modified fibroin, nucleic acid, expression vector, host, product, artificially modified fibroin fibe

专利摘要:
The present invention relates to modified fibroin containing a domain sequence represented by formula 1: [motif (A) n-REP] m or formula 2: [motif (A) n-REP] m- (A) n, being that the domain sequence has an amino acid sequence having a reduced content of glutamine residues in association with the deletion of one or multiple glutamine residues in REP or the replacement of one or multiple glutamine residues in REP with other amino acid residues. [In formula 1 and formula 2, motif (A) n represents an amino acid sequence composed of 4 to 27 amino acid residues, the ratio of the number of alanine residues to the total number of amino acid residues in motif ( A) n is 80% or more; REP represents an amino acid sequence composed of 10 to 200 amino acid residues; m represents an integer from 10 to 300; and multiple parts of (A) n or similar motifs may mutually have the same amino acid sequence or may have mutually different amino acid sequences.]
公开号:BR112020001627A2
申请号:R112020001627-6
申请日:2018-07-25
公开日:2020-07-21
发明作者:Keisuke Morita；Yunosuke Abe；Takehisa Maekawa；Koichi Kotaka；Junichi Sugahara；Keiji Numata
申请人:Spiber Inc.；Riken；
IPC主号:

专利说明:

[001] [001] The present invention relates to a modified fibroin. More particularly, the present invention relates to a modified fibroin with a reduced content of glutamine residue. The present invention also relates to a nucleic acid encoding a modified fibroin, an expression vector including the nucleic acid sequence, a host transformed with the expression vector, and a product made from a modified fibroin. Prior art
[002] [002] Fibroin is a fibrous protein and contains up to 90% residues of glycine, alanine and serine resulting in the formation of a β-pleated leaf (Non-Patent Literature 1). Proteins (silk proteins, hornet silk proteins, and spider silk proteins) and the like making up the thread produced by insects and spiders are known as fibroin.
[003] [003] Silk proteins have excellent mechanical properties, hygroscopic properties and deodorant properties and are widely used as raw materials for garments. In addition, silk thread is a natural immunotolerant fiber and has high biocompatibility, and is therefore also used for surgical sutures.
[004] [004] There are up to seven types of spider silk glands, each
[005] [005] As a specific structure for fibroin, a structure is known in which motifs of amino acids classified as GPGXX, a region rich in alanine residues ((A) n or (GA) n), GGX, and a spacer are repeated ( Non-Patent Literature 2). In addition, it has been reported that replacing motif (GA) n with motif (A) n results in reduced elongation but increased tensile strength, an increase in the number of GPGXX motifs results in increased elongation, and the replacement of several motifs GPGXX for reasons (A) n results in increased tensile strength (Patent Literature 2). In addition, the GGX and GPGXX motifs are considered to have a flexible helical structure that provides elasticity to the thread (Patent Literature 3).
[006] [006] Recombinant spider silk proteins and recombinant silk proteins are produced in various heterologous protein production systems. For example, many cases of recombinant fibroin production have been reported by organisms such as goat, silkworm, plant, mammalian cell, yeast, fungus, gram-negative bacteria, and gram-positive bacteria, and certain results have been reported. obtained. (Non-Patent Literature 3, Patent Literature 4 and 5).
[007] [007] A fiber obtained from spinning has the property of shrinkage when immersed in water or hot water, exposed to a high humidity environment, or the like. This property causes problems in the manufacturing process and production, and also affects the product made from the fiber.
[008] [008] As an anti-shrinkage method to prevent product shrinkage, for example, a method of preventing the shrinking of a silk fabric is described, in which the silk fabric using a strong twisted fiber that has been washed is immersed in water, another solvent, or a mixed solvent thereof in a state of tension and heated for a predetermined time (Patent Literature 6), a method for fixing a shape of an animal fiber product, in which the animal fiber product molded into a required shape is subjected to a treatment of being placed in contact with a high pressure saturated water vapor at 120 ° C to 200 ° C to fix the shape at the time of water vapor treatment (Patent Literature 7 ), and the like.
[009] [009] [Patent Literature 1] Unexamined Japanese Patent Publication No. 2012-55269, [Patent Literature 2] Unexamined Japanese Patent Publication No. 2005-502347, [Patent Literature 3] Unexamined Japanese Patent Publication No. 2009-505668, [Patent Literature 4] Unexamined Japanese Patent Publication No. 2014-502140, [Patent Literature 5] International Patent Publication No. WO2015 / 042164, [Patent Literature 6] Japanese Patent Publication
[0010] [0010] [Non-Patent Literature 1] Asakura et al., “Encyclopedia of Agricultural Science”, Academic Press: New York, NY, 1994, Vol. 4, pp. 1-11, [Non-Patent Literature 2] Microbial Cell Factories, 2004, 3:14, [Non-Patent Literature 3] Science, 2002, Vol. 295, pp. 472-476.
[0011] [0011] Anti-shrinkage methods, as described in Patent Literature 6 and 7, are, on the one hand, industrially disadvantageous because the operation is complicated and the number of steps is increased. On the other hand, it would be industrially useful if the shrinkage of the fiber itself could be suppressed or reduced. However, to date, no fibroin fiber is known to have suppressed or reduced shrinkage, or a modified fibroin constituting the fibroin fiber.
[0012] [0012] An objective of the present invention is to provide a modified fibroin capable of spinning a fibroin fiber with reduced shrinkage. Means for solving problems
[0013] [0013] The present inventors have found that a fibroin fiber with reduced shrinkage can be obtained by reducing the content of glutamine residue present in fibroin. In addition, the present inventors have found that the film obtained by molding fibroin is impermeable to water. The present invention has been completed based on
[0014] That is, the present invention relates, for example, to each of the following inventions.
[1] [1] A modified fibroin including a domain sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m-motif (A) n, in which the domain sequence has an amino acid sequence with a reduced content of an amino acid equivalent of glutamine residue in which one or a plurality of glutamine residues in REP are deleted or replaced by other amino acid residues, compared to the occurring fibroin Natural. [In Formula 1 and Formula 2, motif (A) n represents an amino acid sequence consisting of 4 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in motif (A) n is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer from 10 to 300. A plurality of motifs (A) n can be the same amino acid sequence or different amino acid sequences. A plurality of REPs can be the same amino acid sequence or different amino acid sequences.]
[2] [2] Fibroin modified according to aspect [1], in which the REP contains a GPGXX motif (where X represents an amino acid residue other than a glycine residue) and has a GPGXX motif content of 10% or more .
[3] [3] Fibroin modified according to aspect [1] or [2], in which a glutamine residue content rate is 9% or less.
[4] [4] Fibroin modified according to any aspect [1] to [3], in which the other amino acid residues are amino acid residues selected from the group consisting of isoleucine (I), valine (V), leucine (L ), phenylalanine (F), cysteine (C), methionine (M), alanine (A), glycine
[5] [5] Fibroin modified according to any aspect [1] to [4], in which a REP hydrophobicity is -0.8 or more.
[6] [6] Fibroin modified according to any aspect [1] to [5], including, in addition, an amino acid sequence equivalent to an amino acid sequence in which one or a plurality of amino acid residues are replaced, deleted, inserted and / or added compared to naturally occurring fibroin.
[7] [7] Fibroin modified according to aspect [6], in which the naturally occurring fibroin is a fibroin derived from insects or spiders.
[8] [8] Fibroin modified according to aspect [7], in which naturally occurring fibroin is a major ampule gland araneid protein (MaSp) or spider minor ampule gland araneid protein (MiSp).
[9] [9] A modified fibroin including: an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, or SEQ ID NO: 17; or an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 16, or SEQ ID NO: 17.
[10] [10] Fibroin modified according to either aspect [1] to [9], additionally including a tag sequence at either or both of an N-terminus and a C-terminus.
[11] [11] Fibroin modified according to aspect [10], in which the tag sequence includes an amino acid sequence shown in SEQ ID NO: 7.
[12] [12] A modified fibroin including: a sequence of
[13] [13] A nucleic acid encoding modified fibroin according to aspects [1] to [12.]
[14] [14] A nucleic acid that hybridizes to a complementary strand of the nucleic acid according to aspect [13] under stringent conditions and that encodes a modified fibroin including a domain sequence represented by Formula 1: [motif (A) n-REP ] m or Formula 2: [reason (A) n-REP] m-reason (A) n. [In Formula 1 and Formula 2, motif (A) n represents an amino acid sequence consisting of 4 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in motif (A) n is 80% or more, REP represents an amino acid sequence consisting of 10 to 200 amino acid residues, m represents an integer from 10 to 300, a plurality of reasons (A) n can be the same amino acid sequence or different sequences of amino acids, and a plurality of REPs can be the same amino acid sequence or different amino acid sequences.]
[15] [15] A nucleic acid having 90% or more sequence identity to the nucleic acid according to aspect [13] and which encodes a modified fibroin including a domain sequence represented by Formula 1: [motif (A) n- REP] m or Formula 2: [reason (A) n-REP] m-reason (A) n. [In Formula 1 and Formula 2, motif (A) n represents an amino acid sequence consisting of 4 to 27 amino acid residues and the
[16] [16] An expression vector including: the nucleic acid sequence according to any aspect [13] to [15]; and one or a plurality of regulatory sequences functionally linked to the nucleic acid sequence according to any of the aspects [13] to [15.]
[17] [17] The expression vector according to aspect [16], which is a plasmid vector or a viral vector.
[18] [18] A host transformed with the expression vector according to aspect [16] or [17.]
[19] [19] The host according to aspect [18], which is a prokaryote.
[20] [20] The host according to aspect [19], in which the prokaryote is a microorganism belonging to a genus selected from the group consisting of Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
[21] [21] The host according to aspect [18], which is a eukaryote.
[22] [22] The host according to aspect [21], in which the eukaryote is a yeast, a filamentous fungus, or an insect cell.
[23] [23] The host according to aspect [22], in which yeast is a yeast belonging to a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia, and Hansenula.
[24] [24] The host according to aspect [23], in which the yeast belonging to a genus Saccharomyces is Saccharomyces cerevisiae, the yeast belonging to a genus Schizosaccharomyces is Schizosaccharomyces pombe, the yeast belonging to a genus Kluyveromyces is Kluyveromyces and Kluyveromyces lactis yeast belonging to a genus Trichosporon is Trichosporon pullulans, yeast belonging to a genus Schwanniomyces is Schwanniomyces alluvius, the yeast belonging to a genus Pichia is Pichia pastoris, the yeast belonging to a genus Candida is Candida albicans, the yeast belonging to a genus Yarrowia is Yarrowia lipolytica, and the yeast belonging to a genus Hansenula is Hansenula polymorpha.
[25] [25] The host according to aspect [22], in which the filamentous fungus is a filamentous fungus belonging to a genus selected from the group consisting of Aspergillus, Penicillium, and Mucor.
[26] [26] The host according to aspect [25], in which the filamentous fungus belonging to an Aspergillus genus is Aspergillus oryzae, the filamentous fungus belonging to a Penicillium genus is Penicillium chrysogenum, and the filamentous fungus belonging to a Mucor genus is Mucor fragilis.
[27] [27] The host according to aspect [22], in which the insect cell is a lepidopteran insect cell.
[28] [28] The host according to aspect [27], in which the insect cell is an insect cell derived from Spodoptera frugiperda or an insect cell derived from Trichoplusia ni.
[29] [29] A product including fibroin modified according to any aspect [1] to [12], the product being selected from the group consisting of a fiber, a thread, a film, a foam, a grain, a nanofibril, a gel, and a resin.
[0015] [0015] Furthermore, the present invention also refers to
[30] [30] An artificially modified fibroin fiber including a modified fibroin, in which the artificially modified fibroin fiber elongates when moistened and shrinks when dried from the moistened state.
[31] [31] Fibroin fiber artificially modified according to aspect [30], in which a restoration rate defined by Expression (1) is 95% or more. restoration rate = (length of artificially modified fibroin fiber when dried from the wet state / length of artificially modified fibroin fiber before being wetted) × 100 (%) --- (1)
[32] [32] The artificially modified fibroin fiber according to aspect [30] or [31], in which the artificially modified fibroin fiber is a fiber having a history of irreversibly shrinking being shrunk by contact with water after spinning and a shrinkage rate A defined by Expression (2) of 2% or more. shrinkage rate A = {1 - (fiber length irreversibly shrunk by contact with water after spinning / fiber length before contact with water and after spinning)} × 100 (%) --- (2)
[33] [33] The artificially modified fibroin fiber according to any of the aspects [30] to [32], in which the artificially modified fibroin fiber is a fiber having a history of irreversibly shrinking being shrunk by contact with water after the spinning and then additionally being shrunk by drying and a shrinkage rate B defined by Expression (3) of greater than 7%. shrinkage rate B = {1 - (fiber length irreversibly shrunk by contact with water after spinning and then
[34] [34] Fibroin fiber artificially modified according to any aspect [30] to [33], in which the modified fibroin is fibroin modified according to any aspect [1] to [12.]
[35] [35] Fibroin fiber artificially modified according to any aspect [30] to [34], in which an elongation rate defined by Expression (4) is 17% or less. elongation rate = {(length of artificially modified fibroin fiber when moistened / length of artificially modified fibroin fiber before being moistened) - 1} × 100 (%) --- (4)
[36] [36] Fibroin fiber artificially modified according to any aspect [30] to [35], in which a shrinkage rate C defined by Expression (5) is 15% or less. shrinkage rate C = {1 - (length of artificially modified fibroin fiber when dried from the wet state / length of artificially modified fibroin fiber when wet)} × 100 (%) --- (5)
[37] [37] A method for producing an artificially modified fibroin fiber, including a shrinkage step of placing a raw fiber, before contact with water and after spinning, in contact with water to cause irreversible shrinkage, and then drying the raw fiber to cause additional shrinkage, in which the raw fiber includes a modified fibroin.
[38] [38] The production method according to aspect [37], in which a raw fiber A shrinkage rate defined by Expression (2) is 2% or more. shrinkage rate A = {1 - (fiber length
[39] [39] The production method according to aspect [37] or [38], in which a raw fiber shrinkage rate B defined by Expression (3) is greater than 7%. shrinkage rate B = {1 - (fiber length irreversibly shrunk by contact with water after spinning and then additionally shrunk by drying / fiber length before contact with water and after spinning)} × 100 (%) - - (3)
[40] [40] The production method according to aspects [37] to [39], in which the modified fibroin is the modified fibroin according to any of the aspects [1] to [12.] EFFECTS OF THE INVENTION
[0016] [0016] According to the present invention, it is possible to provide a modified fibroin capable of spinning a fibroin fiber with reduced shrinkage. In addition, it is possible to provide a modified fibroin capable of producing a fibroin film having reduced water absorbance and water impermeability.
[0017] [0017] Furthermore, according to the present invention, it is possible to provide an artificially modified fibroin fiber in which the degree of elongation is approximately the same as the degree of shrinkage (restoration rate is close to 100%) and which has the characteristic of being able to restore itself to its original length, and a method for producing it, in a case in which the size of the same changes (elongation and shrinkage) due to contact with water and subsequent drying. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] [0018] Fig. 1 is a schematic diagram showing a
[0019] [0019] Hereinafter, the modalities for carrying out the present invention will be described in detail. However, the present invention is not limited by the following modalities. [Modified fibroin]
[0020] [0020] The modified fibroin according to the present invention is a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m- reason (A) n. In the modified fibroin, an amino acid sequence (N-terminal sequence and C-terminal sequence) can be additionally added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence, although not limited to them, are typically regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of amino acids of about 100 residues.
[0021] [0021] The term "modified fibroin" as used in the present invention means a fibroin whose domain sequence is different from the naturally occurring amino acid sequence of fibroin. The "naturally occurring fibroin" referred to in the present invention is also a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m- reason (A) n.
