatography. Specific examples of the tag sequence include the amino acid sequence set forth in SEQ ID NO: 11 (amino acid sequence including a His tag sequence and a hinge sequence).
Also, a tag sequence such as glutathione-S-transferase (GST) that specifically binds to glutathione, and a maltose binding protein (MBP) that specifically binds to maltose can also be utilized.
Further, an “epitope tag” utilizing an antigen-antibody reaction can also be utilized. Adding a peptide (epitope) exhibiting antigenicity as a tag sequence allows an antibody against the epitope to be bound. Examples of the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, a myc tag, and a FLAG tag. The modified fibroin can easily be purified with high specificity by utilizing an epitope tag.
Moreover, it is possible to use a tag sequence which can be cleaved with a specific protease. The modified fibroin from which the tag sequence has been cleaved can be recovered by treating a protein adsorbed through the tag sequence with protease.
More specific examples of the modified fibroin including a tag sequence include a modified fibroin including (2-iii) the amino acid sequence set forth in SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799), or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
Each of the amino acid sequences set forth in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 is obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) to the N-terminus of each of the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
The modified fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
The modified fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.
It is preferable that the modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and z/w is 50.9% or more in a case where the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents the amino acid residue other than glycine) in REP is defined as z, and the total number of amino acid residues in REP in the domain sequence is defined as w.
The second modified fibroin may include a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
The domain sequence of the third modified fibroin has an amino acid sequence in which the content of the (A)n motif is reduced, as compared with a naturally derived fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of (A)n motifs are deleted, as compared with a naturally derived fibroin.
The third modified fibroin may have an amino acid sequence corresponding to an amino acid sequence in which 10 to 40% of the (A)n motifs are deleted from a naturally derived fibroin.
The domain sequence of the third modified fibroin may have an amino acid sequence corresponding to an amino acid sequence in which at least one (A)n motif of every one to three (A)n motifs is deleted from the N-terminal side to the C-terminal side, as compared with a naturally derived fibroin.
The third modified fibroin may have a domain sequence having an amino acid sequence corresponding to an amino acid sequence obtained by repeating deletion of at least two consecutive (A)n motifs and deletion of one (A)n motif in this order from the N-terminal side to the C-terminal side, as compared with the naturally derived fibroin.
The third modified fibroin may have a domain sequence having an amino acid sequence corresponding to an amino acid sequence in which at least (A)n motif every other two positions is deleted from the N-terminal side to the C-terminal side.
The third modified fibroin contains a domain sequence represented by Formula 1: [(A)n motif-REP]m, and may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more, or 50% or more in a case where the numbers of amino acid residues in REPs of two adjacent [(A)n motif-REP] units are sequentially compared from the N-terminal side to the C-terminal side, and the number of amino acid residues in one REP having a smaller number of amino acid residues is defined as 1, the maximum value of the total value of the number of amino acid residues in the two adjacent [(A)n motif-REP] units where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 is defined as x, and the total number of amino acid residues in the domain sequence is defined as y. The number of alanine residues with respect to the total number of amino acid residues in the (A)n motif is 83% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, and even still more preferably 100% (meaning that the (A)n motif consists of only alanine residues).
The method of calculating x/y will be described in more detail with reference to
The two adjacent [(A)n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side so as not to overlap. At this time, an unselected [(A)n motif-REP] unit may exist.
Subsequently, the number of amino acid residues of each REP in the selected two adjacent [(A)n motif-REP] units is compared for each pattern. The comparison is performed by determining the ratio of the number of amino acid residues of the other REP in a case where one REP having a smaller number of amino acid residues is defined as 1. For example, in a case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), the ratio of the number of amino acid residues of the second REP is 100/50=2 in a case where the first REP having a smaller number of amino acid residues is defined as 1. Similarly, in a case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), the ratio of the number of amino acid residues of the fifth REP is 30/20=1.5 in a case where the fourth REP having a smaller number of amino acid residues is defined as 1.
In
In each pattern, the number of all amino acid residues of two adjacent [(A)n motif-REP] units indicated by solid lines (including not only the number of amino acid residues of REP but also the number of amino acid residues of the (A)n motif) is combined. Then, the total values combined are compared, and the total value of the pattern whose total value is the maximum (the maximum value of the total value) is defined as x. In the example shown in
Then, x/y (%) can be calculated by dividing x by the total number of amino acid residues y of the domain sequence.
In the third modified fibroin, x/y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, even still more preferably 70% or more, still further preferably 75% or more, and particularly preferably 80% or more. The upper limit of x/y is not particularly limited, but may be, for example, 100% or less. In a case where the Giza ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more; in a case where the Giza ratio is 1:1.8 to 3.4, x/y is preferably 77.1% or more; in a case where the Giza ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more; and in a case where the Giza ratio is 1:1.9 to 4.1, x/y is preferably 64.2% or more.
In a case where the third modified fibroin is a modified fibroin in which at least seven of a plurality of (A)n motifs in the domain sequence consist of only alanine residues, x/y is preferably 46.4% or more, more preferably 50% or more, still more preferably 55% or more, even still more preferably 60% or more, still further preferably 70% or more, and particularly preferably 80% or more. The upper limit of x/y is not particularly limited, but is only required to be 100% or less.
