Modified fibroin

Abstract

This invention relates to a modified fibroin which includes a domain sequence represented by formula 1, [(A)n motif-REP]m, or formula 2, [(A)n motif-REP]m-(A)n motif. In the (A)n motif, the total number of alanine residues in repetitions of six or more residues is at least 20% of the total number of amino acid residues in the entire motif, the hydrophobicity of REP is -1.0 or greater, and the total number of amino acid residues in the entire motif is 580 or greater.

Description

[Technical field]

[0001] The present invention relates to modified fibroins. [Background technology]

[0002] Fibroin is a type of fibrous protein that contains up to 90% glycine, alanine and serine residues that lead to the formation of β-pleated sheets (Non-Patent Document 1). Known examples of fibroin include proteins that constitute the silk produced by insects and spiders (silk proteins, hornet silk proteins, spider silk proteins, etc.).

[0003] Silk protein has excellent mechanical properties, moisture absorption properties, and deodorizing properties, and is widely used as a material for clothing. Silk thread is also used for surgical sutures, etc., because it is a natural fiber with immune tolerance and high biocompatibility.

[0004] Spiders have up to seven types of silk glands, each producing fibroin (spider silk protein) with different properties. Spider silk proteins are named according to their source organs: major ampullate spider protein (MaSp), which has high toughness, minor ampullate spider protein (MiSp), which has high elongation force, and flagelliform (Flag), tubuliform, aggregate, aciniform, and pyriform spider silk proteins. In particular, structural research has been intensively conducted on major ampullate spider protein, which has high toughness due to its excellent strength and elongation (Patent Document 1 and Patent Document 2).

[0005] One of the structures specific to fibroin is the GPGXX, an alanine-rich extension ((A) n Or (GA) n ), GGX, and a structure in which amino acid motifs classified as spacers are repeated are known (Non-Patent Document 2). n Motif (A) n The elongation was increased by increasing the number of GPGXX motifs. n It has been reported that substitution with the motif increases the tensile strength (Patent Document 2). In addition, the GGX and GPGXX motifs are thought to form a flexible helical structure that gives elasticity to the thread (Patent Document 3).

[0006] Recombinant spider silk proteins and recombinant silk proteins have been produced in several heterologous protein production systems. For example, there have been many reports of recombinant fibroin production using recombinant protein production systems with hosts such as goats, silkworms, plants, mammalian cells, yeasts, molds, gram-negative bacteria, and gram-positive bacteria, and certain results have been obtained (Non-Patent Document 3, Patent Documents 4 and 5).

[0007] In recent years, there has been an increasing demand for heat-resistant materials in various industrial fields, including automobiles, electrical and electronics. Silk, which is made of fibroin, has an elegant texture, beautiful luster, and is comfortable to wear, but it has drawbacks such as shrinking and yellowing when wet, and it is recommended to iron it at a medium temperature of up to 160°C, so it is generally considered to be a fiber with low heat resistance. In order to use it as a material for heat-resistant composite materials, it is first necessary to improve the heat resistance of the silk protein itself. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2012-55269 A [Patent Document 2] Special Publication No. 2005-502347 [Patent Document 3] Special Publication No. 2009-505668 [Patent Document 4] Special Publication No. 2014-502140 [Patent Document 5] International Publication No. 2015 / 042164 [Non-patent literature]

[0009] [Non-Patent Document 1] Asakura et al., Encyclopedia of Agricultural Science, Academic Press: New York, NY, 1994, Vol. 4, pp. 1-11 [Non-Patent Document 2] Microbial Cell Factories,2004,3:14 [Non-Patent Document 3] Science, 2002, vol. 295, pp. 472-476 Summary of the Invention [Problem to be solved by the invention]

[0010] Natural spider silk made of fibroin has excellent properties against physical changes such as tensile strength, toughness, and extensibility, has high biocompatibility and biodegradability, and also has excellent heat resistance. However, unlike silkworms, which use natural silk industrially, it is difficult to mass-produce spiders, and therefore attempts have been made to produce artificial synthetic fibers that imitate spider silk and apply them to materials and ingredients such as silk and bedding that have the above-mentioned properties against physical changes, as well as biocompatibility and biodegradability, but no artificial fibers comparable in heat resistance to natural spider silk have been successfully produced.

[0011] Therefore, an object of the present invention is to provide a modified fibroin having improved thermal stability. [Means for solving the problem]

[0012] The present inventors have found that (A) present in fibroin n It has been found that by increasing the alanine content of the alanine-stranded sequence in the motif, a fibroin having improved thermal stability can be obtained. The present invention is based on this novel finding.