[0022] [0022] The "modified fibroin" can be a fibroin whose amino acid sequence has been modified based on the occurring fibroin
[0023] [0023] The term "domain sequence", as used in the present invention, refers to an amino acid sequence that produces a crystalline region (typically equivalent to motif (A) n of an amino acid sequence) and an amorphous region (typically equivalent to the REP of an amino acid sequence) peculiar to fibroin and means an amino acid sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m -motive (A) n. Here, motif (A) n represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues with respect to the total number of amino acid residues in motif (A) n is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer from 10 to 300. A plurality of motifs (A) n can be the same amino acid sequence or different amino acid sequences. A plurality of REPs can be the same amino acid sequence or different amino acid sequences.
[0024] [0024] Reason (A) n may be such that the number of alanine residues is 80% or more with respect to the total number of amino acid residues in reason (A) n, but is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (which means that motif (A) does not consist only of alanine residues). It is preferable that at least seven of
[0025] [0025] Fibroin modified in accordance with the present modality may have an amino acid sequence with a reduced content of glutamine residue, compared to naturally occurring fibroin. Since the modified fibroin according to the present modality has a reduced content of glutamine residues, the fibroin fiber obtained by spinning the modified fibroin has reduced shrinkage. In addition, the film obtained by molding fibroin is impermeable to water.
[0026] [0026] It is preferable that the fibroin modified according to the present modality includes at least one motif selected from the GGX motif or the GPGXX motif (where G represents a glycine residue, P represents a phenylalanine residue, and X represents a residue amino acid other than a glycine residue) in the REP amino acid sequence. Since these motifs are included in REP, the elasticity of the modified fibroin can be improved.
[0027] [0027] In a case in which the fibroin modified according to the present modality includes a GPGXX motif in REP, a rate of GPGXX motif content is usually 1% or more, can be 5% or more, and is preferably 10% or more. With this configuration, the elasticity of the modified fibroin can be further improved. The upper limit of the GPGXX motive content rate is not particularly limited, it can be 50% or less and it can be 30% or less.
[0028] [0028] In this specification, the “GPGXX motive content rate” is a value calculated by the following method.
[0029] [0029] For the calculation of the GPGXX motif content rate, the "sequence excluding a motif sequence (A) n located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is used to exclude the effect that occurs due to the fact that the "motif sequence (A) n located on the most C-terminal side to the C terminal of the domain sequence" (sequence equivalent to REP) can include a sequence that is not correlated with the characteristic sequence of fibroin, which influences the result of calculating the rate of motif content GPGXX in a case in which m is small (that is, in a case in which the domain sequence is short). In a case where a “GPGXX motif” is located at the C-terminal of REP, it is treated as the “GPGXX motif” even if “XX” is, for example, “AA”.
[0030] [0030] Fig. 1 is a schematic diagram showing a domain sequence of a modified fibroin. The method of calculating the GPGXX motif content rate will be specifically described with reference to Fig. 1. First, in a modified fibroin domain sequence shown in Fig. 1 (type [motif (A) n-REP] m- reason (A) n]), since all
[0031] [0031] In the modified fibroin according to the present embodiment, a glutamine residue content rate is preferably 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%. With this configuration, the effect of the present invention is still notably presented.
[0032] [0032] In this specification, the “glutamine residue content rate” is a value calculated by the following method. In a fibroin including a domain sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m-motif (A) n, in a case in which the total number of glutamine residues in all REPs included in a sequence (sequence equivalent to “region A” in Fig. 1) excluding motif sequence (A) n located on the C-terminal side to the C-terminal side of the domain from the domain sequence is defined as w, and the total number of amino acid residues in all REPs excluding the motif sequence
[0033] The modified fibroin domain sequence according to the present embodiment can include an amino acid sequence equivalent to an amino acid sequence in which one or a plurality of glutamine residues in REP are deleted or replaced by other amino acid residues, compared to naturally occurring fibroin.
[0034] [0034] The "other amino acid residue" may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a hydropathy index greater than that of a glutamine residue. Regarding the hydropathy index of amino acid residues, known indexes (“Hydropathy Index”: Kyte J, & Doolittle R (1982) from “A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. 105-132) can be used as a reference. Specifically, the hydropathy index (hereinafter also called “IH”) for each amino acid is as shown in Table 1 below. [Table 1] IH Amino Acid IH Amino Acid Isoleucine (I) 4.5 Tryptophan (W) -0.9 Valine (V) 4.2 Tyrosine (Y) -1.3 Leucine (L) 3.8 Proline (P) - 1.6 Phenylalanine (F) 2.8 Histidine (H) -3.2 Cysteine (C) 2.5 Asparagine (N) -3.5 Methionine (M) 1.9 Aspartic acid (D) -3.5
[0035] [0035] As shown in Table 1, amino acid residues having a hydropathy index higher 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 these, 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 is more preferable an amino acid residue selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
[0036] [0036] In a modified fibroin according to the present embodiment, the hydrophobicity of REP is preferably -0.8 or more, more preferably -0.7 or more, even more preferably 0 or more, still much more preferably 0 , 3 or more, and particularly preferably 0.4 or more. The upper limit of REP hydrophobicity is not particularly limited, it can be 1.0 or less, and it can be 0.7 or less.
[0037] [0037] In this specification, “REP hydrophobicity” is a value calculated by the following method.
[001] [001] In a fibroin including a domain sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m-motif (A) n, in one case in which the sum of the hydropathy indices of each amino acid residue in all REPs included in a sequence (sequence equivalent to “region A” in Fig. 1) excluding the sequence of motif (A) n located on the most C-terminal side to the C-terminal of the domain string from the domain string is defined as z, and the total number
[0038] [0038] Naturally occurring fibroin is a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m-motif (A) n, specifically, for example, a fibroin produced by insects or spiders.
[0039] [0039] Examples of fibroin 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 hornet silk proteins discharged by Vespa simillima xanthoptera larvae.
[0040] [0040] A more specific example of fibroin produced by insects includes a silkworm fibroin L chain (GenBank Accession No. M76430 (sequence of bases), AAA27840.1 (sequence of amino acids)).
[0041] [0041] Examples of fibroin produced by spiders 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 na Neoscona na Neoscona na , Neoscona advances and Neoscona scylloides, spiders belonging to the genus Pronus as Pronous minutes, spiders belonging to the genus Cyrtarachne as Cyrtarachne
[0042] [0042] More specific examples of fibroin produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (AAC47010 GenBank Access Number (amino acid sequence),
[0043] [0043] As another specific example of naturally occurring fibroin, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, its sequences can be confirmed by extracting sequences in which spidroin, ampulla, fibroin, "silk and polypeptide", or "silk and protein" [spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein ”] is described as a DEFINITION keyword within the strings
[0044] [0044] 663 Types of fibroins (among them, 415 types of fibroins derived from spiders) were extracted by confirming fibroins with information on amino acid sequences registered in NCBI GenBank by the exemplified method. Among all fibroins, there were 129 types of naturally occurring fibroin including a domain sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m-motif ( A) n. Among these, there were six types of naturally occurring fibroin whose rate of motif content GPGXX calculated by the method described above is 10% or more, as shown in Table 2 below. The glutamine residue content rate of the six types of naturally occurring fibroin shown in Table 2 was 9.2% or more. [Table 2] Rate of reason content Rate of residue content Hydrophobicity Glutamine GPGXX Access Number REP DQ059135 22.13% 9.27% -1.08 AF350276 22.88% 9.30% -1.08 AF350278 26.28% 13.03% -1.31 NEPFIBPR 22.24% 12.83% -0.29 ADU47855 24.79% 19.21% -1.40 AB829892 14.54% 13.80% -0, 11
[0045] The modified fibroin domain sequence according to the present modality may additionally include an amino acid sequence equivalent to an amino acid sequence in which one or a plurality of amino acid residues are replaced, deleted, inserted and / or added, in addition to a modification corresponding to a modification in which one or a plurality of glutamine residues in REP are deleted and / or one or a plurality of glutamine residues in REP are replaced by other residues of
[0046] [0046] Fibroin modified in accordance with the present modality can be obtained, for example, with respect to a naturally occurring cloned fibroin gene sequence, by deleting one or a plurality of glutamine residues in REP and / or by replacement of one or a plurality of glutamine residues in REP with other amino acid residues. In addition, for example, the modified fibroin according to the present embodiment can also be obtained by designing an amino acid sequence equivalent to an amino acid sequence in which, with respect to the naturally occurring fibroin amino acid sequence, one or a plurality of glutamine residues in REP are deleted and / or one or a plurality of glutamine residues in REP are replaced by other amino acid residues, and by the chemical synthesis of a nucleic acid that encodes the drawn amino acid sequence.
[0047] [0047] A more specific example of the modified fibroin according to the present invention can be a modified fibroin containing (i) an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 21 or SEQ ID NO: 22, or (ii) an amino acid sequence having 90% or more identity of sequence with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 21 or SEQ ID NO: 22.
[0048] [0048] The modified fibroin from (i) will be described.
[0049] [0049] The amino acid sequence shown in SEQ ID NO: 1 (M_PRT410) is a modified amino acid sequence obtained by changing the number of conservative alanine residues in reason (A) n to five, or similar, in order to improve the productivity, based on the base sequence and the amino acid sequence of Nephila clavipes (GenBank Accession Number: P46804.1, GI: 1174415) which is naturally occurring fibroin. However, since M_PRT410 has no modification of glutamine residue (Q), the glutamine residue content rate of the glutamine is equal to the naturally occurring glutamine residue content of fibroin.
[0050] [0050] The amino acid sequence (M_PRT888) presented in SEQ ID NO: 2 is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with VL.
[0051] [0051] The amino acid sequence (M_PRT965) shown in SEQ ID NO: 3 is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with TS and replacing the remaining Q with A.
[0052] [0052] The amino acid sequence (M_PRT889) presented in SEQ ID NO: 4 is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with VL and replacing the remaining Q with I.
[0053] [0053] The amino acid sequence (M_PRT916) presented in SEQ ID NO: 5 is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with VI and replacing the remaining Q with L.
[0054] [0054] The amino acid sequence (M_PRT918) presented in SEQ ID NO: 6 is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with VF and replacing the remaining Q with I.
[0055] [0055] The amino acid sequence (M_PRT525) presented in SEQ ID NO: 15 is obtained, with respect to M_PRT410 (SEQ ID NO: 1), by inserting two alanine residues in a region (A5) in which the residues
[0056] [0056] The amino acid sequence (M_PRT699) presented in SEQ ID NO: 16 is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 15) with VL.
[0057] [0057] The amino acid sequence (M_PRT698) presented in SEQ ID NO: 17 is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 15) with VL and replacing the remaining Q with I.
[0058] [0058] The amino acid sequence (M_PRT917) presented in SEQ ID NO: 21 is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with LI and replacing the remaining Q with V.
[0059] [0059] The amino acid sequence (M_PRT1028) presented in SEQ ID NO: 22 is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with IF and replacing the remaining Q with T.
[0060] [0060] The glutamine residue content rate of any of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID NO: 22 is 9% or less (Table 3). [Table 3] Content rate of modified Fibroin Hydrophobicity content rate motif residue GPGXX REP glutamine M_PRT410 (SEQ ID NO: 1) 17.7% 27.9% -1.52 M_PRT888 (SEQ ID NO: 2) 6.3% 27.9% -0.07 M_PRT965 (SEQ ID NO: 3) 0.0% 27.9% -0.65 M_PRT889 (SEQ ID NO: 4) 0.0% 27.9% 0, 35 M_PRT916 (SEQ ID NO: 5) 0.0% 27.9% 0.47 M_PRT918 (SEQ ID NO: 6) 0.0% 27.9% 0.45 M_PRT525 (SEQ ID NO: 15) 13.7 % 26.4% -1.24 M_PRT699 (SEQ ID NO: 16) 3.6% 26.4% -0.78 M_PRT598 (SEQ ID NO: 17) 0.0% 26.4% -0.03 M_PRT917 (SEQ ID NO: 21) 0.0% 27.9% 0.46 M_PRT1028 (SEQ ID NO: 22) 0.0% 28.1% 0.05
[0061] [0061] Modified fibroin from (i) may consist of the sequence of
[0062] [0062] Modified fibroin from (ii) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 21 or SEQ ID NO: 22. Modified (ii) fibroin is also a protein including a sequence of domain represented by Formula 1: [reason (A) n-REP] m or Formula 2: [reason (A) n-REP] m-reason (A) n. The sequence identity is preferably 95% or more.
[0063] [0063] The modified fibroin of (ii) preferably has a glutamine residue content rate of 9% or less. In addition, the modified fibroin of (ii) preferably has a GPGXX motif content rate of 10% or more.
[0064] [0064] The modified fibroin described above can include a tag sequence at either one or both of the N-terminus and the C-terminus. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
[0065] The tag sequence can, for example, be an affinity tag that uses specific affinity (affinity, binding property) with another molecule. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are arranged and has a property to specifically bind to a metal ion, so that it can be used to isolate modified fibroin by chelation chromatography. metal. A specific example of the tag sequence can be an amino acid sequence shown in SEQ ID NO: 7 (amino acid sequence including the His tag).
[0066] [0066] In addition, a tag sequence such as glutathione-S-transferase (GST) that specifically binds to glutathione or a maltose-binding protein (MBP, Maltose Binding Protein) that specifically binds to maltose can also be used.
[0067] [0067] In addition, an "epitope tag" that uses an antigen-antibody reaction can also be used. By adding a peptide (epitope) that shows antigenicity as a tag sequence, an antibody against the epitope can be attached. Examples of the epitope tag include an HA tag (influenza virus hemagglutinin peptide sequence), a myc tag, and a FLAG tag. Modified fibroin can be easily purified with high specificity by using an epitope tag.
[0068] [0068] It is also possible to use a tag sequence that can be cleaved with a specific protease. By the protease treatment of a protein adsorbed by means of a tag sequence, it is also possible to recover the modified fibroin cleaved from the tag sequence.
[0069] [0069] A more specific example of the modified fibroin having a tag sequence may be a modified fibroin containing (iii) an amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24, or (iv) an amino acid sequence having 90% or more identity sequence with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, or SEQ ID NO: 24.
[0070] [0070] The amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20 , SEQ ID NO: 23 and SEQ ID NO: 24 are respectively amino acid sequences obtained by adding the sequence
[0071] [0071] The modified fibroin from (iii) can consist of the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24.
[0072] [0072] Modified fibroin from (iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24. Modified (iv) fibroin is also a protein including a sequence of domain represented by Formula 1: [reason (A) n-REP] m or Formula 2: [reason (A) n-REP] m-reason (A) n. The sequence identity is preferably 95% or more.
[0073] [0073] Modified fibroin from (iv) preferably has a glutamine residue content rate of 9% or less. In addition, the modified fibroin from (iv) preferably has a GPGXX motif content rate of 10% or more.
[0074] [0074] The modified fibroin, mentioned above, may include a secretory signal for the release of the protein produced in the system of production of recombinant proteins out of a host. The sequence of the secretory signal can be appropriately defined depending on the type of the host. [Nucleic acid]
[0075] [0075] The nucleic acid according to the present invention encodes the modified fibroin according to the present invention. Specific examples of nucleic acid include nucleic acids that encode a modified fibroin including an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 21 or SEQ ID NO: 22, or a protein or the like having an amino acid sequence (tag sequence) shown in SEQ ID NO: 7 linked to any one of or both of the N-terminus and the C-terminus of these amino acid sequences.