Here, x/y in a naturally derived fibroin will be described. First, as described above, 663 types of fibroins (415 types of fibroins derived from spiders among them) were extracted by confirming fibroins with amino acid sequence information registered in NCBI GenBank by an exemplified method. The values of x/y were calculated by the calculation method described above, from amino acid sequences of a naturally derived fibroin consisting of a domain sequence represented by Formula 1: [(A)n motif-REP]m, among all the extracted fibroins. The results in a case where the Giza ratio is 1:1.9 to 4.1 are shown in
The horizontal axis in
The third modified fibroin can be obtained from, for example, a cloned gene sequence of a naturally derived fibroin, by deleting one or a plurality of sequences encoding an (A)n motif so that x/y is 64.2% or more. In addition, for example, the third modified fibroin can also be obtained, from the amino acid sequence of a naturally derived fibroin, by designing an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of (A)n motifs are deleted so that x/y is 64.2% or more, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to deletion of the (A)n motif from the amino acid sequence of the naturally derived fibroin, modification of the amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may be performed.
More specific examples of the third modified fibroin include a modified fibroin having (3-i) an amino acid sequence set forth in SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met-PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (3-i) will be described. The amino acid sequence set forth in SEQ ID NO: 17 is obtained by deleting every other two (A)n motifs from the N-terminal side to the C-terminal side from the amino acid sequence set forth in SEQ ID NO: 10 (Met-PRT313) corresponding to a naturally derived fibroin and further inserting one [(A)n motif-REP] before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described in the second modified fibroin.
The value of x/y in the amino acid sequence set forth in SEQ ID NO: 10 (corresponding to naturally derived fibroin) at a Giza ratio of 1:1.8 to 11.3 is 15.0%. Both the value of x/y in the amino acid sequence set forth in SEQ ID NO: 17 and the value of x/y in the amino acid sequence set forth in SEQ ID NO: 7 are 93.4%. The value of x/y in the amino acid sequence set forth in SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence set forth in SEQ ID NO: 9 is 89.8%. The values of z/w in the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively.
The modified fibroin of (3-i) may consist of the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (3-ii) may consist of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.
The modified fibroin of (3-ii) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and x/y is preferably 64.2% or more, in which when the number of amino acid residues in REP's in two [(A)n motif-REP] units adjacent to each other are sequentially compared from the N-terminus to the C-terminus, and then the number of amino acid residues in REP having a small number of amino acid residues is set as 1, a maximum value of the total value obtained by summing up the number of amino acid residues in the two adjacent [(A)nmotif-REP] units where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 (the Giza ratio is 1:1.8 to 11.3) is x, and the total number of amino acid residues in the domain sequence is y.
The third modified fibroin may have the above-described tag sequence at either or both of the N-terminus and the C-terminus.
More specific examples of the modified fibroin having a tag sequence include a modified fibroin having (3-iii) an amino acid sequence set forth in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799), or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
Each of the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 is obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (having a His tag sequence and a hinge sequence) to the N-terminus of each of the amino acid sequences set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
The modified fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
The modified fibroin of (3-iv) may consist of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.
The modified fibroin of (3-iv) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and x/y is preferably 64.2% or more, in which when the number of amino acid residues in REP's in two [(A)n motif-REP] units adjacent to each other are sequentially compared from the N-terminus to the C-terminus, and then the number of amino acid residues in REP having a small number of amino acid residues is set as 1, a maximum value of the total value obtained by summing up the number of amino acid residues in the two adjacent [(A)n motif-REP] units where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 is x, and the total number of amino acid residues in the domain sequence is y.
The third modified fibroin may include a secretory signal for releasing the protein produced in a recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
The domain sequence of the fourth modified fibroin has an amino acid sequence in which a content of an (A)n motif and a content of glycine residues are reduced, as compared with a naturally derived fibroin. It can be said that the domain sequence of the fourth modified fibroin has an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of (A)n motifs are deleted and at least one or a plurality of glycine residues in REP are substituted with another amino acid residue, as compared with a naturally derived fibroin. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the above-described second modified fibroin and third modified fibroin. Specific aspects and the like of the fourth modified fibroin are as described in the second modified fibroin and the third modified fibroin.
More specific examples of the fourth modified fibroin include a modified fibroin having (4-i) an amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799), or (4-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. Specific aspects of the modified fibroin having the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 are as described above.
The domain sequence of the fifth modified fibroin may have an amino acid sequence including a region locally having a high hydropathy index corresponding to an amino acid sequence in which one or a plurality of amino acid residues in REP are substituted with amino acid residues having a high hydropathy index and/or one or a plurality of amino acid residues having a high hydropathy index are inserted into REP, as compared with the naturally derived fibroin.
It is preferable that the region locally having a high hydropathy index consists of two to four consecutive amino acid residues.
It is more preferable that the above-described amino acid residue having a high hydropathy index is an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A).
The fifth modified fibroin may be further subjected to modification of an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues as compared with the naturally derived fibroin, in addition to modification corresponding to substitution of one or a plurality of amino acid residues in REP with amino acid residues having a high hydropathy index and/or insertion of one or a plurality of amino acid residues having a high hydropathy index into REP, as compared with the naturally derived fibroin.