[0013] That is, the present invention relates to, for example, the following inventions. [1] Formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n A modified fibroin comprising a domain sequence represented by the motif: (A) above n the total number of consecutive alanine residues in the motif is 20% or more of the total number of amino acid residues in the entire motif, The hydrophobicity of REP is -1.0 or more; A modified fibroin having a total number of amino acid residues of 580 or more. [In formula 1 and formula 2, (A) n The motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) n The number of alanine residues relative to the total number of amino acid residues in the motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. Multiple (A) nThe motifs may be identical to each other or different amino acid sequences. The REPs present in multiple locations may be identical to each other or different amino acid sequences. [2] The modified fibroin according to [1], wherein the hydrophobicity of REP is 0 or more. [3] (A) above n The modified fibroin according to [1] or [2], wherein the total number of consecutive alanine residues in the motif that are 7 or more is 20% or more of the total number of amino acid residues. [4] A modified fibroin comprising an amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. [5] The modified fibroin according to any one of [1] to [4], further comprising a tag sequence at either or both of the N-terminus and the C-terminus. [6] The modified fibroin according to [5], wherein the tag sequence comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5. [7] A modified fibroin comprising an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. [8] The modified fibroin according to any one of [1] to [7], which has a thermal decomposition temperature (Td) of 268°C or higher. [9] A nucleic acid encoding the modified fibroin according to any one of [1] to [8].

[10] [9], which hybridizes under stringent conditions with the complementary strand of the nucleic acid according to the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n A nucleic acid encoding a modified fibroin comprising a domain sequence represented by the motif: n The motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) n The number of alanine residues relative to the total number of amino acid residues in the motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. Multiple (A) n The motifs may be identical to each other or different amino acid sequences. The REPs present in multiple locations may be identical to each other or different amino acid sequences.

[11] [9] has a sequence identity of 90% or more with the nucleic acid according to the present invention, and has the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n A nucleic acid encoding a modified fibroin comprising a domain sequence represented by a motif. [In formula 1 and formula 2, (A) n The motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) n The number of alanine residues relative to the total number of amino acid residues in the motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. Multiple (A) nThe motifs may be identical to each other or different amino acid sequences. The REPs present in multiple locations may be identical to each other or different amino acid sequences.

[12] A method for producing modified fibroin, comprising the steps of: expressing the nucleic acid by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence; A method for producing the modified fibroin, wherein the modified fibroin is the modified fibroin according to any one of [1] to [8].

[13] Formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n A method for improving the thermal stability of a modified fibroin comprising a domain sequence represented by a motif, comprising: At least one (A) in the unmodified fibroin n At least one (A) is introduced into the motif or REP by inserting one or more alanine residues or substituting other amino acid residues adjacent to the alanine residue with alanine. n increasing the number of consecutive alanine residues in the motif; A method in which the thermal decomposition temperature (Td) of the modified fibroin is 5°C or more higher than that of the unmodified fibroin. [In formula 1 and formula 2, (A) n The motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) n The number of alanine residues relative to the total number of amino acid residues in the motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. Multiple (A)n The motifs may be identical to each other or different amino acid sequences. The REPs present in multiple locations may be identical to each other or different amino acid sequences.

[14] The method according to

[13] , wherein the unmodified fibroin is naturally occurring fibroin.

[15] The modified fibroin according to

[14] , wherein the naturally occurring fibroin is fibroin derived from insects or arachnids.

[16] The modified fibroin according to

[15] , wherein the naturally occurring fibroin is a major ampullate spider protein (MaSp) or a minor ampullate spider protein (MiSp) of an arachnid.

[17] The modified fibroin according to any one of [1] to [8] is included, 1. An article of manufacture selected from the group consisting of fibers, yarns, films, foams, granules, nanofibrils, gels and resins. Effect of the Invention

[0014] According to the present invention, it is possible to provide a modified fibroin having improved thermal stability. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing an example of a domain arrangement of spider fibroin. [Diagram 2] 1 is a graph showing the results of comparing the thermal decomposition temperatures of PRT380 and PRT525. [Diagram 3] 1 is a graph showing the results of comparing the glass transition points of PRT380 and PRT525. [Figure 4] 1 is a graph showing the results of comparing the thermal decomposition temperatures of PRT1068, PRT1069, and PRT1070. [Diagram 5] 1 is a graph showing the results of comparing the thermal decomposition temperatures of PRT525 and PRT1070. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0017] [Modified fibroin] The modified fibroin according to the present invention is a fibroin represented by the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n The modified fibroin is a protein containing a domain sequence represented by a motif. The modified fibroin may have further amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically, but not limited to, regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of about 100 amino acid residues.

[0018] As used herein, the term "modified fibroin" refers to a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin. As used herein, the term "naturally occurring fibroin" refers to a fibroin whose amino acid sequence is identical to that of fibroin produced by naturally occurring insects or arachnids. Naturally occurring fibroin also has the structure represented by the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n A protein containing a domain sequence represented by a motif.

[0019] A "modified fibroin" may be one whose amino acid sequence has been modified based on naturally occurring fibroin (e.g., one whose amino acid sequence has been modified by modifying the gene sequence of a cloned naturally occurring fibroin), so long as it has an amino acid sequence specified in the present invention, or one whose amino acid sequence has been artificially designed and synthesized without relying on naturally occurring fibroin (e.g., one having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid that codes for a designed amino acid sequence). Note that modified fibroin whose amino acid sequence has been modified is also included in the modified fibroin, so long as the amino acid sequence is different from that of naturally occurring fibroin.