[0076] [0076] Nucleic acid, according to one embodiment, is a nucleic acid that hybridizes to a complementary strand of the nucleic acid that encodes the modified fibroin according to the present invention under stringent conditions and that encodes a modified fibroin including a sequence of domain represented by Formula 1: [reason (A) n-REP] m, or Formula 2: [reason (A) n-REP] m-reason (A) n. The modified fibroin encoded by the nucleic acid preferably has a glutamine residue content rate of 9% or less. In addition, the modified fibroin encoded by the nucleic acid preferably has the GPGXX motif content rate of
[0077] [0077] The term "stringent conditions" refers to the conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. The "stringent conditions" can be any of low stringent conditions, moderately stringent conditions and highly stringent conditions. Low stringent conditions mean that hybridization occurs only in the case where there is at least 85% or more of identity between the sequences, and include, for example, hybridization conditions at 42 ° C using SSC5 × containing 0.5% SDS . Moderately stringent conditions mean that hybridization occurs only in the case where there is at least 90% or more identity between the sequences, and include, for example, hybridization conditions at 50 ° C using SSC5 × containing 0.5% SDS . Highly stringent conditions mean that hybridization occurs only in the case where there is at least 95% or more identity between the sequences, and include, for example, hybridization conditions at 60 ° C using SSC5 × containing 0.5% SDS .
[0078] The nucleic acid according to another embodiment is a nucleic acid having 90% or more sequence identity with the nucleic acid encoding the modified fibroin in accordance with the present invention and encoding a modified fibroin including a domain sequence represented by Formula 1: [reason (A) n-REP] m, or Formula 2: [reason (A) n-REP] m-reason (A) n. The modified fibroin encoded by the nucleic acid preferably has a glutamine residue content rate of 9% or less. In addition, the modified fibroin encoded by the nucleic acid preferably has the GPGXX motif content rate of 10% or more. [Host and expression vector]
[0079] [0079] An expression vector, according to the present invention,
[0080] The host, according to the present invention, is a host that has been transformed with the expression vector according to the present invention. Both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, mammalian cells, and plant cells can be used appropriately as hosts.
[0081] [0081] As the expression vector, an expression vector is suitably used that can replicate autonomously in a host cell or that can be incorporated into a chromosome of a host that contains a promoter in a position capable of transcribing the nucleic acid according to the present invention.
[0082] [0082] In a case in which a prokaryote like a bacterium is used as a host, the expression vector according to the present invention is preferably a vector that is capable of autonomous replication in the prokaryote and at the same time includes a promoter , a ribosome binding site, a nucleic acid according to the present invention and a transcription termination sequence. A gene that controls a promoter can be included.
[0083] [0083] Examples of the prokaryote include microorganisms belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus,
[0084] [0084] Examples of microorganisms belonging to the Escherichia genus include Escherichia coli BL21 (Novagen, Inc.), Escherichia coli BL21 (DE3) (Life Technologies Corporation), Escherichia coli BLR (DE3) (Merck KGaA), Escherichia coli DH1, Escherichia coli GI698, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli K5 (ATCC 23506), Escherichia coli KY3276, Escherichia coli MC1000, Escherichia coli MG1655 (ATCC 47076), Escherichia coli No. 49, Escherichia , Inc.), Escherichia coli TB1, Escherichia coli Tuner (Novagen, Inc.), Escherichia coli Tuner (DE3) (Novagen, Inc.), Escherichia coli W1485, Escherichia coli W3110 (ATCC 27325), Escherichia coli XL1-Blue, and Escherichia coli XL2-Blue.
[0085] [0085] Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus borstelensis, Brevibacillus centrosporus, Brevibacillus formosus, Brevibacillus invocatus, Brevibacillus laterosporus, Brevibacillus limnophilis, Brevibacillus, Brevibacillus, Brevibacillus, Brevibac 1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevibacillus brevis 47-5Q (JCM 8975), Brevibacillus choshinensis HPD31 (FERM BP-1087), Brevibacillus choshinensis HPD31 (FERM BP-6623), Brevibacillus choshinensis HPD31-OK (FERM BP-4573), and the strain Brevibacillus choshinensis SP3 (manufactured by Takara Bio, Inc.).
[0086] [0086] Examples of microorganisms belonging to the Serratia genus include Serratia liquefacience ATCC 14460, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia proteamaculans, Serratia odorifera, Serratia plymuthica, and Serratia rubidaea.
[0087] [0087] Examples of microorganisms belonging to the genus
[0088] [0088] Examples of microorganisms belonging to the Microbacterium genus include Microbacterium ammoniaphilum ATCC 15354.
[0089] [0089] Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC 14067) ATCC 13826, ATCC 14067, Brevibacterium immariophilum ATCC 14069, Bracibacterium ATCC 14068, BrCC 13665, ATCC 13869, Brevibacterium roseum ATCC 13825, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium tiogenitalis ATCC 19240, Brevibacterium album ATCC 15111, and Brevibacterium cerinum ATCC 15112.
[0090] [0090] Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC 6871, ATCC 6872, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 14067, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium ATC 13870, Corynebacterium 15991, Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, Corynebacterium lilium ATCC 15990, Corynebacterium melassecola ATCC 17965, Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), and Corynebacterium herculis ATCC 13868.
[0091] [0091] Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas fulva, and Pseudomonas sp. D-0110.
[0092] [0092] As a method for introducing an expression vector into the host cell mentioned above, any method can be used as long as it introduces DNA into the host cell. Examples of these
[0093] [0093] The transformation of microorganisms belonging to the genus Brevibacillus can be carried out, for example, by the method of Takahashi et al. (J. Bacteriol., 1983, 156: 1130-1134), the method of Takagi et al. (Agric. Biol. Chem., 1989, 53: 3099-3100), or the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61: 202-203).
[0094] [0094] Examples of the vector into which the nucleic acid according to the present invention is introduced (hereinafter, simply called "vector"), include pBTrp2, pBTac1, and pBTac2 (all commercially available from Boehringer Mannheim GmbH), pKK233- 2 (manufactured by Pharmacia Corporation), pSE280 (manufactured by Invitrogen Corporation), pGEMEX-1 (manufactured by Promega Corporation), pQE-8 (manufactured by QIAGEN Corporation), pKYP10 (Unexamined Japanese Patent Publication No. S58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (manufactured by Stratagene Corporation), pTrs30 [built from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [built from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [constructed from Escherichia coli IGHA2 (FERM B-400), Japanese Unexamined Patent Publication No. S60-221091], pGKA2 [constructed from Escherichia coli IGKA 2 (FERM BP-6798), Japanese Unexamined Patent Publication No. 60-221091], pTerm2 (US 4686191, US 4939094, US 5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia Corporation), and pET Systems (manufactured by Novagen, Inc.).
[0095] [0095] In the case in which Escherichia coli is used as a host, pUC18, pBluescriptII, pSupex, pET22b, pCold, or the like can be mentioned as a suitable vector.
[0096] [0096] Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110 or pHY500 (Japanese Unexamined Patent Publication No. H2-31682), pNY700 (Japanese Unexamined Patent Publication No. H4-278091), pHY4831 (J . Bacteriol., 1987, 1239-1245), pNU200 (UDAKA Shigezou, Journal of the Agricultural Chemical Society of Japan, 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80 ), pNU211 (J. Biochem., 1992, 112: 488-491), pNU211R2L5 (Japanese Unexamined Patent Publication No. H7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58: 525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177: 745-749), and pHT210 (Unexamined Japanese Patent Publication No. H6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42: 358-363) , which are known as vectors of Bacillus subtilis; and pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569) which is a bifunctional vector between Escherichia coli and a microorganism belonging to the genus Brevibacillus.
[0097] [0097] The promoter is not limited as long as it functions in a host cell. Examples of the same include promoters derived from Escherichia coli or phage as a trp (Ptrp) promoter, a lac promoter, a PL promoter, a PR promoter, and a T7 promoter. Also, artificially designed and modified promoters can be used, such as a promoter (Ptrp × 2) in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, and a let I promoter.
[0098] [0098] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding site, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18
[0099] [0099] Examples of eukaryotic hosts include yeast, filamentous fungi (molds and the like), and insect cells.
[00100] [00100] Examples of yeast include yeasts belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia, Hansenula, and the like. More specific examples of yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Trichosporon pullulans, Schwanniomyces alluvius, Schwanniomyces occidentalis, Candida utilis, Pichia pastoris, Pichiaiaystica, Pichiaiaymusta, Pichiaia, polymorpha.
[00101] [00101] It is preferable that the expression vector, in the case in which yeast is used as a host cell, usually includes an origin of replication (in the case in which amplification in a host is required), a selection marker for vector propagation in Escherichia coli, a promoter and terminator for expression of recombinant protein in yeast, and a selection marker for yeast.
[00102] [00102] In the case where the expression vector is a non-integrated vector, it is additionally preferable to include an autonomously replicating sequence (ARS). This makes it possible to improve the stability of expression vectors in cells (Myers, A. M., et al. (1986) Gene 45: 299-310).
[00103] [00103] Examples of the vector, in the case in which yeast is used as a host, include YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50
[00104] [00104] The promoter is not limited as long as it can be expressed in yeast. Examples of the promoter include a glycolytic gene promoter such as hexose kinase, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, a gal 1 promoter, a gal 10 promoter, a heat shock polypeptide promoter, an MFα1 promoter , a CUP 1 promoter, a pGAP promoter, a pGCW14 promoter, an AOX1 promoter, and a MOX promoter.
[00105] [00105] As a method for introducing an expression vector in yeast, any method can be used as long as it introduces DNA into yeast. Examples of the same include an electroporation method (Methods Enzymol., 194, 182 (1990)), a spheroplasty method (Proc. Natl. Acad. Sci. USA, 81, 4889 (1984)), a lithium acetate method (J. Bacteriol., 153, 163 (1983)), and a method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
[00106] [00106] Examples of filamentous fungi include fungi belonging to the genera Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, Penicillium, Myceliophtora, Botryts, Magnaporthe, Mucor, Metarhizium, Monascus, Rhizopor, and Rhizomoror.
[00107] [00107] Specific examples of filamentous fungi include Acremonium alabamense, Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus oryzae, Aspergillus sake, Aspergillus soye, Aspergillus tubigensis, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger , Aspergillus flavus, Aspergillus fumigatus, Aspergillus japonicus, Trichoderma viride, Trichoderma harzianum, Trichoderma reseei, Chrysosporium lucknowense, Thermoascus, Sporotrichum, Sporotrichum cellulophilum, Talaromyces, Thielavia terrestris, Thielavia terrestris
[00108] [00108] The promoter in the case in which the host is a filament fungus can be any one of a gene related to a glycolytic system, a gene related to constitutive expression, an enzyme gene related to hydrolysis, and the like. Specific examples thereof include amyB, glaA, agdA, glaB, TEF1, xynF1 tanase gene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, and catB.
[00109] [00109] The introduction of the expression vector in filamentous fungi can be performed by a conventionally known method. Examples of the same include the method by Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)], a protoplast method [Mol. Gen. Genet., 168: 111 (1979)], a competent method [J. Mol. Biol., 56: 209 (1971)], and an electroporation method.
[00110] [00110] Insect cells include, for example, lepidopteran insect cells, more specifically insect cells derived from Spodoptera frugiperda like Sf9 and Sf21, and insect cells derived from Trichoplusia ni like High 5.
[00111] [00111] Examples of the vector in the case in which an insect cell is used as a host include baculovirus such as Autographa Californica nuclear polyhedrosis virus which is a virus that infects insects belonging to the Noctuidae family (“Baculovirus Expression Vectors, A
[00112] [00112] In the case in which an insect cell is used as a host, a polypeptide can be expressed by the method described, for example, in Current Protocols in Molecular Biology, "Baculovirus Expression Vectors, A Laboratory Manual", WH Freeman and Company, New York (1992), USA, or Bio / Technology, 6, 47 (1988). That is, a recombinant gene transfer vector and a baculovirus are co-introduced into an insect cell to obtain a recombinant virus (expression vector) in an insect cell culture supernatant, and then the recombinant virus is further infected inside. of an insect cell, through which the polypeptide can be expressed. Examples of the gene transfer vector used in the above method include pVL1392, pVL1393, and pBlueBacIII (all manufactured by Invitrogen Corporation).
[00113] [00113] As a method for co-introducing a recombinant gene transfer vector and a baculovirus into an insect cell to construct the recombinant virus, for example, a calcium phosphate method (Japanese Patent Publication Unexamined No. H2-227075), a lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), or the like.
[00114] [00114] The recombinant vector according to the present invention preferably contains, in addition, a selection marker gene for the selection of a transformant. For example, in Escherichia coli, genes for resistance to various drugs such as tetracycline, ampicillin, and kanamycin can be used as selectable marker genes. A recessive selection marker gene capable of complementing a genetic mutation involved in auxotrophy may also be used, or a selection marker such as LEU2, URA3, TRP1, or HIS3 may also be used. Examples of the selection marker gene for filamentous fungi include a marker gene
[00115] [00115] Selection of the host transformed with the expression vector, according to the present invention, can be performed by plate hybridization and colony hybridization using a probe that selectively binds to the nucleic acid, in accordance with the present invention. Like the probe, it is possible to use a probe obtained by modifying a partial fragment of amplified DNA by a PCR method based on nucleic acid sequence information, according to the present invention, with a radioisotope or a digoxigenin. [Modified fibroin production]
[00116] [00116] In the host transformed with the expression vector according to the present invention, the modified fibroin according to the present invention can be produced by the expression of the nucleic acid according to the present invention. As for the method of expression, secretory production, expression of fusion protein, or the like, in addition to direct expression, they can be performed according to the method described in “Molecular Cloning”, 2nd edition. In the case in which it is expressed by yeast, an animal cell, or an insect cell, a fibroin
[00117] [00117] The modified fibroin according to the present invention can be produced, for example, by culturing a host transformed with the expression vector according to the present invention in a medium for the culture, production and accumulation of the modified fibroin according to with the present invention in the culture medium, and then collecting the modified fibroin from the culture medium. The method for culturing the host according to the present invention in a culture medium can be carried out according to a method commonly used for culturing a host.
[00118] [00118] In the case in which the host according to the present invention is a prokaryote like Escherichia coli or a eukaryote like yeast, any one of a natural medium and a synthetic medium can be used as a culture medium of the host according to the present invention as long as it contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host and it is capable of efficiently cultivating the host.
[00119] [00119] As the carbon source, any carbon source that can be assimilated by the host can be used. Examples of the carbon source that can be used include carbohydrates such as glucose, fructose, sucrose, and molasses, starch and starch hydrolysates containing them, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol.
[00120] [00120] Examples of the nitrogen source that can be used include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other compounds containing nitrogen, peptone, extract meat, yeast extract, corn macerate whey, casein hydrolyzate, soy bean pie and soy bean pie hydrolyzate, various fermented microbial cells and their digested products.
[00121] [00121] Examples of the inorganic salt that can be used include potassium dihydrogen phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
[00122] [00122] The culture of a prokaryote such as Escherichia coli or a eukaryote such as yeast can be performed under aerobic conditions such as agitated culture or culture with agitation by deep aeration. The culture temperature is, for example, from 15 ° C to 40 ° C. The culture time is usually from 16 hours to 7 days. It is preferable to maintain the pH of the culture medium during culture at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, a solution of alkali, urea, calcium carbonate, ammonia, or the like.
[00123] [00123] In addition, antibiotics such as ampicillin and tetracycline can be added to the culture medium, as needed, during culture. In the case of culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducing agent can be added to the medium as needed. For example, in the case of culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used, and in the case of culturing a microorganism transformed with a vector of expression using a trp promoter, indole-acrylic acid or the like can be added to the culture medium.
[00124] [00124] As a culture medium for insect cells, the commonly used TNM-FH medium (manufactured by Pharmingen Inc.), Sf-900 II SFM medium (manufactured by Life Technologies Corporation), ExCell 400 and ExCell can be used 405 (both manufactured by JRH Biosciences Inc.), Grace Insect Cell Culture Medium (Nature, 195, 788 (1962)), and the like.
[00125] [00125] The culture of insert cells can be carried out, for example, during a culture time of 1 day to 5 days under conditions such as pH 6 to 7 of culture medium and culture temperature of 25 ° C to 30 ° Ç. In addition, an antibiotic such as gentamicin can be added to the culture medium as needed during culture.