The fifth modified fibroin can be obtained by, for example, substituting one or a plurality of hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydropathy index) with hydrophobic amino acid residues (for example, amino acid residues having a positive hydropathy index) from a cloned gene sequence of a naturally derived fibroin, and/or inserting one or a plurality of hydrophobic amino acid residues into REP. In addition, the fifth modified fibroin can be obtained by, for example, designing an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from an amino acid sequence of a naturally derived fibroin, and/or one or a plurality of hydrophobic amino acid residues are inserted into REP, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to modification corresponding to substitution of one or a plurality of hydrophilic amino acid residues in REP with hydrophobic amino acid residues from amino acid sequences of a naturally derived fibroin, and/or insertion of one or a plurality of hydrophobic amino acid residues into REP, modification of an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may be further performed.
The fifth modified fibroin may contain a domain sequence represented by Formula 1: [(A)n motif-REP]m, and may have an amino acid sequence in which p/q is 6.2% or more, in which in all REP's contained in a sequence excluding a sequence from a (A)n motif located the most C-terminal side to the C-terminus of the domain sequence from the domain sequence, a total number of amino acid residues contained in a region where an average value of a hydropathy index of four consecutive amino acid residues is 2.6 or more is p, and a total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located the most C-terminal side to the C-terminus of the domain sequence from the domain sequence is q.
For the hydropathy index of the amino acid residue, a publicly known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. 105-132) is used. Specifically, the hydropathy index (hereinafter, also referred to as “HI”) of each amino acid is as shown in Table 1.
The calculation method of p/q will be described in more detail. In the calculation, a sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence represented by Formula 1 [(A)n motif-REP]m (hereinafter also referred to as “sequence A”) is used. First, in all REPs included in the sequence A, the average values of a hydropathy index of four consecutive amino acid residues are calculated. The average value of a hydropathy index is determined by dividing the sum of HIs of respective amino acid residues included in the four consecutive amino acid residues by 4 (number of amino acid residues). The average value of a hydropathy index is determined for all of the four consecutive amino acid residues (each of the amino acid residues is used for calculating the average value 1 to 4 times). Then, a region where the average value of a hydropathy index of the four consecutive amino acid residues is 2.6 or more is specified. Even in a case where a certain amino acid residue corresponds to the “four consecutive amino acid residues having an average value of a hydropathy index of 2.6 or more” multiple times, the amino acid residue is included as one amino acid residue in the region. The total number of amino acid residues included in the region is p. In addition, the total number of amino acid residues contained in the sequence A is q.
For example, in a case where the “four consecutive amino acid residues having an average value of a hydropathy index of 2.6 or more” are extracted from 20 places (no overlap), in the region where the average value of a hydropathy index of four consecutive amino acid residues is 2.6 or more, the number of the four consecutive amino acid residues (no overlap) is 20, and thus p is 20×4=80. Furthermore, in a case where, for example, only one amino acid residue overlaps within two sets of “four consecutive amino acid residues of which the average value of a hydropathy index is 2.6 or higher”, the region in which the average value of a hydropathy index of four consecutive amino acid residues is 2.6 or higher contains seven amino acid residues (p=2×4−1=7. “−1” is a deduction of the overlapping amino acid residue). For example, in a case of the domain sequence shown in
In the fifth modified fibroin, p/q is preferably 6.2% or more, more preferably 7% or more, still more preferably 10% or more, still more preferably 20% or more, and still more preferably 30% or more. An upper limit of p/q is not particularly limited, but may be, for example, 45% or less.
The fifth modified fibroin can be obtained by, for example, substituting one or a plurality of hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydropathy index) with hydrophobic amino acid residues (for example, amino acid residues having a positive hydropathy index) so that a cloned amino acid sequence of a naturally derived fibroin satisfies the condition of p/q, and/or modifying the cloned amino acid sequence of a naturally derived fibroin with an amino acid sequence including a region locally having a high hydropathy index by inserting one or a plurality of hydrophobic amino acid residues into REP. In addition, the fifth modified fibroin can also be obtained by, for example, designing an amino acid sequence satisfying the condition of p/q from the amino acid sequence of a naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, modification corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may also be performed, in addition to modification corresponding to substitution of one or a plurality of amino acid residues in REP with amino acid residues having a high hydropathy index, and/or insertion of one or a plurality of amino acid residues having a high hydropathy index into REP, as compared with the naturally derived fibroin.
The amino acid residue having a high hydropathy index is not particularly limited, but is preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A), and more preferably valine (V), leucine (L), and isoleucine (I).
More specific examples of the fifth modified fibroin include a modified fibroin having (5-i) an amino acid sequence set forth in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665), or SEQ ID NO: 21 (Met-PRT666), or (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
The modified fibroin of (5-i) will be described. The amino acid sequence set forth in SEQ ID NO: 19 is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at two sites for each REP into the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410), except for the domain sequence at the end on the C-terminal side, and further substituting a part of glutamine (Q) residues with serine (S) residues, and deleting a part of amino acids on the C-terminal side. The amino acid sequence set forth in SEQ ID NO: 20 is obtained by inserting the amino acid sequence consisting of three amino acid residues (VLI) at one site for each REP into the amino acid sequence set forth in SEQ ID NO: 8 (Met-PRT525). The amino acid sequence set forth in SEQ ID NO: 21 is obtained by inserting the amino acid sequence consisting of three amino acid residues (VLI) at two sites for each REP into the amino acid sequence set forth in SEQ ID NO: 8.