[0020] As used herein, the term "domain sequence" refers to a crystalline region specific to fibroin (typically, the (A) n A motif) and an amorphous region (typically corresponding to REP of an amino acid sequence), and the amino acid sequence is represented by the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n The amino acid sequence represented by the motif (A) n The motif shows an amino acid sequence mainly consisting of alanine residues, and the number of amino acid residues is 4 to 27. (A) n The number of amino acid residues in the motif may be 4 to 20, 8 to 20, 10 to 20, 4 to 16, 5 to 10, 6 to 12, 6 to 18, 7 to 10, 7 to 14, 8 to 16, or 11 to 16. n The ratio of the number of alanine residues to the total number of amino acid residues in the motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. There are multiple (A) n The motifs may be identical to each other or different from each other in amino acid sequence, and the multiple REPs may be identical to each other or different from each other in amino acid sequence.

[0021] (A)n The motif is (A) n The number of alanine residues relative to the total number of amino acid residues in the motif may be 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that the motif is composed only of alanine residues). n It is preferable that at least seven of the motifs are composed of only alanine residues. n The motif is (Ala) k (Ala represents an alanine residue, and k represents an integer of 4 to 27, preferably an integer of 4 to 20, and more preferably an integer of 4 to 16).

[0022] The modified fibroin according to this embodiment is (A) n The total number of consecutive alanine residues of 6 or more in the motif is 20% or more of the total number of all amino acid residues, the hydrophobicity of REP is -1.0 or more, and the total number of all amino acid residues is 580 or more.

[0023] The modified fibroin according to this embodiment is (A) n The total number of consecutive alanine residues in the motif is 20% or more of the total number of amino acid residues. (A) n The total number of six or more consecutive alanine residues in a motif means the total number of alanine residues when only the number of six or more consecutive alanine residues is counted. Therefore, the number of five or more consecutive alanine residues is not counted. n In the motif, even if there are six or more consecutive alanine residues due to the presence of other amino acid residues between the alanine residues, the number of the alanine residues is not counted.

[0024] (A) nThe total number of 6 or more consecutive alanine residues in the motif may be, for example, 21% or more, 22% or more, or 23% or more of the total number of all amino acid residues. n The total number of 7 or more consecutive alanine residues in the motif may be, for example, 20% or more, 21% or more, 22% or more, or 23% or more of the total number of all amino acid residues.

[0025] The modified fibroin according to this embodiment has a total number of amino acid residues of 580 or more. The total number of amino acid residues may be, for example, 590 or more, 600 or more, 610 or more, or 620 or more.

[0026] The modified fibroin according to this embodiment preferably contains at least one motif selected from the GGX motif and the GPGXX motif (G represents a glycine residue, P represents a phenylalanine residue, and X represents an amino acid residue other than a glycine residue) in the amino acid sequence of REP. By containing these motifs in REP, the extensibility of the modified fibroin can be improved.

[0027] When the modified fibroin according to the present embodiment contains a GPGXX motif in the REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and is preferably 10% or more. This allows the elongation of the modified fibroin to be further improved. There is no particular upper limit to the GPGXX motif content, and it may be 50% or less, or 30% or less.

[0028] As used herein, the "GPGXX motif content" is a value calculated by the following method.

[0029] Formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n In the fibroin containing the domain sequence represented by the motif, it is located at the most C-terminal side (A).n For all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, the total number of GPGXX motifs contained in that region is multiplied by three (i.e., the total number of G and P in the GPGXX motif), and the number of GPGXX motifs located at the most C-terminus side (A) is n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and further (A) n When the total number of amino acid residues of all REPs excluding the motif is y, the GPGXX motif content is calculated as x / y.

[0030] In calculating the GPGXX motif content, the most C-terminal (A) n The target sequence is the sequence excluding the sequence from the motif to the C-terminus of the domain sequence, which is the sequence located at the most C-terminus (A). n The "sequence from the motif to the C-terminus of the domain sequence" (sequence corresponding to REP) may contain sequences with low correlation with sequences characteristic of fibroin, and when m is small (i.e., when the domain sequence is short), this affects the calculation results of the GPGXX motif content, so this is to eliminate this influence. Note that when the "GPGXX motif" is located at the C-terminus of REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

[0031] FIG. 1 is a schematic diagram showing the domain sequence of a modified fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. 1. First, the domain sequence of the modified fibroin shown in FIG. n Motif-REP] m -(A) n In the 1990s, all REPs were located at the C-terminus (A) n Since the number of GPGXX motifs is 7, x is 7 × 3 = 21. Similarly, all REPs are located at the C-terminus (A) nSince the domain sequence is excluded from the sequence from the motif to the C-terminus of the domain sequence (the sequence shown as "Region A" in Figure 1), the sequence is further divided into (A) n The total number of amino acid residues in all REPs excluding motifs, y, is 50 + 40 + 10 + 20 + 30 = 150. Next, x / y (%) can be calculated by dividing x by y, which is 21 / 150 = 14.0% in the case of the modified fibroin in Figure 1.

[0032] The modified fibroin according to the present embodiment preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%, which allows the effects of the present invention to be exhibited more significantly.