[00126] [00126] In the case where the host is a plant cell, the transformed plant cell can be directly cultivated, or it can be differentiated into a plant organ and then cultivated. As the culture medium for the culture of a plant cell, it can be used, for example the Murashige and Skoog (MS) medium commonly used, White medium, or a medium in which a plant hormone such as auxin or cytokinin is added to these means.
[00127] [00127] The culture of animal cells can be carried out, for example, during a culture time of 3 days to 60 days under conditions such as pH of 5 to 9 of the culture medium and culture temperature of 20 ° C to 40 ° C . In addition, an antibiotic such as kanamycin or hygromycin can be added to the medium as needed during culture.
[00128] [00128] As a method for the production of a modified fibroin using a host transformed with the expression vector according to the present invention, there is a method for the production of the modified fibroin in a host cell, a method for the secretion of fibroin modified out of the host cell, and a method for producing the modified fibroin on the outer membrane of the host cell. Each of these methods can be selected depending on the host cell to be used and the structure of the modified fibroin to be produced.
[00129] [00129] For example, in the case where a modified fibroin is produced in the host cell or on the outer membrane of the host cell, the production method can be changed to actively secrete
[00130] [00130] The modified fibroin produced by the host transformed with the expression vector according to the present invention can be isolated and purified by a method commonly used for protein isolation and purification. For example, in the case in which the modified fibroin is expressed in a state dissolved in cells, the host cells are recovered by centrifugation after the completion of the culture, suspended in an aqueous buffering solution, and then broken up using an ultrasound machine, a French press, a Manton-Gaulin homogenizer, a Dyno-Mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified preparation can be obtained by a method commonly used for the isolation and purification of proteins, that is, a method of solvent extraction, a method of separation by salting using ammonium sulfate or the like, a desalination method, a precipitation method using an organic solvent, an anion exchange chromatography method using a resin such as DiEtilAminoEtil (DEAE) -Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Kasei Kogyo Kabushiki Kaisha ), a cation exchange chromatography method using a resin such as S-Sepharose FF (Pharmacia
[00131] [00131] As chromatography, column chromatography using Phenyl-TOYOPEARL (available from Tosoh Corporation), DEAE-TOYOPEARL (available from Tosoh Corporation), and Sephadex G-150 (available from Pharmacia Biotech Inc.) ).
[00132] [00132] In the case in which the modified fibroin is expressed by the formation of an insoluble matter in the cell, the host cells are recovered, disrupted and centrifuged to recover the insoluble matter of the modified fibroin as a precipitated fraction. The insoluble matter recovered from the modified fibroin can be solubilized with a protein denaturing agent. After this operation, a purified modified fibroin preparation can be obtained by the same isolation and purification method as described above.
[00133] [00133] In the case in which a modified fibroin or a derivative in which a sugar chain has been added to the modified fibroin is secreted extracellularly, the modified fibroin or derivative thereof can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above. [Wiring]
[00134] [00134] The modified fibroin according to the present invention can be produced and purified as described above, and then spun by a
[00135] [00135] The spinning liquid is prepared by adding a solvent to the modified fibroin and adjusting its viscosity to allow spinning. Any solvent can be used as long as it can dissolve the modified fibroin. Examples of the solvent include an aqueous solution or the like, containing hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide, calcium chloride, lithium thiocyanate.
[00136] [00136] An inorganic salt can be added to the spinning liquid as needed. Inorganic salts include, for example, inorganic salts consisting of the following Lewis acids and Lewis bases. Examples of the Lewis base include an oxoacid ion (such as a nitrate ion and a perchlorate ion), a metal oxoacid ion (such as a permanganate ion), a halide ion, a thiocyanate ion, a cyanate ion, and the like. Examples of Lewis acid include metal ions like an alkali metal ion and an alkaline earth metal ion, a polyatomic ion like an ammonium ion, and a complex ion. Specific examples of inorganic salts consisting of a Lewis acid and a Lewis base include: lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate; calcium salts such as calcium chloride, calcium bromide, calcium iodide, calcium nitrate, calcium perchlorate and calcium thiocyanate; iron salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron thiocyanate; aluminum salts such as aluminum chloride, sodium bromide
[00137] [00137] The viscosity of the spinning liquid can be properly adjusted according to the spinning method and can be, for example, from 100 to
[00138] [00138] The spinning method is not particularly limited as long as it is a method capable of spinning the modified fibroin according to the present invention, and examples thereof include dry spinning, melt spinning, and wet spinning. A preferred method of spinning is wet spinning.
[00139] [00139] In wet spinning, an unstretched yarn with a yarn shape can be obtained by extrusion, from a spinner (nozzle), a solvent in which a modified fibroin is dissolved in a coagulation liquid (liquid bath). coagulation) in which the modified fibroin is solidified. The coagulation liquid can be any solution that can be desolvated, and examples of it include lower alcohols having 1 to 5
[00140] [00140] The unstretched yarn (or the pre-stretched yarn) obtained by the method described above can be transformed into a stretched yarn (fibroin fiber) by means of a stretching step. Examples of the drawing method include hot-wet drawing and hot-dry drawing.
[00141] [00141] Hot-wet drawing can be carried out in hot water, in a solution obtained by adding an organic solvent or similar to hot water, or in heated water vapor. The temperature can be, for example, 50 ° C to 90 ° C and preferably 75 ° C to 85 ° C. In hot-wet drawing, the unstretched yarn (or pre-stretched yarn) can be stretched, for example, 1 to 10 times and preferably 2 to 8 times.
[00142] [00142] Hot-dry drawing can be carried out using an electric tubular oven, a dry heating plate, or the like. The temperature can be, for example, from 140 ° C to 270 ° C and preferably from 160 ° C to 230 ° C. In hot-dry drawing, the unstretched yarn (or pre-stretched yarn) can be stretched, for example, 0.5 to 8 times and preferably 1 to 4 times.
[00143] [00143] Hot-wet drawing and hot-dry drawing can be performed independently or in combination, or can be performed in multiple stages. That is, the hot-wet stretch and the hot-dry stretch can be performed in a suitable combination, for example, in a way in which a first-stage stretch is performed by the hot-wet stretch and a second-stage stretch. is performed by hot-dry drawing, or in a manner in which the first stage drawing is performed by hot-wet drawing, the second stage drawing is performed by hot-wet drawing, and a third stage drawing is performed by hot-dry drawing.
[00144] [00144] The final drawing ratio in the drawing step is, for example, 5 to 20 times and preferably 6 to 11 times with respect to the unstretched yarn (or the pre-stretched yarn).
[00145] [00145] The modified fibroin according to the present invention can be stretched into a fibroin fiber and then chemically cross-linked between polypeptide molecules in the fibroin fiber. Examples of functional groups that can be cross-linked include an amino group, a carboxyl group, a thiol group, and a hydroxyl group. For example, an amino group on a lysine side chain contained in the polypeptide can be cross-linked by condensation amide bonding by dehydration with a carboxyl group on a glutamic acid or aspartic acid side chain. Reticulation can be accomplished by performing a
[00146] [00146] Crosslinking between polypeptide molecules can be performed using a crosslinking agent such as carbodiimides or glutaraldehyde, or it can be performed using an enzyme such as transglutaminase. Carbodiimides are compounds represented by the general formula R1N = C = NR2 (where each R1 and R2, independently, represent an organic group containing an alkyl group having from 1 to 6 carbon atoms or a cycloalkyl group). Specific examples of carbodiimides include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N, N′-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3- ( 2-morpholinoethyl) carbodiimide, and diisopropylcarbodiimide (DIC). Among these, EDC and DIC are preferable because they have a high capacity to form an amide bond between polypeptide molecules and easily carry out a crosslinking reaction.
[00147] [00147] The crosslinking treatment is preferably carried out by applying a crosslinking agent to the fibroin fiber and carrying out crosslinking with heating and vacuum drying. As the crosslinker, a pure product can be applied to the fibroin fiber, or a product diluted with a lower alcohol having from 1 to 5 carbon atoms, a buffering solution, or the like to a concentration of 0.005% by mass to 10% en masse can be applied to fibroin fiber. The crosslinking treatment is preferably carried out at a temperature of 20 ° C to 45 ° C for 3 hours to 42 hours. Higher stress (resistance) can be given to the fibroin fiber by the crosslinking treatment. [Evaluation of the shrinking capacity of the fibroin fiber]
[00148] [00148] Fig. 2 is a graph showing an example of a change in the length of a fibroin fiber due to contact with water or the like. Fibroin fiber has the property to shrink
[00149] [00149] The second shrinkage can be evaluated using a second shrinkage rate obtained by the following method as an index. <Second shrinkage rate>
[00150] [00150] A plurality of fibroin fibers having a length of about 30 cm are bundled together to form a bundle of fibers having a fineness of 150 denier. With a lead weight of 0.8 g being attached to this fiber bundle, the fiber bundle is left immersed in water at 40 ° C for 10 minutes to cause the first shrinkage, and the length of the fiber bundle is measured in water .
[00151] [00151] The bundle of fibers shrunk by the first shrinkage is removed from the water and dried at room temperature for 2 hours with the lead weight of 0.8 g attached. After drying, the length of the fiber bundle is measured. Again, wetting and drying are repeated at least three times, and an average length when moistened (Wet) and an average length when dried (Cseco) are determined. The second rate of shrinkage is calculated according to the following expression. Expression: second shrinkage rate (%) = (1− (Dry / Wet)) * 100
[00152] [00152] A naturally occurring fibroin stretched fibroin fiber usually has a second shrinkage rate of 11% to 20%, while the stretched fibroin fiber modified according to the present invention may have a second shrinkage rate reduced by 8% or less. [Movie]
[00153] [00153] The film according to the present invention can be obtained by preparing a spinning liquid in which the modified fibroin according to the present invention is dissolved in a solvent, casting by casting the spinning liquid on the surface of a base material, and drying and / or desolvation.
[00154] [00154] Examples of the solvent include the same solvents as those exemplified for the doping liquid. The solvent is preferably a polar solvent such as formic acid, hexafluoro-2-propanol (HFIP), or dimethyl sulfoxide. An inorganic salt can be added to the spinning liquid as needed. Examples of inorganic salt include the same salts as those exemplified for the doping liquid.
[00155] [00155] The viscosity of the spinning liquid is preferably 15 cP to 80 cP (centipoise) (mPa.s) and more preferably 20 cP to 70 cP (mPa.s).
[00156] [00156] The concentration of the modified fibroin according to the present invention is preferably from 3% by weight to 50% by weight, more preferably from 3.5% by weight to 35% by weight, and even more preferably 4.2 % by mass to 15.8% by mass in a case in which the spinning liquid is adjusted to 100% by mass.
[00157] [00157] When preparing the spinning liquid, heating can be carried out from 30 ° C to 60 ° C. Flouring and stirring can be performed to favor dissolution.
[00158] [00158] The base material can be a resin substrate, a glass substrate, a metal substrate, or the like. The base material is preferably a resin substrate from the point of view that the film after casting can be easily detached from the mold. The resin substrate can be, for example, a poly (ethylene terephthalate) film (PET, PolyEthylene Terephthalate), a fluororesin film such as polytetrafluoroethylene, a polypropylene (PP) film, or a removable film in which a compound of silicone is immobilized on the surface of these films. It is more preferable that the base material is stable with respect to the solvent such as HFIP and DMSO, be stably molded by casting with the spinning liquid, and from the point of view that the film after molding can be easily detached from the mold, either a removable film in which the silicone compound is immobilized on the PET film or on the surface of the PET film.
[00159] [00159] The specific procedure is as follows. First, the spinning liquid is poured over the surface of the base material, and a wet film having a predetermined thickness (for example, a thickness of 1 µm at
[00160] [00160] Drying and / or desolvation can be carried out by a dry method or by a wet method. Examples of the dry method include vacuum drying, hot air drying, and air drying. Examples of the wet method include a method in which a cast film is immersed in a desolvation liquid (also called a coagulation liquid) to remove the solvent. Examples of the desolvation liquid include water, alcoholic liquids such as lower alcohols having 1 to 5 carbon atoms including methanol, ethanol, and 2-propanol, and a mixed liquid of water and alcohol. The temperature of the
[00161] [00161] The film not stretched after drying and / or desolvation can be uniaxially or biaxially stretched in water. The biaxial stretch can be sequential stretch or simultaneous biaxial stretch. Multi-stage stretching of two or more stages can be performed. The stretch ratio is preferably 1.01 to 6 times and more preferably 1.05 to 4 times in both length and width. Within this range, it is easy to balance the stress with the deformation. The stretching in water is preferably carried out at a water temperature of 20 ° C to 90 ° C. The stretched film is preferably thermally fixed by dry heat at 50 ° C to 200 ° C for 5 seconds to 600 seconds. This thermal fixation provides dimensional stability to the film at room temperature. A uniaxially stretched film becomes a uniaxially aligned film, and a biaxially stretched film becomes a biaxially aligned film. [Evaluation of the film's water-tightness]
[00162] [00162] The film's water tightness can be assessed by measuring the degree of moisture absorption under high humidity using a saturated salt method using a saturated aqueous solution of salts.
[00163] [00163] Examples of the salts include potassium sulfate, potassium chloride, sodium chloride, sodium bromide, potassium carbonate, and magnesium chloride.
[00164] [00164] The water impermeability of the film can be assessed, for example, by positioning a film, cut to a suitable size so that the film does not remain immersed in the aqueous solution, inside an air-impervious container, such as a Falcon® tube. , containing a saturated aqueous solution of potassium sulphate, and, for example, leaving the film for 20 hours to 48 hours in air balanced in high humidity with 98% relative humidity, and by measuring the weight and moisture content
[00165] [00165] The fibroin fiber formed from the modified fibroin according to the present invention can be applied to a weaving fabric, a knitted fabric, a folded fabric, a non-woven fabric, and the like, as a fiber (such as a long fiber, short fiber, multifilament, and monofilament) or yarn (such as spun yarn, twisted yarn, false-twisted yarn, processed yarn, mixed yarn, and spun mixed yarn). This fibroin fiber can also be applied in high strength applications such as a rope, a surgical suture, a flexible switch for electrical components, and a physiologically active material for implantation (for example, artificial ligament and aortic band).
[00166] [00166] In addition to the film, the modified fibroin according to the present invention can also be applied to a foam, a grain, a nanofibril, a gel (such as a hydrogel), a resin and equivalents thereof, which can be produced with the method described in Japanese Unexamined Patent Publication No. 2009-505668, in Japanese Patent No. 5678283, in Japanese Patent No. 4638735, or the like. [Artificially modified fibroin fiber]
[00167] [00167] The fibroin fiber artificially modified according to the present modality includes a modified fibroin, which elongates when moistened, and shrinks when dried from the wetted state (corresponds to stretching after the “second shrinkage” in Fig. 2 ).
[00168] [00168] The modified fibroin in the fibroin fiber artificially modified according to the present modality is not limited to the modified fibroin having an amino acid sequence with a reduced content of glutamine residue, as long as it falls within the modified fibroin
[00169] [00169] The first modified fibroin includes a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m. In the first modified fibroin, the number of motif amino acid residues (A) n is preferably an integer from 3 to 20, more preferably an integer from 4 to 20, even more preferably an integer from 8 to 20, thankfully more preferably an integer from 10 to 20, much more preferably an integer from 4 to 16, particularly preferably an integer from 8 to 16, and with the
[00170] [00170] The first modified fibroin can be a polypeptide including an amino acid sequence unit represented by Formula 1: [motif (A) n-REP] m, and including the C-terminal sequence which is the amino acid sequence shown in any one of SEQ ID NOs: 25 to 27 or the C-terminal sequence which is an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 25 to 27.