The modified fibroin of (5-i) may consist of the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
The modified fibroin of (5-ii) may consist of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.
The modified fibroin of (5-ii) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and p/q is preferably 6.2% or more, in which in all REP's contained in a sequence excluding a sequence from a (A)n motif located the most C-terminal side to the C-terminus of the domain sequence from the domain sequence, a total number of amino acid residues contained in a region where an average value of a hydropathy index of four consecutive amino acid residues is 2.6 or more is p, and a total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located the most C-terminal side to the C-terminus of the domain sequence from the domain sequence is q.
The fifth modified fibroin may have a tag sequence at either or both of the N-terminus and the C-terminus.
More specific examples of the modified fibroin having a tag sequence include a modified fibroin having (5-iii) an amino acid sequence set forth in SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665), or SEQ ID NO: 24 (PRT666), or (5-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
Each of the amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 is obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (having a His tag sequence and a hinge sequence) to the N-terminus of each of the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
The modified fibroin of (5-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
The modified fibroin of (5-iv) may consist of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.
The modified fibroin of (5-iv) preferably has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and p/q is preferably 6.2% or more, in which in all REP's contained in a sequence excluding a sequence from a (A)n motif located the most C-terminal side to the C-terminus of the domain sequence from the domain sequence, a total number of amino acid residues contained in a region where an average value of a hydropathy index of four consecutive amino acid residues is 2.6 or more is p, and a total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located the most C-terminal side to the C-terminus of the domain sequence from the domain sequence is q.
The fifth modified fibroin may include a secretory signal for releasing the protein produced in a recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
The sixth modified fibroin has an amino acid sequence in which a content of glutamine residues is reduced, as compared with the naturally derived fibroin.
The sixth modified fibroin preferably contains at least one motif selected from a GGX motif and a GPGXX motif in the amino acid sequence of REP.
In a case where the sixth modified fibroin contains the GPGXX motif in REP, a content rate of the GPGXX motif is generally 1% or more, may be 5% or more, and is preferably 10% or more. An upper limit of the content rate of the GPGXX motif is not particularly limited, but may be 50% or less or 30% or less.
In the present specification, the “content rate of the GPGXX motif” is a value calculated by the following method. In fibroin (modified fibroin or naturally derived fibroin) containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif, the content rate of the GPGXX motif is calculated as s/t, in which the number obtained by tripling the total number of GPGXX motifs in the regions of all REP's contained in a sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence (that is, corresponding to the total number of G and P in the GPGXX motifs) is s, and the total number of amino acid residues in all REP's excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence and further excluding the (A)n motifs is t.
For the calculation of the content rate of the GPGXX motif, the “sequence excluding a sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence” is used to exclude the effect occurring due to the fact that the “sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence” (sequence corresponding to REP) may have a sequence having a low correlation with the sequence characteristic of fibroin, which influences the calculation result of the content rate of the GPGXX motif in a case where m is small (that is, in a case where the domain sequence is short). In a case where the “GPGXX motif” is located at the C-terminus of REP, it is regarded as the “GPGXX motif” even in a case where “XX” is, for example, “AA”.
In the sixth modified fibroin, a content rate of glutamine residues is preferably 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%.
In the present specification, the “content rate of the glutamine residue” is a value calculated by the following method. The content rate of the glutamine residue in fibroin (modified fibroin or naturally derived fibroin) including a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif is calculated as u/t, in a case where the total number of glutamine residues included in regions of all REPs included in a sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence (a sequence corresponding to the “region A” in
The domain sequence of the sixth modified fibroin may have an amino acid sequence corresponding to deletion of one or a plurality of glutamine residues in REP, or substitution of one or a plurality of glutamine residues with another amino acid residue, as compared with the naturally derived fibroin.
“Another amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a higher hydropathy index than that of a glutamine residue. The hydropathy index of the amino acid residue is shown in Table 1.
As shown in Table 1, examples of the amino acid residue having a higher hydropathy index than that of a glutamine residue include an amino acid residue 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, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A) is more preferable, and an amino acid residue selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) is still more preferable.
In the sixth modified fibroin, the hydrophobicity of REP is preferably −0.8 or more, more preferably −0.7 or more, still more preferably 0 or more, even still more preferably 0.3 or more, and particularly preferably 0.4 or more. An upper limit of the hydrophobicity of REP is not particularly limited, but may be 1.0 or less or 0.7 or less.
In the present specification, the “hydrophobicity of REP” is a value calculated by the following method. The hydrophobicity of REP in fibroin including a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif (modified fibroin or naturally derived fibroin) is calculated as v/t, in a case where the sum of hydropathy indices of amino acid residues in regions of all REPs included in a sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence (a sequence corresponding to the “region A” in
The sixth modified fibroin may have a domain sequence that is further subjected to modification of an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues, in addition to modification corresponding to deletion of one or a plurality of glutamine residues in REP, and/or substitution of one or a plurality of glutamine residues in REP with another amino acid residue, as compared to a naturally derived fibroin.