[0033] In this specification, the "glutamine residue content" is a value calculated by the following method.

[0034] Formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n In the fibroin containing the domain sequence represented by the motif, it is located at the most C-terminal side (A). n In all REPs contained in the sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence (the sequence corresponding to "Region A" in Figure 1), the total number of glutamine residues contained in the region is w, and the glutamine residue located most C-terminally is (A) n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and further (A) n When the total number of amino acid residues of all REPs excluding the motif is y, the glutamine residue content is calculated as w / y. n The reason for targeting "the sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as that mentioned above.

[0035] The modified fibroin according to this embodiment has a domain sequence in which one or more glutamine residues in REP have been deleted or replaced with other amino acid residues, compared to the fibroin before modification. It may have an amino acid sequence corresponding to that.

[0036] The "other amino acid residue" may be any amino acid residue other than glutamine residue, but is preferably an amino acid residue with a higher hydrophobicity index than glutamine residue. The hydrophobicity index of amino acid residues is determined by a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132). Specifically, the hydrophobicity index (Hydropathy index, hereinafter also referred to as "HI") of each amino acid is as shown in Table 1 below.

[0037] [Table 1]

[0038] As shown in Table 1, examples of amino acid residues having a higher hydrophobicity index than glutamine residues include amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A) are more preferred, and amino acid residues selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are even more preferred.

[0039] In the modified fibroin according to the present embodiment, the hydrophobicity of REP may be -1.0 or more, preferably -0.9 or more, more preferably -0.8 or more, even more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.2 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of REP, and it may be, for example, 1.0 or less, or 0.7 or less. The hydrophobicity of REP may be, for example, -1.0 or more and 1.0 or less, or 0 or more and 0.7 or less.

[0040] In this specification, the "hydrophobicity of REP" is a value calculated by the following method.

[0041] Formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) In fibroin containing a domain sequence represented by the n motif, (A) is located at the most C-terminal end n For all REPs contained in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence (the sequence corresponding to "Region A" in Figure 1), the sum of the hydrophobicity indexes of each amino acid residue in that region is z, and the REP located at the most C-terminus side (A) n The sequence from the motif to the C-terminus of the domain sequence is removed from the domain sequence, and further (A) n When the total number of amino acid residues of all REPs excluding the motif is y, the hydrophobicity of the REP is calculated as z / y. n The reason for targeting "the sequence obtained by excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as that mentioned above.

[0042] In the modified fibroin according to the present embodiment, the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin is calculated, and then the sum is divided by the total number of amino acid residues (average HI) is preferably -1.0 or more, more preferably -0.9 or more, even more preferably -0.8 or more, even more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.2 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the average HI of the modified fibroin, and it may be, for example, 1.0 or less, or 0.7 or less. The average HI of the modified fibroin may be, for example, -1.0 or more and 1.0 or less, or 0 or more and 0.7 or less.

[0043] Naturally occurring fibroin has the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n A protein containing a domain sequence represented by a motif, specifically, for example, fibroin produced by insects or arachnids.

[0044] Examples of fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, and hornet silk proteins excreted by the larvae of the Japanese hornet (Vespa simillima xanthoptera).

[0045] A more specific example of fibroin produced by an insect is, for example, silkworm fibroin L chain (GenBank accession numbers M76430 (base sequence), AAA27840.1 (amino acid sequence)).

[0046] Examples of fibroin produced by spiders include spiders belonging to the Araneus genus, such as the Japanese squirrel spider, the Japanese garden squirrel spider, the red squirrel spider, the green squirrel spider, and the Japanese bean squirrel spider; spiders belonging to the Neoscona genus, such as the Japanese mountain squirrel spider, the Japanese house squirrel spider, and the Japanese Satsuma squirrel spider; spiders belonging to the Pronus genus, such as the Japanese squirrel spider; spiders belonging to the Cyrtarachne genus, such as the Japanese squirrel spider and the Japanese squirrel spider; spiders of the genus Gasteracantha such as the genus Acanthurus and the genus Siberian siberiana; spiders of the genus Ordgarius such as the genus Orbgarius and the genus Argiope such as the orb-weaver spider, the orb-weaver spider and the genus Arachnura such as the orb-weaver spider; spiders of the genus Acusilas such as the scraping spider; spiders of the genus Centipede such as the orb-weaver spider, the orb-weaver spider and the genus Centipede; spider silk proteins produced by spiders belonging to the genus Poltys such as the Japanese bush spider, the Japanese bush spider, the Japanese bush spider and the Japanese black bush spider, and spiders belonging to the genus Cyclosa such as the Japanese bush spider, the Japanese bush spider, the Japanese black bush spider and the Japanese black bush spider, and spiders belonging to the genus Chorizopes such as the Japanese black bush spider, and spiders belonging to the genus Tetragnatha such as the Japanese long-legged spider, the Japanese long-legged spider, the Japanese bush spider and the Japanese long-legged spider, and spiders belonging to the genus Tetragnatha such as the Japanese long-legged spider, the Japanese long-legged spider, the Japanese black bush spider and the Japanese black bush spider, spiders belonging to the genus Leucauge such as the medium-sized orb-weaver spider and the small orb-weaver spider; spiders belonging to the genus Nephila such as the orb-weaver spider and the giant orb-weaver spider; spiders belonging to the genus Menosira such as the golden spider; spiders belonging to the genus Dyschiriognatha such as the long-legged spider; spiders belonging to the genus Latrodectus such as the black widow spider, the redback spider, the gray widow spider and the three-spotted widow spider;Spider silk proteins include spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), etc.