[00171] [00171] The amino acid sequence shown in SEQ ID NO: 25 is identical to the amino acid sequence consisting of 50 amino acid residues at the C-terminus of the ADF3 amino acid sequence (GI: 1263287, NCBI). The amino acid sequence shown in SEQ ID NO: 26 is identical to the amino acid sequence obtained by removing 20 C-terminal residues from the amino acid sequence shown in SEQ ID NO: 25. The amino acid sequence shown in SEQ ID NO: 27 is identical to the amino acid sequence obtained by removing 29 C-terminal residues from the amino acid sequence shown in SEQ ID NO: 25.
[00172] [00172] More specific examples of the first modified fibroin may include a modified fibroin including (1-i) the amino acid sequence shown in SEQ ID NO: 28 (recombinant silk protein
[00173] [00173] The amino acid sequence shown in SEQ ID NO: 28 is an amino acid sequence obtained by approximately duplication of the repeat regions from the first repeat region to the 13th repeat and mutation region so that the translation is completed at the 1154th amino acid residue in an amino acid sequence obtained by adding the amino acid sequence (SEQ ID NO: 29) consisting of an initiation codon, a His10 tag, and a recognition site for HRV3C protease (human rhinovirus protease 3C) to the N-terminal of ADF3. The C-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO: 28 is identical to the amino acid sequence shown in SEQ ID NO: 27.
[00174] [00174] The modified fibroin of (1-i) can consist of the amino acid sequence shown in SEQ ID NO: 28.
[00175] [00175] The second modified fibroin domain sequence has an amino acid sequence with a reduced content of glycine residue, compared to naturally occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence equivalent to an amino acid sequence in which at least one or a plurality of glycine residues in REP are replaced by other amino acid residues, compared to naturally occurring fibroin.
[00176] [00176] The modified second fibroin domain sequence may have an amino acid sequence equivalent to an amino acid sequence in which a glycine residue in at least one or in the plurality of motif sequences, at least one of which is selected from among GGX and GPGXX (where G represents a glycine residue,
[00177] [00177] In the second modified fibroin, the proportion of the motif sequences in which the glycine residue described above is replaced by another amino acid residue may be 10% or more with respect to the entire motif sequences.
[00178] [00178] The second modified fibroin may include a domain sequence represented by Formula 1: [motif (A) n-REP] m has 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) in all REPs in a sequence excluding the sequence of motif (A) n located on the most C-terminal side to the C-terminal 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 motif sequence ( A) n located on the most C-terminal side to the C-terminal of the domain string from the domain string is defined as w. The number of alanine residues is 83% or more with respect to the total number of amino acid residues in reason (A) n, preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and yet much more preferably 100% (meaning that motif (A) does not consist of alanine residues only).
[00179] [00179] In the second modified fibroin, the content ratio of an amino acid sequence consisting of XGX is preferably increased by replacing a glycine residue in GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of an amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, still
[00180] [00180] In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, still much more preferably 70% or more, and with maximum preference 80% or more. The upper limit of z / w is not particularly limited, but it can be, for example, 95% or less.
[00181] [00181] The second modified fibroin can be obtained, for example, by modifying the cloned naturally occurring fibroin gene sequence so that at least part of the base sequence encoding a glycine residue is replaced by another amino acid residue for encode the other amino acid residue. At the same time, a glycine residue in GGX motif and GPGXX motif can be selected as the glycine residue to be modified, or can be replaced so that z / w is 50.9% or more. Alternatively, the second modified fibroin according to the present embodiment can also be obtained, for example, by designing an amino acid sequence that satisfies the aspect described above on the naturally occurring fibroin amino acid sequence and by chemical synthesis of an acid nucleic encoding the drawn amino acid sequence. In any case, with respect to the naturally occurring fibroin amino acid sequence, in addition to the modification equivalent to the substitution of glycine residue in REP with another amino acid residue, additional modification of the amino acid sequence equivalent to the substitution, deletion, insertion can be performed and / or adding one or a plurality of amino acid residues.
[00182] [00182] The other amino acid residue described above is not particularly limited as long as it is an amino acid residue other than glycine residue, but is preferably a hydrophobic amino acid residue such as valine residue (V), leucine residue (L) , isoleucine residue (I), methionine residue (M), proline residue (P), phenylalanine residue (F), and tryptophan residue (W), or a hydrophilic amino acid residue such as glutamine residue (Q) , asparagine residue (N), serine residue (S), lysine residue (K), and glutamic acid residue (E), more preferably valine residue (V), leucine residue (L), isoleucine residue (I), phenylalanine residue (F), and glutamine residue (Q), and even more preferably glutamine residue (Q).
[00183] [00183] More specific examples of the second modified fibroin may include a modified fibroin including (2-i) the amino acid sequence shown in SEQ ID NO: 30 (Met-PRT380), SEQ ID NO: 31 (Met-PRT410), SEQ ID NO: 32 (Met-PRT525), or SEQ ID NO: 33 (Met-PRT799), and (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.
[00184] [00184] The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 30 is obtained by replacing all GGXs in REP from the amino acid sequence shown in SEQ ID NO: 34 (Met-PRT313) equivalent to naturally occurring fibroin with GQX. The amino acid sequence shown in SEQ ID NO: 31 is obtained by deleting one of every two motifs (A) n from the N-terminal side to the C-terminal side in the amino acid sequence shown in SEQ ID NO: 30 and additional insertion of a [reason (A) n-REP] just before the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 32 is obtained by inserting two alanine residues on the C-terminal side of each motif (A) n of the amino acid sequence shown in SEQ ID
[00185] [00185] The z / w value in the amino acid sequence presented in SEQ ID NO: 34 (equivalent to naturally occurring fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 30, in the amino acid sequence shown in SEQ ID NO: 31, in the amino acid sequence shown in SEQ ID NO: 32, and in the amino acid sequence shown in SEQ ID NO: 33 is 58.7%, 70.1%, 66.1%, and 70.0%, respectively. In addition, x / y values with a Giza ratio (described below) of 1: 1.8 to 11.3 in the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32 and SEQ ID NO: 33 are 15.0%, 15.0%, 93.4%, 92.7%, and 89.8%, respectively.
[00186] [00186] The modified (2-i) fibroin can consist of the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.
[00187] [00187] Modified (2-ii) fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 , or SEQ ID NO: 33. Modified (2-ii) fibroin is also a
[00188] [00188] The modified (2-ii) fibroin preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, and in a case in which 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 z, and the total number of amino acid residues in REP the domain sequence is w, z / w is preferably 50.9% or more.
[00189] [00189] The second modified fibroin can include a tag sequence at either one or both of the N-terminus and the C-terminus. This makes it possible to isolate, immobilize, detect, and visualize the modified fibroin.
[00190] [00190] The tag sequence can be, for example, an affinity tag that uses specific affinity (affinity, binding property) with another molecule. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are arranged and has a property to specifically bind to a metal ion such as nickel, so that it can be used to isolate modified fibroin by chromatography of metal chelation. A specific example of the tag sequence may include the amino acid sequence shown in SEQ ID NO: 35 (amino acid sequence including a His tag sequence and a hinge sequence).
[00191] [00191] In addition, a tag sequence such as glutathione-S-transferase (GST) that specifically binds to glutathione or a maltose-binding protein (MBP) that specifically binds to maltose can be used.
[00192] [00192] In addition, an "epitope tag" that uses an antigen-antibody reaction can also be used. By adding a peptide (epitope) that shows antigenicity as a tag sequence, an antibody against the epitope can be attached. Examples of the epitope tag include an HA tag (influenza virus hemagglutinin peptide sequence), a myc tag, and a FLAG tag. Modified fibroin can be easily purified with high specificity by using an epitope tag.
[00193] [00193] It is also possible to use a tag sequence that can be cleaved with a specific protease. By protease treatment of a protein adsorbed by the tag sequence, it is also possible to recover the modified fibroin cleaved from the tag sequence.
[00194] [00194] A more specific example of the modified fibroin including a tag sequence may be a modified fibroin including (2-iii) the amino acid sequence shown in SEQ ID NO: 36 (PRT380), SEQ ID NO: 37 (PRT410), SEQ ID NO: 38 (PRT525), or SEQ ID NO: 39 (PRT799), or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 36 , SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39.
[00195] [00195] The amino acid sequences shown in SEQ ID NO: 40 (PRT313), SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39 are respectively amino acid sequences obtained by adding from the amino acid sequence (including a His tag sequence and a hinge sequence) shown in SEQ ID NO: 35 to the N-terminus of the amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
[00196] [00196] The modified (2-iii) fibroin may consist of the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID
[00197] [00197] Modified (2-iv) fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 , or SEQ ID NO: 39. Modified (2-iv) fibroin is also a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m. The sequence identity is preferably 95% or more.
[00198] [00198] The modified (2-iv) fibroin preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39, and 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 z, and the total number of amino acid residues in REP the domain sequence is w, z / w is preferably 50.9% or more.
[00199] [00199] The second modified fibroin may include a secretory signal to release the protein produced in the recombinant protein production system out of a host. The sequence of the secretory signal can be appropriately defined depending on the type of the host.
[00200] [00200] The modified third fibroin domain sequence has an amino acid sequence in which motif content (A) n is reduced compared to naturally occurring fibroin. It can be said that the modified third fibroin domain sequence has an amino acid sequence equivalent to an amino acid sequence in which at least one or a plurality of (A) n motifs are deleted, compared to naturally occurring fibroin.
[00201] [00201] The third modified fibroin can have a sequence of
[00202] The modified third fibroin domain sequence can have an amino acid sequence equivalent to an amino acid sequence obtained by deleting one of each of the three (A) n motifs on at least one of the N-terminal side and the C-terminal side compared to naturally occurring fibroin.
[00203] The modified third fibroin domain sequence may have an amino acid sequence equivalent to an amino acid sequence obtained by repeatedly deleting at least two consecutive (A) n motifs and deleting one (A) n motif in the order of a from the N-terminal side to the C-terminal side, compared to naturally occurring fibroin.
[00204] The modified third fibroin domain sequence can have an amino acid sequence equivalent to an amino acid sequence obtained by deleting one of every two (A) n motifs on at least one of the N-terminal side and the terminal side -Ç.
[00205] [00205] The third modified fibroin may include a domain sequence represented by Formula 1: [motif (A) n-REP] m, and have an amino acid sequence in a case in which the number of amino acid residues of two units [ motif (A) n-REP] mutually adjacent is sequentially compared from the N-terminal side to the C-terminal side, so the number of REP amino acid residues having a small number of amino acid residues is defined as 1, the maximum total value of the number of amino acids residues of two units [reason (A) n-REP] mutually adjacent, where the ratio (Gaza ratio) of the number of amino acid residues of 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, x / y can be 20% or more, 30% or more, 40% or more, or 50% or
[00206] [00206] In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, even more preferably 70% or more, still much more preferably 75% or more , and particularly preferably 80% or more. The upper limit of x / y is not particularly limited, but it can 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 more preferably 77.1% or more. In a case where the Giza ratio is 1: 1.9 to 8.4, x / y is even more preferably 75.9% or more. In a case where the Giza ratio is 1: 1.9 to 4.1, x / y is much more preferably 64.2% or more.
[00207] [00207] In a case in which the third modified fibroin is a modified fibroin in which at least seven of the multiple motifs (A) n present in the domain sequence are composed of only alanine residues, x / y is preferably 46.4% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, still much more preferably 70% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited as long as it is 100% or less.
[00208] [00208] The third modified fibroin, for example, can be obtained by deleting one or a plurality of sequences encoding the motif (A) from a naturally occurring cloned fibroin gene sequence so that x / y is 64.2% or more. Alternatively, the third modified fibroin can also be obtained, for example, by
[00209] [00209] More specific examples of the third modified fibroin may include a modified fibroin including (3-i) the amino acid sequence shown in SEQ ID NO: 41 (Met-PRT399), SEQ ID NO: 31 (Met-PRT410), SEQ ID NO: 32 (Met-PRT525), or SEQ ID NO: 33 (Met-PRT799), and (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 41, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.
[00210] [00210] The modified fibroin of (3-i) will be described. The amino acid sequence shown in SEQ ID NO: 41 is obtained by deleting one out of two motifs (A) from the N-terminal side to the C-terminal side in the amino acid sequence shown in SEQ ID NO: 34 (Met -PRT313) equivalent to naturally occurring fibroin and the additional insertion of a [motif (A) n-REP] immediately before the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 is as described in the second modified fibroin.
[00211] [00211] The value of x / y with a Giza ratio of 1: 1.8 to 11.3 in the amino acid sequence presented in SEQ ID NO: 34 (equivalent to naturally occurring fibroin) is 15.0%. Both the x / y value in the amino acid sequence shown in SEQ ID NO: 41 and the x / y value in the
[00212] [00212] The modified (3-i) fibroin can consist of the amino acid sequence shown in SEQ ID NO: 41, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.
[00213] [00213] Modified (3-ii) fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 41, SEQ ID NO: 31, SEQ ID NO: 32 , or SEQ ID NO: 33. Modified (3-ii) fibroin is also a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m. The sequence identity is preferably 95% or more.
[00214] [00214] Modified (3-ii) fibroin preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 41, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, and in a case in which the number of amino acid residues of two units [motif (A) n-REP] mutually adjacent is sequentially compared from the N-terminal side to the C-terminal side, then the number of amino acid residues of a REP having a small number of amino acid residues is defined as 1, the maximum total value of the number of mutually adjacent two unit [motif (A) n-REP], where the ratio ( 1: 1.8 to 11.3 as a Giza 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 sequence of
[00215] [00215] The third modified fibroin may include a tag sequence described above at either or both the N-terminus and the C-terminus.
[00216] [00216] A more specific example of the modified fibroin including a tag sequence may be a modified fibroin including (3-iii) the amino acid sequence shown in SEQ ID NO: 42 (PRT399), SEQ ID NO: 37 (PRT410), SEQ ID NO: 38 (PRT525), or SEQ ID NO: 39 (PRT799), or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 42 , SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39.
[00217] [00217] The amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39 are respectively amino acid sequences obtained by adding the amino acid sequence (including a sequence of His tag and a hinge sequence) shown in SEQ ID NO: 35 to the N-terminus of the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
[00218] [00218] The modified (3-iii) fibroin may consist of the amino acid sequence shown in SEQ ID NO: 42, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39.
[00219] [00219] Modified (3-iv) fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 42, SEQ ID NO: 37, SEQ ID NO: 38 , or SEQ ID NO: 39. Modified (3-iv) fibroin is also a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m. The sequence identity is preferably 95% or more.
[00220] [00220] The modified (3-iv) fibroin preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 42, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39, and in a case in which the number of amino acid residues of two units [motif (A) n-REP] mutually adjacent is sequentially compared from the N-terminal side to the C-terminal side, then the number of amino acid residues from a REP having a small number of amino acid residues is defined as 1, the maximum total value of the number of mutually adjacent two unit [motif (A) n-REP], 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, x / y is preferably 64.2% or more.
[00221] [00221] The third modified fibroin may include a secretory signal for the release of the protein produced in the recombinant protein production system out of a host. The sequence of the secretory signal can be appropriately defined depending on the type of the host.
[00222] [00222] The modified fourth fibroin domain sequence has an amino acid sequence with not only a reduced content of motif (A) n but also a reduced content of glycine residue, compared to naturally occurring fibroin. It can be said that the modified fourth fibroin domain sequence has an amino acid sequence equivalent 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 they are additionally replaced by other amino acid residues, compared to naturally occurring fibroin. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the second modified fibroin and the
[00223] [00223] More specific examples of the fourth modified fibroin may include a modified fibroin including (4-i) the amino acid sequence shown in SEQ ID NO: 31 (Met-PRT410), SEQ ID NO: 32 (Met-PRT525), SEQ ID NO: 33 (Met-PRT799), SEQ ID NO: 37 (PRT410), SEQ ID NO: 38 (PRT525), or SEQ ID NO: 39 (PRT799), and (4-ii) an amino acid sequence having 90 % or more of sequence identity with the amino acid sequence shown in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39 Specific aspects of the modified fibroin including the amino acid sequence shown in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39 are as described above.