The sixth modified fibroin can be obtained by, for example, deleting one or a plurality of glutamine residues in REP from a cloned gene sequence of a naturally derived fibroin, and/or substituting one or a plurality of glutamine residues in REP with another amino acid residue. In addition, the sixth modified fibroin can be obtained by, for example, designing an amino acid sequence corresponding to deletion of one or a plurality of glutamine residues in REP from an amino acid sequence of a naturally derived fibroin, and/or substitution of one or a plurality of glutamine residues in REP with another amino acid residue, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
More specific examples of the sixth modified fibroin include a modified fibroin having (6-i) an amino acid sequence set forth in SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917), or SEQ ID NO: 42 (Met-PRT1028), and a modified fibroin having (6-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42.
The modified fibroin of (6-i) will be described. The amino acid sequence set forth in SEQ ID NO: 25 is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410) with VL. The amino acid sequence set forth in SEQ ID NO: 26 is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 7 with TS and substituting the remaining Q with A. The amino acid sequence set forth in SEQ ID NO: 27 is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 7 with VL and substituting the remaining Q with I. The amino acid sequence set forth in SEQ ID NO: 28 is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 7 with VI and substituting the remaining Q with L. The amino acid sequence set forth in SEQ ID NO: 29 is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 7 with VF and substituting the remaining Q with I.
The amino acid sequence set forth in SEQ ID NO: 30 is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence set forth in SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 8 with VL and substituting the remaining Q with I.
The amino acid sequence set forth in SEQ ID NO: 32 is obtained by substituting, with VF, all QQs in a sequence obtained by repeating a region of 20 domain sequences present in the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410) two times and substituting the remaining Q with I.
The amino acid sequence set forth in SEQ ID NO: 41 (Met-PRT917) is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 7 with LI and substituting the remaining Q with V. The amino acid sequence set forth in SEQ ID NO: 42 (Met-PRT1028) is obtained by substituting all QQs in the amino acid sequence set forth in SEQ ID NO: 7 with IF and substituting the remaining Q with T.
The content rate of the glutamine residues in each of the amino acid sequences set forth 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 is 9% or less (Table 2).
The modified fibroin of (6-i) may consist of the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42.
The modified fibroin of (6-ii) may consist of the amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42. The modified fibroin of (6-ii) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP], or Formula 2: [(A)n motif-REP]m-(A)nmotif. The sequence identity is preferably 95% or more.
It is preferable that a content rate of glutamine residues in the modified fibroin of (6-ii) is 9% or less. In addition, it is preferable that a content rate of a GPGXX motif in the modified fibroin of (6-ii) is 10% or more.
The sixth modified fibroin may have a tag sequence at either or both of the N-terminus and the C-terminus. This enables the modified fibroin to be isolated, immobilized, detected, and visualized.
More specific examples of the modified fibroin having a tag sequence include a modified fibroin having (6-iii) an amino acid sequence set forth in SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917), or SEQ ID NO: 44 (PRT1028), or a modified fibroin having (6-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth 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
Each of the amino acid sequences set forth 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 is obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (having a His tag sequence and a hinge sequence) to the N-terminus of each of the amino acid sequences set forth 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. Since only the tag sequence is added to the N-terminus, the content rate of the glutamine residues is not changed, and the content rate of the glutamine residues in each of the amino acid sequences set forth 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 is 9% or less (Table 3).
The modified fibroin of (6-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 44.
The modified fibroin of (6-iv) may consist of the amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 44. The modified fibroin of (6-iv) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP], or Formula 2: [(A)n motif-REP]n-(A)n motif. The sequence identity is preferably 95% or more.
It is preferable that a content rate of glutamine residues in the modified fibroin of (6-iv) is 9% or less. In addition, it is preferable that a content rate of a GPGXX motif in the modified fibroin of (6-iv) is 10% or more.
The sixth modified fibroin may include a secretory signal for releasing the protein produced in a recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set depending on the type of the host.
The modified fibroin may be a modified fibroin having at least two or more characteristics of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin.
The modified fibroin may be a hydrophilic modified fibroin or a hydrophobic modified fibroin. In the present specification, the “hydrophilic modified fibroin” means a modified fibroin having an average value (average HI) of a hydropathy index of 0 or less. In the present specification, the “hydrophobic modified fibroin” means a modified fibroin having an average HI of a hydropathy index of more than 0. The average HI means a value obtained by determining the total sum of the hydropathy indices (HI) of all amino acid residues constituting the modified fibroin, and then dividing the sum by the total number of amino acid residues. The hydropathy index is as shown in Table 1. The hydrophilic modified fibroin has a cool touch feeling property and is excellent in a water-absorbing and quick-drying property. The hydrophobic modified fibroin is excellent in a cool touch feeling property and has a sufficient water-absorbing and quick-drying property.
Examples of the hydrophilic modified fibroin include a modified fibroin including the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
Examples of the hydrophobic modified fibroin include a modified fibroin including the amino acid sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43, or the amino acid sequence set forth in SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 44.