[0047] More specific examples of fibroins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession numbers AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), major angu11ate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major anpullate spidroin 1 [Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank accession numbers CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence) and the like.

[0048] More specific examples of naturally derived fibroins include fibroins whose sequence information is registered in NCBI GenBank. For example, from among the sequence information registered in NCBI GenBank, sequences that include INV as DIVISION can be confirmed by extracting sequences with spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" as keywords in DEFINITION, sequences with a specific product character string in CDS, and sequences with a specific character string in TISSUE TYPE in SOURCE.

[0049] When fibroins whose amino acid sequence information is registered in NCBI GenBank were identified by the method described above, 663 types of fibroins (of which 415 were spider-derived fibroins) were extracted. Of all the extracted fibroins, those with the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n There were 129 types of naturally occurring fibroins containing domain sequences represented by the motif. Among these, naturally occurring fibroins with a GPGXX motif content of 10% or more calculated by the above-mentioned method were The loins were of six types as shown in Table 2. The glutamine residue content of each of the six naturally derived fibroins shown in Table 2 was 9.2% or more.

[0050] [Table 2]

[0051] The modified fibroin according to this embodiment may have, for example, the following domain sequence in the unmodified fibroin: (A) nIt can be obtained by inserting one or more alanine residues into the motif or REP, or by making a modification equivalent to substituting another amino acid residue adjacent to the alanine residue with alanine. Furthermore, there may be a modification of the amino acid sequence equivalent to substituting, deleting, inserting and / or adding one or more amino acid residues. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, it can be performed according to the methods described in literature such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0052] The modified fibroin according to the present embodiment has, for example, the following structure with respect to the gene sequence of a cloned naturally-occurring fibroin: (A) n It can be obtained by inserting one or more alanine residues into the motif or REP, or by adding a mutation corresponding to substituting another amino acid residue adjacent to the alanine residue with alanine. n It can also be obtained by inserting one or more alanine residues into the motif or REP, or by designing an amino acid sequence corresponding to the alanine residue by substituting other amino acid residues adjacent to the alanine residue with alanine, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0053] The molecular weight of the modified fibroin according to the present invention is not particularly limited, and may be, for example, 40 kDa or more and 700 kDa or less. The molecular weight of the modified fibroin according to the present invention may be, for example, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, and may be 600 kDa or less, 500 kDa or less, 400 kDa or less, 300 kDa or less, or 200 kDa or less.

[0054] More specific examples of the modified fibroin of the present invention include (i) a modified fibroin comprising the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0055] The modified fibroin (i) will be described. The amino acid sequence (Met-PRT1069) shown in SEQ ID NO: 1 is based on the base sequence and amino acid sequence of the naturally-occurring fibroin Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), and is (A) n The amino acid sequence of consecutive alanine residues in the motif was modified by increasing the number of consecutive alanine residues to six, substituting all QQ with VF, and substituting the remaining Q with I. The hydrophobicity of REP is 0.21.

[0056] The amino acid sequence (Met-PRT1070) shown in SEQ ID NO:2 is a sequence similar to that shown in SEQ ID NO:1, except that each of (A) n In the motif, one alanine residue was inserted to increase the number of consecutive alanine residues, and the hydrophobicity of REP is 0.21.

[0057] The amino acid sequence shown in SEQ ID NO:3 (Met-PRT1076) is obtained by deleting some amino acid residues in REP of the amino acid sequence shown in SEQ ID NO:2, and the hydrophobicity of REP is 0.21.

[0058] The amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 all have a content of 6 or more consecutive alanines of 20% or more (Table 3).

[0059] [Table 3]

[0060] The modified fibroin of (ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The modified fibroin of (ii) also ... n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n It is a protein comprising a domain sequence represented by a motif. The sequence identity may be 80% or more, or 85% or more, and is preferably 95% or more.

[0061] The modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows the isolation, immobilization, detection, visualization, etc. of the modified fibroin.

[0062] An example of a tag sequence is an affinity tag that utilizes specific affinity (binding ability, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). The His tag is a short peptide consisting of about 4 to 10 histidine residues lined up, and has the property of specifically binding to metal ions such as nickel, and can be used to isolate modified fibroin by metal chelating chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 (amino acid sequence containing a His tag).

[0063] Furthermore, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione, and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

[0064] Furthermore, an "epitope tag" utilizing an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, and FLAG tag. By using an epitope tag, the modified fibroin can be easily purified with high specificity.

[0065] Furthermore, a tag sequence that can be cleaved with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with the protease, the modified fibroin from which the tag sequence has been cleaved can be recovered.