[00224] The domain sequence of the modified fifth fibroin may include the domain sequence having an amino acid sequence locally containing a region with a high hydropathy index equivalent to an amino acid sequence in which one or a plurality of amino acid residues in REP are replaced by amino acid residues with a high hydropathic index and / or one or a plurality of amino acid residues with a high hydropathic index are inserted in REP as compared to naturally occurring fibroin.
[00225] [00225] It is preferable that the region locally having a high rate of hydropathy consists of two to four amino acid residues.
[00226] [00226] It is more preferable that amino acid residues with a high hydropathy index are selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A).
[00227] [00227] The fifth modified fibroin may additionally include
[00228] [00228] The modified fifth fibroin can be obtained by, with respect to a naturally occurring cloned fibroin gene sequence, replacing one or a plurality of hydrophilic amino acid residues in REP (for example, amino acid residues having an index of negative hydropathy) by a hydrophobic amino acid residue (e.g., amino acid residues having a positive hydropathy index), and / or insertion of one or a plurality of hydrophobic amino acid residues in REP. In addition, for example, the modified fifth fibroin can also be obtained by designing an amino acid sequence equivalent to an amino acid sequence in which, with respect to the naturally occurring fibroin amino acid sequence, one or a plurality of hydrophilic amino acid residues in REP they are replaced by hydrophobic amino acid residues and / or one or a plurality of hydrophobic amino acid residues are inserted in REP, and by the chemical synthesis of a nucleic acid that encodes the drawn amino acid sequence. In any case, with respect to the naturally occurring fibroin amino acid sequence, in addition to the modification equivalent to replacing one or a plurality of hydrophilic amino acid residues in REP with hydrophobic amino acid residues and / or the insertion of one or a plurality of residues of hydrophobic amino acids
[00229] [00229] The fifth modified fibroin may include a domain sequence represented by Formula 1: [reason (A) n-REP] m and have an amino acid sequence in which p / q is 6.2% or more, in one case in which in all REPs included in a sequence excluding a sequence from a motif (A) n located on the most C-terminal side to the C-terminal of the domain sequence of the domain sequence, the total number of amino acid residues contained in a region where an average hydropathy index value of four consecutive amino acid residues is 2.6 or more is defined as p, and the total number of amino acid residues contained in the sequence excluding the sequence of motif (A) n located on the most terminal side -C to the C-terminal of the domain string from the domain string is defined as q.
[00230] [00230] Regarding the hydropathy index of amino acid residues, known indexes (“Hydropathy Index”: Kyte J, & Doolittle R (1982) from “A simple method for displaying the hydropathic character of a protein” can be used as references) , J. Mol. Biol., 157, pp. 105-132). Specifically, the hydropathy index (hereinafter also called “IH”) for each amino acid is as shown in Table 1 above.
[00231] [00231] The method of calculating p / q will be described in more detail. In the calculation, the sequence (hereinafter also called “sequence A”) is used excluding the sequence of the motif (A) n located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the Formula 1: [reason (A) n-REP] m. First, in all REPs included in sequence A, the average hydropathy index values for four consecutive amino acid residues are calculated. The average value
[00232] [00232] For example, in a case in which the characteristic “four consecutive amino acid residues whose average hydropathy index value is 2.6 or more” is extracted from 20 places (without overlapping), in the region in which the average value of the hydropathy indices of four consecutive amino acid residues is 2.6 or more, the number of the four consecutive amino acid residues (without overlap) is 20, and therefore p is 20 × 4 = 80. In addition, for example, when two of the "four consecutive amino acid residues having an average hydropathy index value of 2.6 or more" overlap by an amino acid residue, in the region where the average hydropathy index value of the four consecutive amino acid residues is 2, 6 or more, the number of amino acid residues being included is 7 [ = (2 × 4) - 1 = 7]. “- 1” is the deduction for overlap).
[00233] [00233] In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, even more preferably 20% or more, and even more
[00234] [00234] The modified fifth fibroin can be obtained, for example, by modifying a naturally occurring fibroin amino acid sequence cloned into an amino acid sequence containing a region having a high hydropathy index by replacing one or a plurality of hydrophilic amino acid residues in REP (eg, amino acid residues having a negative hydropathy index) by hydrophobic amino acid residues (eg, amino acid residues having a positive hydropathy index), and / or insertion of one or a plurality of hydrophobic amino acid residues in REP, so that the p / q condition is satisfied. Alternatively, the modified fifth fibroin can also be obtained, for example, by designing an amino acid sequence that satisfies the p / q condition based on the naturally occurring fibroin amino acid sequence and by chemical synthesis of a nucleic acid that encodes the sequence amino acids drawn. In any case, in addition to the modification equivalent to replacing one or a plurality of amino acid residues in REP with amino acid residues with a high hydropathy index and / or the insertion of one or a plurality of amino acid residues with a high index of hydropathy in REP, compared to naturally occurring fibroin, additional modification equivalent to the substitution, deletion, insertion, and / or addition of one or a plurality of amino acid residues can be performed.
[00235] [00235] Amino acid residues with a high hydropathic index are preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A) , and more preferably valine (V), leucine (L), and isoleucine (I), but not particularly refer to them.
[00236] [00236] More specific examples of the modified fifth fibroin may include a modified fibroin including (5-i) the sequence of
[00237] [00237] The modified fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 43 is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at two sites in each REP with respect to the amino acid sequence shown in SEQ ID NO: 31 (Met -PRT410), except the domain sequence at the end of the C-terminal side, and additionally replacing a part of glutamine residues (Q) with serine residues (S) and deletion of amino acids on the C-terminal side. The amino acid sequence shown in SEQ ID NO: 44 is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at one site in each REP with respect to the amino acid sequence shown in SEQ ID NO: 42 (Met -PRT525). The amino acid sequence shown in SEQ ID NO: 45 is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at two sites in each REP with respect to the amino acid sequence shown in SEQ ID NO: 32.
[00238] [00238] The modified (5-i) fibroin can consist of the amino acid sequence shown in SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45.
[00239] [00239] Modified (5-ii) fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45. Modified (5-ii) fibroin is also a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m. The sequence identity is preferably 95% or more.
[00240] [00240] The modified fibroin of (5-ii) preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45, and preferably has an amino acid sequence in which p / q is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from a (A) n motif located on the most C-terminal side of the terminal -C of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average hydropathy index value of four consecutive amino acid residues is 2.6 or more is defined as p, and the total number of amino acid residues contained in the sequence excluding the sequence from motif (A) n located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q.
[00241] [00241] The modified fifth fibroin may include a tag sequence at either one or both of the N-terminus and the C-terminus.
[00242] [00242] A more specific example of the modified fibroin including a tag sequence may be a modified fibroin including (5-iii) the amino acid sequence shown in SEQ ID NO: 46 (PRT720), SEQ ID NO: 47 (PRT665), or SEQ ID NO: 48 (PRT666), or (5-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.
[00243] [00243] The amino acid sequences shown in SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48 are respectively amino acid sequences obtained by adding the amino acid sequence (including a His tag and a hinge sequence) shown in SEQ ID NO: 35 to the N-terminus of the amino acid sequences shown in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45.
[00244] [00244] Modified (5-iii) fibroin may consist of the sequence
[00245] [00245] Modified (5-iv) fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48. Modified (5-iv) fibroin is also a protein including a domain sequence represented by Formula 1: [motif (A) n-REP] m. The sequence identity is preferably 95% or more.
[00246] [00246] The modified fibroin of (5-iv) preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, and preferably has an amino acid sequence in which p / q is 6.2% or more, in a case where in all REPs included in a sequence excluding a sequence from a (A) n motif located on the most C-terminal side of the terminal -C of the domain sequence from the domain sequence, the total number of amino acid residues contained in a region where an average hydropathy index value of four consecutive amino acid residues is 2.6 or more is defined as p, and the total number of amino acid residues contained in the sequence excluding the sequence from motif (A) n located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q.
[00247] [00247] The fifth modified fibroin may include a secretory signal for the release of the protein produced in the recombinant protein production system out of a host. The sequence of the secretory signal can be appropriately defined depending on the type of the host.
[00248] [00248] The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residue, compared to naturally occurring fibroin. Preferred and similar aspects of the modified sixth fibroin are as described above.
[00249] [00249] As the modified fibroin in the fibroin fiber artificially modified according to the present modality, the sixth modified fibroin is preferable due to the fact that when the dimension changes (elongation and shrinkage) due to contact with water and subsequent drying, the degree of elongation is approximately equal to the degree of shrinkage (restoration rate is close to 100%) and the modified sixth fibroin has the characteristic of being able to restore itself to its original length, and the change is reduced dimension of the sixth modified fibroin due to contact with water and subsequent drying.
[00250] [00250] The fibroin fiber artificially modified according to the present modality can be obtained by a production method including a shrinkage step of shrinking a raw fiber obtained by spinning the modified fibroin with water. The shrinkage step may include, for example, a step (contact step) of irreversibly shrinking the raw fiber (the raw fiber before contacting the water after spinning) when placing the raw fiber in contact with the water. The shrinkage step may include a step (drying step) of drying and additionally shrinking the fiber after the contact step.
[00251] [00251] Raw fiber can be produced using a known spinning method. Specifically, for example, the raw fiber can be produced according to the fibroin fiber spinning method described above.
[00252] [00252] Fig. 3 is an illustrative view schematically showing an example of a spinning device for the production of raw fiber. A spinning device 10 shown in Fig. 3 is an example of a spinning device for wet-dry spinning, and includes an extrusion device 1, an unstretched yarn production device 2, a hot-draw device wet 3, and a drying device 4.
[00253] [00253] A wiring method using the 10 spinning device will be
[00254] [00254] Coagulation liquid 11 can be any solvent that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone. The clotting liquid 11 may suitably contain water. The temperature of the coagulation liquid 11 is preferably 0 ° C to 30 ° C. In a case in which a syringe pump having a nozzle with a diameter of 0.1 mm to 0.6 mm is used as the spinner 9, the extrusion speed is preferably 0.2 ml / h per hole at 6, 0 ml / h per well and more preferably from 1.4 ml / h per well to 4.0 ml / h per well. The distance that the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the wire guide 18a to the wire guide 18b) can be a length that allows desolvation, for example, from 200 mm to 500 mm. The speed of removal of the unstretched yarn can be, for example, from 1 m / min to 20 m / min and preferably from 1 m / min to 3 m / min. THE
[00255] [00255] As the stretching carried out when raw fiber is obtained, for example, a pre-stretching carried out in the coagulation liquid bath 20 and a hot-wet stretching carried out in the stretching bath 21 are used, and also a hot-dry stretch.
[00256] [00256] Hot-humid stretching can be performed in hot water, in a solution obtained by adding an organic solvent or similar to hot water, or in heated water vapor. The temperature can be, for example, 50 ° C to 90 ° C and preferably 75 ° C to 85 ° C. In hot-wet drawing, the unstretched yarn (or the pre-stretched yarn) can be stretched, for example 1 to 10 times and preferably 2 to 8 times.
[00257] [00257] Hot-dry drawing can be carried out using an electric tubular oven, a dry heating plate,, or the like. The temperature can be, for example, from 140 ° C to 270 ° C and preferably from 160 ° C to 230 ° C. In hot-dry drawing, the unstretched yarn (or the pre-stretched yarn) can be stretched, for example, 0.5 to 8 times and preferably 1 to 4 times.
[00258] [00258] Hot-wet drawing and hot-dry drawing can be performed independently or in combination, or can be performed in multiple stages. That is, the hot-wet stretch and the hot-dry stretch can be carried out in a suitable combination, for example, in a way in which a first stretch
[00259] [00259] The lower limit value of the final stretch ratio with respect to the unstretched yarn (or the pre-stretched yarn) is preferably any greater than 1 time, 2 times or more, 3 times or more, 4 times or more , 5 times or more, 6 times or more, 7 times or more, 8 times or more, or 9 times or more, and the upper limit value is preferably 40 times or less, 30 times or less, 20 times or less , 15 times or less, 14 times or less, 13 times or less 12 times or less, 11 times or less, or 10 times or less. In a case in which the raw fiber is a spun fiber at a stretch ratio of 2 times or more, the shrinkage rate, when the raw fiber is moistened when brought into contact with water, becomes higher.
[00260] [00260] As shown in Fig. 2, the raw fiber (fiber containing the modified fibroin) has a shrinkage characteristic (change in length indicated by the “first shrinkage” in Fig. 2) by contact (wetting) with water. After the first shrinkage, the raw fiber shrinks even further when dried (change in length indicated by the “second shrinkage” in Fig. 2). After the second shrinkage, the fibroin fiber stretches back to the same or similar length before the second shrinkage by contact with water, and then repeats the shrinkage and stretching over a length range (indicated by the “rate stretch ”in Fig. 2) similar to the length range in the second shrinkage in a case of subsequent repetition of drying and wetting. Consequently, fiber
[00261] [00261] It is considered that irreversible shrinkage (“first shrinkage” in Fig. 2) of the raw fiber (of the fiber containing the modified fibroin) occurs in the contact step, for example, due to the following reasons. That is, it is considered a reason due to the secondary structure and the tertiary structure of the raw fiber (of the fiber containing modified fibroin). Another reason is considered that, for example, in the raw fiber (in the fiber containing modified fibroin) having a residual tension due to stretching or similar in the production process, the residual tension is relaxed by the water entering between the fibers or into the fiber . Consequently, it is considered that it is possible to freely control the shrinkage rate of the raw fiber (of the fiber containing modified fibroin) in the shrinking step, for example, according to the size of the stretch ratio in the manufacturing process of the raw fiber (of the fiber containing modified fibroin) described above.
[00262] [00262] In the contact stage, the raw fiber before contact with water and after spinning is placed in contact with water to make the raw fiber moist. The wet state means a state in which at least part of the raw fiber is moistened with water. As a result, the raw fiber can be shrunk without external force. This shrinkage is irreversible (corresponds to the “first shrinkage” in Fig. 2).
[00263] [00263] The temperature of the water contacted with the raw fiber in the contact step may be lower than the boiling point. As a result, handling and applicability in the shrinking step are improved. In addition, from the point of view of shortening the shrinking time, the lower limit value of the water temperature is preferably 10 ° C or higher, more preferably 40 ° C or higher,
[00264] [00264] In the contact step, the method of putting water in contact with the raw fiber is not particularly limited. Examples of the method include a method of immersing the raw fiber in water, a method of spraying water on the raw fiber at room temperature, a method of exposing the raw fiber to heated water vapor, or the like, and a method of exposure of the raw fiber to a high humidity environment filled with water vapor. Among these methods, the method of immersing the raw fiber in water is preferable in the contact step, because the shrinking time can be effectively shortened and the processing equipment can be simplified.
[00265] [00265] In a case in which the raw fiber is brought into contact with water in a relaxed state in the contact step, the raw fiber can not only be shrunk but also curled to become wavy. For the purpose of preventing the occurrence of curling, for example, the contact step can be performed in a state in which the raw fiber is not relaxed, for example in a state in which the raw fiber is brought into contact with water while is being tensioned (pulled) in an axial direction of the fiber.
[00266] [00266] The method for producing the artificially modified fibroin fiber according to the present modality may additionally include the drying step. The drying step is a drying step and additionally shrinks the raw fiber (or the artificially modified fibroin fiber obtained through the contact step) that has passed through the contact step (corresponds to the “second shrinkage” in Fig. 2). Drying can be, for example, natural drying or forced drying using drying equipment. Like drying equipment, any known drying equipment of the type with
[00267] [00267] The fibroin fiber artificially modified according to the present modality is obtained, for example, by the production method described above, and therefore substantially does not contain residual stress generated by stretching in the spinning process.