The modified fibroin according to the present embodiment can be produced by an ordinary method using a nucleic acid encoding the modified fibroin. The nucleic acid encoding the modified fibroin may be chemically synthesized based on base sequence information, or may be synthesized using a PCR method or the like. Isolation and purification of the produced modified fibroin can be performed by a commonly used method.
(Agent for Imparting a Cool Touch Feeling Property and a Water-Absorbing and Quick-Drying Property)
The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment may contain one type of modified fibroins alone or a combination of two or more types of modified fibroins.
The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment may have a heat flow peak value (q max, W/cm2) evaluated by a contact cooling sensation measuring instrument of 0.055 W/cm2 or more, 0.060 W/cm2 or more, 0.065 W/cm2 or more, or 0.070 W/cm2 or more. The upper limit of the heat flow peak value is not particularly limited, but is usually 0.2 W/cm2 or less. The heat flow peak value can be evaluated, for example, by a method described in Examples described later.
The water-absorbing property of the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment as evaluated in accordance with JIS L 1907 may be 60 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, or 5 seconds or less. The water-absorbing property can be evaluated, for example, by a method described in Examples described later.
The quick-drying property of the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment as evaluated through measurement of the diffusible residual moisture content may be 100 minutes or less, 90 minutes or less, 80 minutes or less, or 70 minutes or less. The diffusible residual moisture content (%) is a value calculated by the following equation. The diffusible residual moisture content (%)=weight of water (g) at each time/weight of water (g) at the start of measurement×100 quick-drying property means a time required until the diffusible residual moisture content becomes 10% or less. The quick-drying property can be evaluated, for example, by a method described in Examples described later.
The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment may further contain other additives (components other than the active ingredient) according to its form, application, and the like. Examples of the additive include a plasticizer, a leveling agent, a crosslinking agent, a nucleating agent, an antioxidant, an ultraviolet absorber, a coloring agent, a filler, and a synthetic resin. The content of the additive can be set within a range in which the effect of the present invention is not impaired.
The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment may be in any form of, for example, a powder, a paste, and a liquid (for example, a suspension or solution). The agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment may also be in the form of, for example, a fiber, a film, a gel, a porous body, a particle, or the like. The form of the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment may be appropriately set according to the types and uses of the article, the object to which a cool touch feeling property and a water-absorbing and quick-drying property are imparted.
Since the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment contains a modified fibroin as a main component, the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property can be formed into any form described above. The formed body may be formed of a modified fibroin itself, or may be formed of a combination of a modified fibroin and another material.
When the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment is prepared in the form of a powder, for example, a protein obtained by the above-described method for producing a modified fibroin may be dried into a powder. The protein powder may contain other additives as necessary.
When the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment is prepared in the form of a liquid (for example, a solution), for example, a protein obtained by the above-described method for producing a modified fibroin may be dissolved in a solvent that can dissolve a modified fibroin to obtain a liquid (modified fibroin solution). The modified fibroin solution may contain other additives as necessary. Examples of the solvent that can dissolve a modified fibroin include dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP). An inorganic salt may be added to the solvent as a dissolution promoter.
When the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment is prepared in the form of a fiber, for example, the modified fibroin solution may be used as a dope solution, and the dope solution is spun into fibers (modified fibroin fibers) by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning. The form of the fiber may be a single yarn, may be a composite yarn such as a blended yarn, a combined filament yarn, a union yarn, a mix-weaved yarn, a piled yarn, and a covering yarn, or may be a nonwoven fabric or the like.
The modified fibroin fiber may be a short fiber or a long fiber. The modified fibroin fiber may also be a modified fibroin fiber alone or may be combined with other fibers. That is, a single yarn composed of only modified fibroin fibers, or a composite yarn composed of a combination of modified fibroin fibers and other fibers may be used singly or in combination. The single yarn and the composite yarn may be spun yarns in which short fibers are twisted, or may be filament yarns in which long fibers are twisted or not twisted. The modified fibroin fiber may be either a short fiber or a long fiber, and may be used as a fiber alone or in combination with other fibers without being processed into a yarn. Examples of the other fibers include synthetic fibers such as nylon and polyester, regenerated fibers such as cupra and rayon, and natural fibers such as cotton, hemp, animal hair fibers, and silk. When the modified fibroin fiber is used in combination with other fibers, the content of the modified fibroin fiber is preferably 20 mass % or more, more preferably 30 mass % or more, still more preferably 40 mass % or more, and even still more preferably 50 mass % or more, based on the total amount of fibers.
When the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment is prepared in the form of a film, a gel, a porous body, a particle, or the like, the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property can be produced, for example, in accordance with the methods described in JP 2009-505668 A, JP 2009-505668 A, JP 5678283 B2, JP 4638735 B2, and the like.
[Method of Imparting a Cool Touch Feeling Property and a Water-Absorbing and Quick-Drying Property to an Article]
The method of imparting a cool touch feeling property and a water-absorbing and quick-drying property to an article according to the present embodiment includes the step of incorporating a modified fibroin into the article. Since the modified fibroin according to the present invention has both a cool touch feeling property and a water-absorbing and quick-drying property, it is possible to impart a cool touch feeling property and a water-absorbing and quick-drying property to an article by incorporating the modified fibroin into the article.