[0066] More specific examples of modified fibroins containing a tag sequence include (iii) modified fibroins containing the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or (iv) modified fibroins containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0067] The amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 has an amino acid sequence in which the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5 (including a His tag) is added to the N-terminus of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, respectively.

[0068] The modified fibroin (iii) may consist of the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0069] The modified fibroin of (iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. The modified fibroin of (iv) also ... n Motif-REP] m Or formula 2: [(A)n Motif-REP] m -(A) n It is a protein comprising a domain sequence represented by a motif. The sequence identity may be 80% or more, or 85% or more, and is preferably 95% or more.

[0070] The amino acid sequences shown in SEQ ID NO:6 (PRT1069), SEQ ID NO:7 (PRT1070), and SEQ ID NO:8 (PRT1076) all have a content of 6 or more consecutive alanines of 20% or more (Table 4). In addition, the hydrophobicity of REP in each sequence is 0.21.

[0071] [Table 4]

[0072] The modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of the host.

[0073] [Thermal Stability] Examples of indicators for evaluating thermal stability (heat resistance) include thermal decomposition temperature (Td) and glass transition point (Tg). Thermal decomposition temperature is the temperature at which thermal decomposition of a material starts, and glass transition point is the temperature at which a material changes from crystal to liquid. It can be said that the higher the thermal decomposition temperature and / or glass transition point, the higher the thermal stability. The thermal decomposition temperature of a protein such as the modified fibroin of the present invention can be measured, for example, by thermogravimetric analysis, which observes the mass change of a sample accompanying heating. In addition, the glass transition point of a protein such as the modified fibroin of the present invention can be measured, for example, by differential scanning calorimetry, which is an analytical method for detecting the glass transition, crystallization, and melting phenomena of a polymer as changes in heat flow. The thermal decomposition temperature and / or glass transition point can be measured, for example, using a differential thermal-thermogravimetric simultaneous measurement device (DTG-60H, manufactured by Shimadzu Corporation). For example, general properties are maintained even at high temperatures, and general properties are maintained even when exposed to high temperatures for a long period of time. The thermal stability of protein fibers such as the modified fibroin of the present invention can be measured, for example, by thermomechanical analysis (TMA), which observes the changes in physical properties of a sample that accompany long-term high-temperature treatment and measures the magnitude of resistance when a test piece is subjected to a certain elongation.

[0074] The modified fibroin of this embodiment may have a thermal decomposition temperature of 268° C. or more, 270° C. or more, 272° C., 280° C., or 290° C. or more. The thermal decomposition temperature of the modified fibroin of this embodiment may be 5° C. or more, 8° C. or more, 10° C. or more, or 12° C. or more higher than that of the fibroin before modification.

[0075] The modified fibroin of this embodiment may have a glass transition point (Tg) of 185° C. or more, 190° C. or more, 192° C. or more, or 195° C. or more. The glass transition point of the modified fibroin of this embodiment may be 5° C. or more, 8° C. or more, 10° C. or more, or 12° C. or more higher than that of the fibroin before modification.

[0076] [Nucleic Acid] The nucleic acid according to the present invention encodes the modified fibroin according to the present invention. Specific examples of the nucleic acid include nucleic acids encoding modified fibroins containing the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or modified fibroins having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 (tag sequence) bound to either or both of the N-terminus and C-terminus of these amino acid sequences.

[0077] The nucleic acid according to one embodiment hybridizes under stringent conditions with a complementary strand of the nucleic acid encoding the modified fibroin according to the present invention and has the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n The modified fibroin encoded by the nucleic acid is a nucleic acid encoding a modified fibroin comprising a domain sequence represented by the motif (A) above. n It is preferable that the total number of consecutive alanine residues of 6 or more residues in the motif is 20% or more of the total number of all amino acid residues. In addition, it is preferable that the hydrophobicity of REP of the modified fibroin encoded by the nucleic acid is -1.0 or more. The modified fibroin encoded by the nucleic acid preferably has a total number of amino acid residues of 580 or more.

[0078] "Stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. "Stringent conditions" may be low stringent conditions, medium stringent conditions, or high stringent conditions. Low stringent conditions mean that hybridization occurs only when there is at least 85% or more identity between sequences, for example, 5×SSC containing 0.5% SDS is used and hybridization is performed at 42°C. Medium stringent conditions mean that hybridization occurs only when there is at least 90% or more identity between sequences, for example, 5×SSC containing 0.5% SDS is used and hybridization is performed at 50°C. High stringent conditions mean that hybridization occurs only when there is at least 95% or more identity between sequences, for example, 5×SSC containing 0.5% SDS is used and hybridization is performed at 60°C.

[0079] In another embodiment, the nucleic acid has a sequence identity of 90% or more with the nucleic acid encoding the modified fibroin of the present invention and has the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n The modified fibroin encoded by the nucleic acid is a nucleic acid encoding a modified fibroin comprising a domain sequence represented by the motif (A) above. n It is preferable that the total number of 6 or more consecutive alanine residues in the motif is 20% or more of the total number of all amino acid residues. Furthermore, it is preferable that the hydrophobicity of REP in the modified fibroin encoded by the nucleic acid is -1.0 or more. Furthermore, it is preferable that the total number of all amino acid residues in the modified fibroin encoded by the nucleic acid is 580 or more. The above sequence identity may be 80% or more, or may be 85% or more, and is preferably 95% or more.