[00268] [00268] Fibroin fiber artificially modified according to the present modality can have a restoration rate defined by Expression (1) of 95% or more. Expression (1): restoration rate = (length of the artificially modified fibroin fiber when dried from the wet state / length of the artificially modified fibroin fiber before it is wetted) × 100 (%)
[00269] [00269] It can be said that the higher the restoration rate defined by Expression (1), the more the artificially modified fibroin fiber can be restored to its original length when moistened / dried. In the fibroin fiber artificially modified according to the present modality, the restoration rate defined by Expression (1) is preferably 96% or more, more preferably 97% or more, still
[00270] [00270] Fibroin fiber artificially modified according to the present modality may have an elongation rate defined by Expression (4) of 17% or less. The elongation rate defined by Expression (4) is an index of elongation when the artificially modified fibroin fiber is in a moistened state. Expression (4): elongation rate = {(length of the artificially modified fibroin fiber when wetted / length of the artificially modified fibroin fiber before being wetted) - 1} × 100 (%)
[00271] [00271] The upper limit of the stretching rate defined by Expression (4) is, for example, 15% or less, 13% or less, 10% or less, or 5% or less, and the lower limit is, for example , more than 0%, 1% or more, 2% or more, 5% or more, 10% or more, or 13% or more. The elongation rate defined by Expression (4) can be, for example, more than 0% and 17% or less, more than 0% and 15% or less, 2% or more and 15% or less, 5% or more and 15% or less, 5% or more and 13% or less, 5% or more and 10% or less, more than 0% and 10% or less, or more than 0% and 5% or less.
[00272] [00272] Fibroin fiber artificially modified according to the present modality may have a shrinkage rate C defined by Expression (5) of 17% or less. The shrinkage rate C defined by Expression (5) is an index of shrinkage capacity when the artificially modified fibroin fiber is dried from a moistened state. Expression (5): shrinkage rate C = {1 - (length of artificially modified fibroin fiber when dried from the wet state / length of artificially fibroin fiber
[00273] [00273] The upper limit of the shrinkage rate C defined by Expression (5) is, for example, 15% or less, 13% or less, 10% or less, or 5% or less, and the lower limit is, for example, more than 0%, 1% or more, 2% or more, 5% or more, 10% or more, or 13% or more. The shrinkage rate C defined by Expression (5) can be, for example, more than 0% and 17% or less, more than 0% and 15% or less, 2% or more and 15% or less, 5% or more than 15% or less, 5% or more and 13% or less, 5% or more and 10% or less, more than 0% and 10% or less, or more than 0% and 5% or less.
[00274] [00274] The fibroin fiber artificially modified according to the present modality is preferably a fiber having a history of shrinkage of irreversibly being shrunk by contact with water after spinning and a shrinkage rate A defined by Expression (2) of 2% or more. The shrinkage rate A defined by Expression (2) is an index that indicates the characteristics related to the first shrinkage of the raw fiber (see Fig. 2). In a case where the shrinkage rate A defined by Expression (2) is 2% or more, the restoration rate defined by Expression (1) becomes higher. Expression (2): shrinkage rate A = {1 - (fiber length irreversibly shrunk by contact with water after spinning / fiber length before contact with water and after spinning)} × 100 (%)
[00275] [00275] The rate of shrinkage A defined by Expression (2) can be 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 10% or more, 15% or more, 20% or more, or 25% or more. The upper limit of the shrinkage rate A defined by Expression (2) is not particularly limited, but it is 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or
[00276] [00276] The fibroin fiber artificially modified according to the present modality is preferably of a fiber having a history of shrinkage of irreversibly being shrunk by contact with water after spinning and then additionally being shrunk by drying and a shrinkage rate B defined by Expression (3) of 7% or more. The shrinkage rate B defined by Expression (3) is an index that indicates the characteristics related to the first shrinkage and the second shrinkage of the raw fiber (see Fig. 2). In a case in which the shrinkage rate B defined by Expression (3) is more than 7%, the restoration rate defined by Expression (1) becomes higher. Expression (3): shrinkage rate B = {1 - (fiber length irreversibly shrunk by contact with water after spinning and then additionally shrunk by drying / fiber length before contact with water and after spinning)} × 100 (%)
[00277] [00277] The shrinkage rate B defined by Expression (3) can be 10% or more, 15% or more, more than 25%, 32% or more, 40% or more, 48% or more, 56% or more , 64% or more, or 72% or more. The upper limit of the shrinkage rate B defined by Formula (3) is not particularly limited, but it is usually 80% or less. EXAMPLES
[00278] [00278] Hereinafter, the present invention will be described more specifically based on the Examples. However, the present invention is not limited to the following Examples. [Test Example 1] [(1) Synthesis of nucleic acid encoding the modified fibroin and expression vector construction]
[00279] [00279] Based on the base sequence and the amino acid sequence of Nephila clavipes (GenBank Access Number of:
[00280] [00280] The amino acid sequence shown in SEQ ID NO: 8 (PRT410: Test Example 1-1) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 1 (M_PRT410). M_PRT410 (SEQ ID NO: 1) is a modified amino acid sequence obtained by changing the number of consecutive alanine residues in reason (A) n to five, or similar, in order to improve productivity, based on the base sequence and the amino acid sequence of Nephila clavipes (GenBank Accession Number: P46804.1, GI: 1174415) which is naturally occurring fibroin. However, since M_PRT410 (SEQ ID NO: 1) has no modification of glutamine residue (Q), the glutamine residue content rate of the same is the same as the naturally occurring glutamine residue content of fibroin.
[00281] [00281] The amino acid sequence shown in SEQ ID NO: 9 (PRT888: Test Example 1-2) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 2 (M_PRT888). M_PRT888 (SEQ ID NO: 2) is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with VL.
[00282] [00282] The amino acid sequence shown in SEQ ID NO: 10 (PRT965: Test Example 1-3) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 3 (M_PRT965). M_PRT965 (SEQ ID NO: 3) is
[00283] [00283] The amino acid sequence shown in SEQ ID NO: 11 (PRT889: Test Example 1-4) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 4 (M_PRT889). M_PRT889 (SEQ ID NO: 4) is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with VL and replacing the remaining Q with I.
[00284] [00284] The amino acid sequence shown in SEQ ID NO: 12 (PRT916: Test Example 1-5) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 5 (M_PRT916). M_PRT916 (SEQ ID NO: 5) is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with VI and replacing the remaining Q with L.
[00285] [00285] The amino acid sequence shown in SEQ ID NO: 13 (PRT918: Test Example 1-6) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 6 (M_PRT918). M_PRT918 (SEQ ID NO: 6) is obtained by replacing all QQs in M_PRT410 (SEQ ID NO: 1) with FV and replacing the remaining Q with I.
[00286] [00286] The amino acid sequence shown in SEQ ID NO: 14 (PRT720: Test Example 1-7) is obtained by inserting an amino acid sequence consisting of three amino acid residues (VLI) at two sites in each REP with respect to PRT410 (SEQ ID NO: 8), and by deleting a part of the amino acids on the N-terminal side of it so that its molecular weight is adjusted to approximately the same as
[00287] [00287] The amino acid sequence shown in SEQ ID NO: 18 (PRT525: Test Example 1-8) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 15 (M_PRT525). M_PRT525 (SEQ ID NO: 15) is obtained, with respect to M_PRT410 (SEQ ID NO: 1), by inserting two alanine residues in a region (A5) in which the alanine residues are consecutive, and by deleting two domain sequences on the C-terminal side and by replacing 13 glutamine residues (Q) with serine residue (S) or proline residue (P) so that its molecular weight becomes the same as that of M_PRT410 .
[00288] [00288] The amino acid sequence shown in SEQ ID NO: 19 (PRT699: Test Example 1-9) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 16 (M_PRT699). M_PRT699 (SEQ ID NO: 16) is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 15) with VL.
[00289] [00289] The amino acid sequence shown in SEQ ID NO: 20 (PRT698: Test Example 1-10) is obtained by adding the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) to the terminal -N of the amino acid sequence shown in SEQ ID NO: 17 (M_PRT698). M_PRT698 (SEQ ID NO: 17) is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 15) with VL and replacing the remaining Q with I.
[00290] [00290] Each of the nucleic acids designed to encode proteins having amino acid sequences presented in SEQ ID NOs: 8 to 14 and SEQ ID NOs: 18 to 20 were synthesized. In the nucleic acid, a site
[00291] [00291] Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector including each of the nucleic acids encoding proteins having the amino acid sequences shown in SEQ ID NOs: 8 to 14 and SEQ ID NOs: 18 to 20. The transformed Escherichia coli was grown in 2 ml of an LB medium containing ampicillin for 15 hours. The culture solution was added to 100 ml of a seed culture medium (Table 5) containing ampicillin so that the DO600 was 0.005. While maintaining the temperature of the culture solution at 30 ° C, the flask culture was performed (for about 15 hours) until the DO600 reached 5, thus obtaining a seed culture solution. [Table 5] Seed culture medium (per 1 L at the start of the culture) Glucose 5g KH2PO4 4g K2HPO4 10g Yeast Extract 6g
[00292] [00292] The seed culture solution was added to a vessel fermenter containing 500 ml of production medium (Table 6) so that the transformed Escherichia coli was inoculated at 0.05 OD600. The culture was performed while maintaining the temperature of the culture solution at 37 ° C and controlling the constant pH at 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
[00293] [00293] Immediately after the complete consumption of glucose in the production medium, a feed solution (455 g / 1L of glucose and 120 g / 1L of Yeast Extract) was added at a rate of 1 mL / min. The culture was performed while maintaining the temperature of the culture solution at 37 ° C and controlling the constant pH at 6.9. In addition, the concentration of oxygen dissolved in the culture solution was maintained at 20% of the saturation concentration of dissolved oxygen, and the culture was performed for 20 hours. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target protein. 20 hours after adding IPTG, the culture solution was centrifuged to recover the bacterial cell pellet. SDS-PAGE was performed using bacterial cell pellets prepared from the culture solution before the addition of IPTG and after the addition of IPTG, and the expression of the target protein was checked for the appearance, dependent on the addition of IPTG, of an equivalent band to a size of the target protein. [(3) Protein purification]
[00294] [00294] The bacterial cell pellets recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer solution (pH 7.4). The bacterial cell pellets after washing were suspended in a 20 mM Tris-HCl buffer solution (pH 7.4) containing 1 mM PMSF, and the cells in the suspension were ruptured with a high pressure homogenizer (available from GEA Niro Soavi SpA ). The disrupted cells were centrifuged to obtain a precipitate. The precipitate obtained was
[00295] [00295] Using as a DMSO solvent containing 4 wt% dissolved lithium chloride, the lyophilized protein powder of PRT410 (SEQ ID NO: 8: Test Example 1-1), PRT888 (SEQ ID NO: 9: Example of Test 1-2), PRT965 (SEQ ID NO: 10: Test Example 1-3), PRT889 (SEQ ID NO: 11: Test Example 1-4), PRT916 (SEQ ID NO: 12: Test Example 1 -5), PRT918 (SEQ ID NO: 13: Test Example 1-6), or PRT720 (SEQ ID NO: 14: Test Example 1-7) was added to the solvent so that its concentration was 24% in large scale. After dissolving with an aluminum block heater at 90 ° C for 1 hour, insoluble materials and bubbles were removed to obtain a doping liquid. [(5) Wiring]
[00296] [00296] The spinning liquid was added to a reserve tank until it was filled and was discharged from a single orifice nozzle, having a diameter of 0.1 mm or 0.2 mm, into a coagulation bath containing 100% mass methanol using a gear pump. The discharge rate was adjusted from 0.01 ml / min to 0.08 ml / min. After
[00297] [00297] The raw yarn obtained was arranged to be about 30 cm long and bundled to form a bundle of fibroin fibers having a fineness of 150 denier. With a lead weight of 0.8 g being attached to each bundle of fibroin fibers, the bundle of fibers was left immersed in water at 40 ° C for 10 minutes to cause the first shrinkage, and the length of the bundle of fibroin fibers was measured in the water. The bundle of fibroin fibers shrunk by the first shrinkage was removed from the water and dried at room temperature for 2 hours with the lead weight of 0.8 g being trapped. After drying, the length of each bundle of fibroin fibers was measured. This wetting and drying operation was repeated three times, and then the average length when wetted (Wet: unit cm) and the average length when dried (Cseco: unit cm) were determined to calculate the second shrinkage rate according to following expression. The results are shown in the Table. 7. Second shrinkage rate (%) = {1 - (Dry / Wet)} * 100 [Table 7] Second Protein Hydrophobicity content rate shrinkage residue
[00298] [00298] PRT720 (Test Example 1-7) has the amino acid sequence obtained by inserting hydrophobic amino acid residues in PRT410 (Test Example 1-1). Due to the insertion of hydrophobic amino acid residues, the glutamine residue content of PRT720 (Test Example 1-7) is slightly low and the hydrophobicity of REP is notably increased compared to PRT410 (Test Example 1-1). However, there was no difference in the second shrinkage rate between the PRT720 spun fiber (Test Example 1-7) and the PRT410 spun fiber (Test Example 1-1) (Test Example 1-1 and Sample of Test 1-7 in Table 7). From this result, it was found that the second shrinkage rate cannot be assumed to be reduced in a case in which only hydrophobicity is increased.
[00299] [00299] On the other hand, in PRT888 (SEQ ID NO: 9) in which REP was designed to have approximately the same hydrophobicity as that of REP in PRT720 (Test Example 1-7) by replacing glutamine residue (Q) in dominance by another amino acid residue so that the glutamine residue content was reduced (6.3%), a remarkable effect of reducing the second shrinkage rate was observed (Test Example 1-2 in Table 7). This effect of reducing the second shrinkage rate was observed even in a case in which the hydrophobicity of REP was not increased as in the case of PRT888, by further reducing the glutamine residue content (0%) (Test Example 1-3 in Table 7). In addition, the effect of reducing the second shrinkage rate was most notable in the case of further reduction of the glutamine residue content (0%) and replacement of the glutamine residue with an amino acid residue having higher hydrophobicity (Test Example 1 -4 to Test Example 1-6 in Table 7). [(7) Preparation of spinning liquid for film production]
[00300] [00300] PRT410 lyophilized protein powder (SEQ ID NO: 8: Test example 1-1), PRT525 (SEQ ID NO: 18: Test example 1-8), PRT699 (SEQ ID NO: 19: Example Test 1-9), or PRT698 (SEQ ID NO: 20: Test Example 1-10) was added to 99% hexafluoro-2-propanol (HFIP) at a concentration of 10% by mass, stirred at 400 rpm and at 55 ° C for 20 minutes, and dissolved to obtain a spinning liquid. [(8) Film leak molding]
[00301] [00301] A removable film (manufactured by Mitsui Chemicals, Inc., product number “SP-PET-01-75-BU”) using a silicone compound immobilized on the surface of a poly film ( ethylene terephthalate) (PET) having a thickness of 75 μm. Using a batch coating machine (manufactured by Imoto Seisakusho), the spinning liquid prepared above was cast by casting over the substrate surface under conditions of a feed speed of 20 mm / s and a slot width of 0.18 mm to prepare a wet film. [(9) Drying and desolvation]
[00302] [00302] The wet molded film was allowed to be left for 12 hours in a thermostatic chamber (manufactured by ESPEC CORP.) At 55 ° C and dried. Then, the dried film was removed from the substrate and left immersed in methanol for 12 hours. The molded wet film was again allowed to be left for 12 hours in a thermostatic chamber (manufactured by ESPEC CORP.) At 60 ° C and dried. The film obtained was cut into a 33 mm square and subjected to the following water impermeability assessment. [(10) Evaluation of the film's water tightness]
[00303] [00303] The 30 mm square cut film was placed inside a Falcon® tube containing a saturated aqueous solution of potassium sulfate (K2SO4 · H2O) so as not to be immersed inside the aqueous solution, and was allowed to be left in the humidity 98% high for 48 hours. So, the moisture content of the film was determined as a moisture content rate,
[00304] [00304] It has been found that the water absorbance of the film is reduced when the glutamine residue content is reduced and the impermeability to water is improved. [Test Example 2] [(1) Modified fibroin production]
[00305] [00305] The lyophilized powder of the modified fibroin was obtained in the same procedure as in Test Example 1 except that a modified fibroin having the amino acid sequence shown in SEQ ID NO: 23 (PRT917) and a modified fibroin having the sequence of amino acids shown in SEQ ID NO: 24 (PRT1028) as the modified fibroin. [(2) Raw fiber production]
[00306] [00306] Using as a DMSO solvent containing 4% by weight of dissolved lithium chloride, the lyophilized powder of modified fibroin was added to the solvent so that its concentration was 24% by weight. After dissolving with an aluminum block heater at 90 ° C for 1 hour, insoluble materials and bubbles were removed to obtain a spinning liquid (raw fiber spinning liquid).