The article is not particularly limited as long as it is an article to which a cool touch feeling property and a water-absorbing and quick-drying property should be imparted. Specific examples thereof include fibers, woven fabrics, knitted fabrics, nonwoven fabrics, cotton, sponges, films, resins, composite materials (the form of the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present embodiment is not limited), and various articles produced using these materials.
The step of incorporating the modified fibroin may be a step of incorporating the modified fibroin by blending the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property according to the present invention described above. The method for incorporating the modified fibroin is not particularly limited, and may be a method of mixing the modified fibroin with the material (raw material), or a method of forming an article by combining the agent for imparting a cool touch feeling property and a water-absorbing and quick-drying property prepared in the form of the above-described formed body with another material (formed body or the like). Alternatively, a method of forming an article (formed body) by forming a modified fibroin itself (other additives may be included as necessary) may be used.
The content of the modified fibroin in the article is preferably 20 mass % or more, more preferably 30 mass % or more, still more preferably 40 mass % or more, and even still more preferably 50 mass % or more, based on the total amount of the article. The upper limit of the content of the modified fibroin may be 100 mass % or 90 mass % or less, based on the total amount of the article. By adjusting the content of the modified fibroin in the article, the cool touch feeling property and the water-absorbing and quick-drying property of the article can be controlled. That is, since the modified fibroin according to the present invention has both a cool touch feeling property and a water-absorbing and quick-drying property, the cool touch feeling property and the water-absorbing and quick-drying property of the article can be further enhanced as the content of the modified fibroin in the article is increased.
The present invention described above can also be regarded as use of a modified fibroin for imparting a cool touch feeling property and a water-absorbing and quick-drying property to an article, including the step of incorporating the modified fibroin into the article.
Hereinafter, the present invention will be described more specifically based on Examples and the like. However, the present invention is not limited to the following Examples.
(1) Production of Expression Vector
A modified fibroin having the amino acid sequence set forth in SEQ ID NO: 40 (PRT966) and a modified fibroin having the amino acid sequence shown in SEQ ID NO: 15 (PRT799) were designed. A nucleic acid encoding the designed modified fibroin was synthesized. The nucleic acid had an NdeI site added at the 5′ end and an EcoRI site added downstream from the stop codon. The nucleic acid was cloned in a cloning vector (pUC118). Thereafter, the nucleic acid was enzymatically cleaved by treatment with NdeI and EcoRI, and then recombined into a protein expression vector pET-22b(+) to obtain an expression vector.
(2) Expression of Protein
Escherichia coli BLR (DE3) was transformed with the obtained expression vector. The transformed Escherichia coli was cultured in 2 mL of an LB culture medium containing ampicillin for 15 hours. The culture solution was added to a 100 mL seed culture medium containing ampicillin (Table 4) so that OD600 was 0.005. The temperature of the culture solution was maintained at 30° C., and the flask culture was performed (for about 15 hours) until the OD600 reached 5, thus obtaining a seed culture solution.
The seed culture solution was added to a jar fermenter to which a 500 mL production culture medium (Table 5) was added so that OD600 was 0.05. The culture was performed while maintaining the culture solution temperature at 37° C. and constantly controlling the pH to 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
Immediately after glucose in the production culture medium was completely consumed, a feed solution (455 g/1 L of glucose, 120 g/1 L of Yeast Extract) was added at a rate of 1 mL/min. The culture was performed while maintaining the culture solution temperature at 37° C. and constantly controlling the pH to 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was performed for 20 hours. Thereafter, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the modified fibroin. The culture solution was centrifuged 20 hours after addition of IPTG, and bacterial cells were recovered. SDS-PAGE was conducted using the bacterial cells prepared from the culture solutions obtained before the addition of IPTG and after the addition of IPTG. The expression of the target modified fibroin which depended on the addition of IPTG was confirmed by the appearance of a band of the size of the target modified fibroin.
(3) Purification of Protein
The bacterial cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer solution (pH 7.4). The bacterial cells after washing were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until the purity of the precipitate became high. The precipitate after washing was suspended in a 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so that the concentration of the precipitate was 100 mg/mL, and dissolved by stirring with a stirrer for 30 minutes at 60° C. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32, manufactured by Sanko Junyaku Co., Ltd.). A white aggregated protein obtained after dialysis was recovered by centrifugation, and moisture was removed in a lyophilizer to recover lyophilized powder, thereby obtaining modified fibroins (PRT966 and PRT799).
PRT966 is a hydrophobic modified fibroin having an average HI of more than 0. PRT799 is a hydrophilic modified fibroin having an average HI of 0 or less.
(4) Production of Modified Fibroin Fiber
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0 mass % was prepared as a solvent, and a lyophilized powder of the modified fibroin was added thereto so as to have a concentration of 24 mass %, and dissolved for 3 hours using a shaker. Thereafter, insoluble matters and foams were removed to obtain a modified fibroin solution (spinning dope).