[0080] [Host and Expression Vector] In this embodiment, the expression vector has the nucleic acid sequence according to the present invention and one or more regulatory sequences operably linked to the nucleic acid sequence. The regulatory sequence is a sequence that controls the expression of a recombinant protein in a host (e.g., a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of the host. The type of the expression vector can be appropriately selected depending on the type of the host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, etc.

[0081] The host according to the present invention is transformed with the expression vector according to the present invention. As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.

[0082] As the expression vector, one which is capable of autonomous replication in a host cell or can be integrated into a host chromosome and contains a promoter at a position where the nucleic acid according to the present invention can be transcribed is preferably used.

[0083] When a prokaryote such as a bacterium is used as a host, the expression vector according to the present invention is preferably a vector capable of autonomous replication in the prokaryote and containing a promoter, a ribosome binding sequence, the nucleic acid according to the present invention, and a transcription termination sequence. A gene for controlling the promoter may also be contained.

[0084] Examples of prokaryotes include microorganisms belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas.

[0085] Examples of microorganisms belonging to the genus Escherichia include Escherichia coli BL21 (Novagen), Escherichia coli BL21(DE3) (Life Technologies), Escherichia coli BLR(DE3) (Merck Millipore), 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 coli Rosetta(DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W3110 (ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, and the like.

[0086] Examples of the microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus borstelensis, Brevibacillus centroporas, Brevibacillus formosus, Brevibacillus invocatus, Brevibacillus lathyrosporus, Brevibacillus limnophilus, Brevibacillus parabrevis, Brevibacillus reuszeri, Brevibacillus thermolvar, Brevibacillus brevis 47 (FERM BP-1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevibacillus brevis 47-5Q (JCM8975), Brevibacillus choshinensis HPD31 (FERM BP-1087), Brevibacillus choshinensis HPD31-S (FERM BP-6623), Brevibacillus choshinensis HPD31-OK (FERM BP-4573), Brevibacillus choshinensis SP3 strain (manufactured by Takara Co., Ltd.), and the like.

[0087] Examples of microorganisms belonging to the genus Serratia include Serratia liquefacience ATCC14460, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia proteamaculans, Serratia odorifera, Serratia plymuthica, and Serratia rubidaea.

[0088] Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis and Bacillus amyloliquefaciens.

[0089] An example of a microorganism belonging to the genus Microbacterium is Microbacterium ammoniaphilum ATCC15354.

[0090] Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC14067) ATCC13826, ATCC14067, Brevibacterium immariophilum ATCC14068, Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC13869) ATCC13665, ATCC13869, Brevibacterium roseum ATCC13825, Brevibacterium saccharolyticum (Brevibacterium saccharolyticum ATCC14066, Brevibacterium thiogenitalis ATCC19240, Brevibacterium album ATCC15111, Brevibacterium serinum ATCC15112, etc.

[0091] Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC6871, ATCC6872, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium acetoacidophilum ATCC14067, and Corynebacterium acetoacidophilum) ATCC13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanolyticum ATCC21511, Corynebacterium callunae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060, Corynebacterium lilium ATCC15990, Corynebacterium melassecora ATCC17965, Corynebacterium thermoaminogenes AJ12340 (FERMBP-1539), Corynebacterium herculis ATCC13868, and the like can be mentioned.

[0092] Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas fulva, and Pseudomonas sp. D-0110.

[0093] The method for introducing an expression vector into the host cell can be any method for introducing DNA into the host cell, such as a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP Patent Publication 63-248394), or the methods described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979).

[0094] 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).

[0095] Examples of vectors into which the nucleic acid according to the present invention is introduced (hereinafter simply referred to as "vectors") include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP10 (JP Patent Publication 58-110600), 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(-) (Stratagene), pTrs30 [Escherichia coli JM109 / pTrS3 pTrs32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM Examples of such vectors include those prepared from pGEX (prepared from pGEX-6798, JP-A-60-221091), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), and the pET system (Novagen).

[0096] When Escherichia coli is used as the host, suitable vectors include pUC18, pBluescriptII, pSupex, pET22b, and pCold.

[0097] Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (JP Patent Publication No. 2-31682), pNY700 (JP Patent Publication No. 4-278091), pHY4831 (J. Bacteriol., 1987, pp. 1239-1245), pNU200 (Udaka Shigezo, Journal of the Nogeikagaku Society of Japan, 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211 (J. Biochem., 1992, 112: 488-491), pNU2 Examples of such vectors include 11R2L5 (JP Patent Publication No. 7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58:525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177:745-749), pHT210 (JP Patent Publication No. 6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42:358-363), and pNCO2 (JP Patent Publication No. 2002-238569), which is a shuttle vector between Escherichia coli and microorganisms belonging to the genus Brevibacillus.