[00307] [00307] The spinning liquid was added to a reserve tank until
[00308] [00308] Each raw fiber was arranged to be about 30 cm long and bundled to form a bundle of raw fibers having a fineness of 150 denier. With a lead weight of 0.8 g being trapped in each bundle of raw fibers, the bundle of raw fibers was left immersed in water at 40 ° C for 10 minutes to be shrunk (contact step) and was removed from the water. With the lead weight of 0.8 g being trapped in each bundle of raw fibers, the bundle of raw fibers was dried at room temperature for 2 hours (drying step) to obtain artificially modified fibroin fibers in Test Example 2- 1 and Test Example 2-2, each of which had different types of proteins. [(4) Evaluation of artificially modified fibroin fiber (water-elasticity)]
[00309] [00309] The lengths of the artificially modified fibroin fibers when wetted (lengths of artificially modified fibroin fibers before being wetted) in Test Example 2-1 and Test Example 2-2 obtained in (3). With a lead weight of 0.8 g being trapped in each artificially modified fibroin fiber, the bundle of raw fibers was left immersed in water at 4 ° C for 10 minutes. Then, the length of each artificially modified fibroin fiber was measured (fiber length artificially
[00310] [00310] As shown in Table 9, artificially modified fibroin fiber including modified fibroin having a reduced content of glutamine residues (Test Example 2-1 and Test Example 2-2) has the characteristic that it stretches when moistened and then restored to its original length when dried (restoration rate is 98.2% to 99%). In addition, the artificially modified fibroin fiber (Test Example 2-1 and Test Example 2-2) has both a low elongation rate and a low C shrinkage rate, and a dimensional change is suppressed due to contact with the water (and subsequent drying). [Test Example 3] [(1) Modified fibroin production]
[00311] [00311] The lyophilized powder of the modified fibroin was obtained in the same procedure as in Test Example 1 except that a modified fibroin having the amino acid sequence shown in SEQ ID NO: 42 (PRT399), a modified fibroin having the sequence of amino acids shown in SEQ ID NO: 36 (PRT380), a modified fibroin having the amino acid sequence shown in SEQ ID NO: 37 (PRT410), and a modified fibroin having the amino acid sequence shown in SEQ ID NO: 39 (PRT799) like modified fibroin. [(2) Raw fiber production]
[00312] [00312] Using as a DMSO solvent containing 4.0% by weight of dissolved lithium chloride, the lyophilized powder of modified fibroin was added to the solvent so that its concentration was 18% by weight or 24% by weight (see to Table 10), and dissolved for 3 hours using a stirrer. Then, insoluble materials and bubbles were removed to obtain a modified fibroin liquid.
[00313] [00313] Using the modified fibroin liquid obtained as a
[00314] [00314] The artificially modified fibroin fiber was produced by subjecting each raw fiber obtained in Production Examples 1 to 19 to the contact step of putting the raw fiber in contact with water, or to the drying step of drying the raw fiber. at room temperature after the contact step is complete. <Evaluation of the shrinkage rate A in the contact stage>
[00315] [00315] A plurality of raw fibers each having a length of 30 cm were cut from the coiled raw fibers obtained in Production Examples 1 to 19. The plurality of raw fibers were bundled together to form a bundle of raw fibers having a fineness of 150 denier. With a lead weight of 0.8 g being trapped in each bundle of raw fibers, each bundle of raw fibers was left immersed in water for 10 minutes at a temperature shown in Tables 11 to 14 (contact step). Then, the length of each bundle of raw fibers in water was measured. The measurement of the length of the raw fiber bundle in water was carried out with a lead weight of 0.8 g being trapped in the raw fiber bundle with the purpose of eliminating the crunch of the raw fiber bundle. Next, the shrinkage rate A (%) of each raw fiber was calculated according to Expression (2). In Expression (2), C0 represents the length of the fiber before contact with water and after spinning, and here C0 is 30 cm. Similarly, in Expression (2), Humid represents the length of the fiber irreversibly shrunk due to contact with water after spinning, and here C0 is the length of each bundle of raw fibers measured in water. Expression (2): shrinkage rate A = {1− (Wet / C0)} × 100 (%) <Evaluation of shrinkage rate B in the drying step>
[00316] [00316] After the contact stage was complete, the bundle of raw fibers was removed from the water. The bundle of raw fibers removed from the water was dried
[00317] [00317] The results are shown in Tables 11 to 14.
[00318] [00318] 1… extrusion device, 2… unstretched yarn production device, 3… hot-wet drawing device, 4 ... drying device, 6 ... spinning liquid, 10 ... device spinning, 20 ... coagulation liquid bath, 21 ... stretch bath, 36 ... raw fiber

权利要求:
Claims (40)
[1]
1. Modified fibroin, characterized by the fact that it comprises: a domain sequence represented by Formula 1: [motif (A) n-REP] m or Formula 2: [motif (A) n-REP] m-motif (A) n, where the domain sequence has an amino acid sequence with a reduced content of an amino acid equivalent of glutamine in which one or a plurality of glutamine residues in REP are deleted or replaced by other amino acid residues, in comparison with naturally occurring fibroin, where in Formula 1 and Formula 2, motif (A) n represents an amino acid sequence consisting of 4 to 27 amino acid residues and the number of alanine residues with respect to the total number of residues of amino acids in motif (A) n is 80% or more, REP represents a sequence of amino acids consisting of 10 to 200 amino acid residues, m represents an integer from 10 to 300, a plurality of motifs (A) n can be the same amino acid sequence or different sequences number of amino acids, and a plurality of REPs can be the same amino acid sequence or different amino acid sequences.
[2]
2. Modified fibroin according to claim 1, characterized by the fact that the REP contains a GPGXX motif (where X represents an amino acid residue other than a glycine residue) and has a GPGXX motif content of 10% or more.
[3]
Modified fibroin according to claim 1 or 2, characterized by the fact that a glutamine residue content rate is 9% or less.
[4]
Modified fibroin according to any one of claims 1 to 3, characterized in that the other amino acid residues are amino acid residues selected from the group consisting of 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).
[5]
Modified fibroin according to any one of claims 1 to 4, characterized by the fact that a REP hydrophobicity is -0.8 or more.
[6]
Modified fibroin according to any one of claims 1 to 5, characterized in that it further comprises: an amino acid sequence equivalent to an amino acid sequence in which one or a plurality of amino acid residues are substituted, deleted, inserted and / or added compared to naturally occurring fibroin.
[7]
7. Modified fibroin according to claim 6, characterized by the fact that naturally occurring fibroin is a fibroin derived from insects or spiders.
[8]
8. Modified fibroin according to claim 7, characterized by the fact that naturally occurring fibroin is a spider larger ampule gland (MaSp) protein or spider smaller ampule gland (MiSp) araneid protein.
[9]
9. Modified fibroin, characterized by the fact that it comprises: an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, or SEQ ID NO: 17; or an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 16, or SEQ ID NO: 17.
[10]
Modified fibroin according to any one of claims 1 to 9, characterized in that it further comprises: a tag sequence at either or both of an N-terminus and a C-terminus.
[11]
11. Modified fibroin according to claim 10, characterized in that the tag sequence includes an amino acid sequence shown in SEQ ID NO: 7.
[12]
12. Modified fibroin, characterized by the fact that it comprises: an amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, or SEQ ID NO: 20; or an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 19, or SEQ ID NO: 20.
[13]
13. Nucleic acid, characterized by the fact that it encodes the modified fibroin as defined in any one of claims 1 to
12.
[14]
14. Nucleic acid, characterized by the fact that it hybridizes to a complementary strand of the nucleic acid as defined in claim 13 under stringent conditions, and encodes a modified fibroin including a domain sequence represented by Formula 1: [motif (A) n-REP ] m or Formula 2: [motif (A) n-REP] m-motif (A) n, in which in Formula 1 and Formula 2, motif (A) n represents an amino acid sequence consisting of 4 to 27 residues number of amino acids and the number of alanine residues with respect to the total number of amino acid residues in reason (A) n is 80% or more, REP represents a sequence of amino acids consisting of 10 to 200 amino acid residues, m represents a number integer from 10 to 300, a plurality of motifs (A) n can be the same sequence of amino acids or different sequences of amino acids, and a plurality of REPs can be the same sequence of amino acids or different sequences of amino acids.
[15]
15. Nucleic acid, characterized by the fact that it has 90% or more sequence identity with the nucleic acid, as defined in claim 13, and encodes a modified fibroin including a domain sequence represented by Formula 1: [reason (A) n-REP] m or Formula 2: [motif (A) n-REP] m-motif (A) n, in which in Formula 1 and Formula 2, motif (A) n represents an amino acid sequence consisting of 4 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in reason (A) n is 80% or more, REP represents an amino acid sequence consisting of 10 to 200 amino acid residues, m represents an integer from 10 to 300, a plurality of motifs (A) n can be the same sequence of amino acids or different sequences of amino acids, and a plurality of REPs can be the same sequence of amino acids or different sequences of amino acids.
[16]
16. Expression vector, characterized by the fact that it comprises: the nucleic acid sequence, as defined in any one of claims 13 to 15; and one or a plurality of regulatory sequences functionally linked to the nucleic acid sequence as defined in any one of claims 13 to 15.
[17]
17. Expression vector according to claim 16, characterized by the fact that it is a plasmidial vector or a viral vector.
[18]
18. Host, characterized by the fact that it is transformed with the expression vector as defined in claim 16 or 17.
[19]
19. Host according to claim 18, characterized by the fact that he is a prokaryote.
[20]
20. Host according to claim 19, characterized by the fact that the prokaryote is a microorganism belonging to a genus selected from the group consisting of Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
[21]
21. Host according to claim 18, characterized by the fact of being a eukaryote.
[22]
22. Host according to claim 21, characterized by the fact that the eukaryote is a yeast, a filamentous fungus, or an insect cell.
[23]
23. Host according to claim 22, characterized by the fact that yeast is a yeast belonging to a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia, and Hansenula.
[24]
24. Host according to claim 23, characterized by the fact that the yeast belonging to a genus Saccharomyces is Saccharomyces cerevisiae, the yeast belonging to a genus Schizosaccharomyces being Schizosaccharomyces pombe, the yeast belonging to a genus Kluyveromyces is Kluyveromyces lactose, Kluyveromyces lactis belonging to a genus Trichosporon is Trichosporon pullulans, yeast belonging to a genus Schwanniomyces is Schwanniomyces alluvius, yeast belonging to a genus Pichia is Pichia pastoris, yeast belonging to a genus Candida is Candida albicans, the yeast belonging to a genus Yarrowia is Yarrowia lipolytica, and yeast belonging to a genus Hansenula is Hansenula polymorpha.
[25]
25. Host according to claim 22, characterized by the fact that the filamentous fungus is a filamentous fungus belonging to a genus selected from the group consisting of Aspergillus, Penicillium, and Mucor.
[26]
26. Host according to claim 25, characterized by the fact that the filamentous fungus belonging to a genus Aspergillus is Aspergillus oryzae, the filamentous fungus belonging to a genus Penicillium is Penicillium chrysogenum, and the filamentous fungus belonging to a genus Mucor is Mucor fragilis.
[27]
27. Host according to claim 22, characterized by the fact that the insect cell is a lepidopteran insect cell.
[28]
28. Host according to claim 27, characterized in that the insect cell is an insect cell derived from Spodoptera frugiperda or an insect cell derived from Trichoplusia ni.
[29]
29. Product comprising the modified fibroin as defined in any one of claims 1 to 12, the product characterized by the fact that it is selected from the group consisting of a fiber, a thread, a film, a foam, a grain, a nanofibril, a gel, and a resin.
[30]
30. Artificially modified fibroin fiber, characterized by the fact that it comprises a modified fibroin, with the artificially modified fibroin fiber stretching when moistened and shrinking when dried from the wetted state.
[31]
31. Artificially modified fibroin fiber according to claim 30, characterized by the fact that a restoration rate defined by Expression (1) is 95% or more, where restoration rate = (length of artificially modified fibroin fiber when dried from the moistened state / length of the artificially modified fibroin fiber before being moistened) × 100 (%) --- (1).
[32]
32. Artificially modified fibroin fiber according to claim 30 or 31, characterized by the fact that the artificially modified fibroin fiber is a fiber having a history of irreversibly shrinking being shrunk by contact with water after spinning, and a shrinkage rate A defined by Expression (2) of 2% or more, where shrinkage rate A = {1 - (fiber length irreversibly shrunk by contact with water after spinning / fiber length before contact with water and after wiring)} × 100 (%) --- (2).
[33]
33. Artificially modified fibroin fiber according to any of claims 30 to 32, characterized by the fact that the artificially modified fibroin fiber is a fiber having a history of irreversibly shrinking being shrunk by contact with water after spinning and then additionally being shrunk by drying, and a shrinkage rate B defined by Expression (3) of greater than 7%, where shrinkage rate B = {1 - (length of the fiber irreversibly shrunk by contact with water after spinning and then further shrunk by drying / length of the fiber before contact with water and after spinning)} × 100 (%) --- (3).
[34]
34. Artificially modified fibroin fiber according to any one of claims 30 to 33, characterized in that the modified fibroin is the modified fibroin as defined in any one of claims 1 to 12.
[35]
35. Fibroin fiber artificially modified according to any of claims 30 to 34, characterized by the fact that an elongation rate defined by Expression (4) is 17% or less, where elongation rate = {(length of artificially modified fibroin fiber when moistened / length of artificially modified fibroin fiber before being moistened) - 1} × 100 (%) --- (4).
[36]
36. Fibroin fiber artificially modified according to any of claims 30 to 35, characterized by the fact that a shrinkage rate C defined by Expression (5) is 17% or less, where shrinkage rate C = {1 - (length of artificially modified fibroin fiber when dried from the wet state / length of artificially modified fibroin fiber when wet)} × 100 (%) --- (5).
[37]
37. Method for the production of an artificially modified fibroin fiber, the method characterized by the fact that it comprises: a shrinking step of placing a raw fiber, before contact with water and after spinning, in contact with water for cause irreversible shrinkage, and then dry the raw fiber to cause additional shrinkage, the raw fiber including a modified fibroin.
[38]
38. Production method according to claim 37, characterized in that the raw fiber A shrinkage rate defined by Expression (2) is 2% or more, wherein the shrinkage rate A = {1 - (fiber length irreversibly) shrunk by contact with water after spinning / fiber length before contact with water and after spinning)} × 100 (%) --- (2).
[39]
39. The production method of claim 37 or
38, characterized by the fact that a shrinkage rate B of the raw fiber defined by Expression (3) is greater than 7%, in which the shrinkage rate B = {1 - (length of the fiber irreversibly shrunk by contact with water after spinning and then further shrunk by drying / length of the fiber before contact with water and after spinning)} × 100 (%) --- (3).
[40]
40. Production method according to any one of claims 37 to 39, characterized in that the modified fibroin is the modified fibroin as defined in any one of claims 1 to
12.

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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JP2017-144586|2017-07-26|

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JP2017144586|2017-07-26|

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PCT/JP2018/027974|WO2019022163A1|2017-07-26|2018-07-25|Modified fibroin|

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