The prepared spinning dope at 60° C. was filtrated with a metal filter having an opening of 5 μm. Thereafter, the filtrate was allowed to stand in a 30 mL-stainless steel syringe to remove foams. The resulting spinning dope was discharged from a solid nozzle having a needle diameter of 0.2 mm into a 100 mass % methanol coagulation bath. The discharge temperature was 60° C. After completion of the coagulation, the obtained original yarn was wound up, and naturally dried to obtain a modified fibroin fiber (raw material fiber).
(1) Production of Knitted Fabric
Knitted fabrics were produced by weft-knitting respective raw material fibers prepared in Test Example 1 by using a weft-knitting machine. For comparison, commercially available silk fibers, cotton fibers, and polyester fibers were prepared as raw material fibers.
The knitted fabric formed by using PRT966 fibers as raw material fibers had a fineness of 1/30 Nm (metric count, single yarn) and a gauge number of 18. The knitted fabric formed by using PRT799 fibers as raw material fibers had a fineness of 1/30 Nm (metric count, single yarn) and a gauge number of 16. The fineness and gauge number of each of the knitted fabrics formed by using other raw material fibers were adjusted so as to have a cover factor approximately the same as those of the knitted fabrics using PRT966 fibers and PRT799 fibers. Details are as follows. Silk fineness: 2/60 Nm (two folded yarn), gauge number: 14; Cotton fineness: 2/34 Nm (two folded yarn), gauge number: 14; Polyester fineness: 1/60 Nm (single yarn), gauge number: 14
(2) Evaluation of Water-Absorbing Property
The water-absorbing property was evaluated by a test in accordance with JIS L 1907 (Testing methods for water-absorbing properties of textiles/dropping method). Specifically, under a standard environment (temperature 20±2° C./humidity 65±4% RH), one drop of water was dropped from the burette onto the surface of the knitted fabric prepared above, and the time until the dropped water droplet was absorbed by the knitted fabric (specular reflection disappeared) was measured (maximum measurement time was 60 seconds). The results are shown in Table 6.
(3) Evaluation of Quick-Drying Property
The quick-drying property was evaluated by measuring the diffusible residual moisture content. Specifically, under a standard environment (temperature 20±2° C./humidity 65±4% RH), 0.6 mL of tap water was dropped onto the back side of the knitted fabric, and the weight of water was determined by measuring the weight of the knitted fabric at every lapse of a certain time (every 5 minutes), and the diffusible residual moisture content was calculated by the following equation. The measurement was performed until the diffusible residual moisture content (%)=weight of water (g) at each time/weight of water (g) at the start of measurement×100, the diffusible residual moisture content reached 10% or less (that is, the weight of water reached 10% of 0.6 mL=60 μL (60 mg)), and the time required until the diffusible residual moisture content reached 10% was determined. The results are shown in Table 6.
It can be understood that the modified fibroins (PRT799 and PRT966) are excellent in a water-absorbing and quick-drying property. In particular, it can be understood that the hydrophilic modified fibroin (PRT799) is excellent in both a water-absorbing property and a quick-drying property.
(1) Production of Knitted Fabric
Knitted fabrics were produced by weft-knitting respective raw material fibers prepared in Test Example 1 by using a weft-knitting machine. For comparison, commercially available wool fibers, cashmere fibers, silk fibers, cotton fibers, rayon fibers, and polyester fibers were prepared as a raw material fiber.
The knitted fabric formed by using PRT966 fibers as raw material fibers had a fineness of 1/30 Nm (metric count, single yarn) and a gauge number of 18. The knitted fabric formed by using PRT799 fibers as raw material fibers had a fineness of 1/30 Nm (metric count, single yarn) and a gauge number of 16. The fineness and gauge number of each of the knitted fabrics formed by using other raw material fibers were adjusted so as to have a cover factor approximately the same as those of the knitted fabrics formed by using PRT966 fibers and PRT799 fibers. Details are as follows.
Wool fineness: 2/30 Nm (two folded yarn), gauge number: 14
Cashmere fineness: 1/32 Nm (single yarn), gauge number: 14
Silk fineness: 2/60 Nm (two folded yarn), gauge number: 14
Cotton fineness: 2/34 Nm (two folded yarn), gauge number: 14
Rayon fineness: 1/38 Nm (single yarn), gauge number: 14
Polyester fineness: 1/60 Nm (single yarn), gauge number: 14
(2) Evaluation of Cool Touch Feeling Property
Knitted fabrics were cut into test pieces having a size of 10 cm×10 cm (samples), which were left to stand under a test environment (temperature: 20±2° C., relative humidity: 65±4%) for 4 hours or more. Thereafter, using a contact cooling sensation measuring instrument (KES-F7 Thermolab type II, manufactured by KATO TECH CO., LTD.) set at 30° C., the sensor was laid on the hot plate to make the sensor temperature constant, then the sensor was brought into contact with the back surface of test pieces, and the heat flow peak value (q max, W/cm2) of each test piece was measured under the condition of a temperature difference (ΔT) from the test piece of 10° C. The test piece having a larger heat flow peak value has a larger amount of heat transfer from the contact object (skin or the like) to the test piece, and has a higher cool touch feeling property. The results are shown in Table 7.
It can be understood that the modified fibroins (PRT799 and PRT966, in particular, PRT966) are excellent in a cool touch feeling property.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-179937 | Sep 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/036538 | 9/28/2020 | WO |