[0098] The promoter is not limited as long as it functions in the host cell. Examples of promoters include promoters derived from Escherichia coli or phages, such as the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. Artificially designed and modified promoters such as a promoter with two Ptrp promoters in series (Ptrp×2), tac promoter, lacT7 promoter, and let I promoter can also be used.

[0099] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). In the expression vector according to the present invention, a transcription termination sequence is not necessarily required for the expression of the nucleic acid according to the present invention, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.

[0100] Eukaryotic hosts include, for example, yeast, filamentous fungi (such as molds), and insect cells.

[0101] Examples of yeast include yeasts belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia, and Hansenula. More specifically, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Trichosporon pullulans, Schwanniomyces alluvius, Schwanniomyces occidentalis, Candida utilis, Pichia pastoris, Pichia angusta, Pichia methanolica, Pichia polymorpha, Examples of such fungi include Pichia stipitis, Yarrowia lipolytica, and Hansenula polymorpha.

[0102] When yeast is used as a host cell, the expression vector usually preferably contains an origin of replication (if amplification in the host is required) and a selectable marker for propagation of the vector in E. coli, a promoter and terminator for expression of the recombinant protein in yeast, and a selectable marker for yeast.

[0103] When the expression vector is a non-integrative vector, it is preferable that the vector further contains an autonomously replicating sequence (ARS), which can improve the stability of the expression vector in cells (Myers, AM, et al. (1986) Gene 45:299-310).

[0104] Examples of vectors used when yeast is used as a host include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, and pPICZ B.

[0105] The promoter is not limited as long as it can be expressed in yeast, and examples thereof include promoters of glycolytic genes such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP 1 promoter, pGAP promoter, pGCW14 promoter, AOX1 promoter, MOX promoter, etc.

[0106] As a method for introducing an expression vector into yeast, any method for introducing DNA into yeast can be used, such as electroporation (Methods Enzymol., 194, 182 (1990)), spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), lithium acetate method (J. Bacteriol., 153, 163 (1983)), and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

[0107] Examples of filamentous fungi include fungi belonging to the genera Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, Penicillium, Myceliophtora, Botryts, Magnaporthe, Mucor, Metarhizium, Monascus, Rhizopus, and Rhizomucor.

[0108] Specific examples of filamentous fungi include Acremonium alabamense, Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus oryzae, Aspergillus sake, Aspergillus sojae, Aspergillus tubigensis, Aspergillus niger, Aspergillus nidulans, Aspergillus parasiticaus, Aspergillus parasiticus, Aspergillus ficuum, Aspergillus phoeicus, Aspergillus foetidus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus japonicus, Trichoderma viride, Trichoderma harzianum, Trichoderma reseei, Chrysosporium ruechnoense lucknowense, Thermoascus, Sporotrichum, Sporotrichum cellulophilum, Talaromyces, Thielavia terrestris, Thielavia, Neurospora crassacrassa, Fusarium oxysporus, Fusarium graminearum, Fusarium venenatum, Humicola insolens, Penicillium chrysogenum, Penicillium camemberti, Penicillium canescens, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium perprogenum purpurogenum, Penicillium roqueforti, Myceliophtaora thermophilum, Mucor ambiguus, Mucor circinelloides, Mucor fragilis, Mucor hiemalis, Mucor inaequisporus, Mucor oblongiellipticus, Mucor la Examples of such species include Mucor racemosus, Mucor recurvus, Mucor saturninus, Mucor subtilissmus, Ogataea polymorpha, Phanerochaete chrysosporium, Rhizomucor miehei, Rhizomucor pusillus, and Rhizopus arrhizus.

[0109] When the host is a filamentous fungus, the promoter may be any of genes related to the glycolysis pathway, genes related to constitutive expression, and enzyme genes related to hydrolysis, and specific examples include amyB, glaA, agdA, glaB, TEF1, xynF1 tannasegene, No.8AN, gpdA, pgkA, enoA, melO, sodM, catA, catB, and the like.

[0110] The expression vector can be introduced into filamentous fungi by using a conventional method, such as the method of Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69:2110 (1972)], the protoplast method [Mol. Gen. Genet., 168:111 (1979)], the competent method [J. Mol. Biol., 56:209 (1971)], and the electroporation method.

[0111] Examples of insect cells include Lepidoptera insect cells, more specifically, insect cells derived from Spodoptera frugiperda, such as Sf9 and Sf21, and insect cells derived from Trichoplusia ni, such as High 5.

[0112] When insect cells are used as hosts, examples of vectors include baculoviruses such as Autographa californica nuclear polyhedrosis virus, which is a virus that infects insects of the family Noctuidae (Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992)).

[0113] When insect cells are used as hosts, the polypeptide can be expressed by the method described in, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Bio / Technology, 6, 47 (1988), etc. That is, a recombinant gene transfer vector and a baculovirus are co-transfected into insect cells to obtain a recombinant virus (expression vector) in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the polypeptide. Examples of gene transfer vectors used in this method include pVL1392, pVL1393, and pBlueBacIII (both manufactured by Invitrogen).

[0114] Methods for co-introducing a recombinant gene transfer vector and a baculovirus into insect cells to prepare a recombinant virus include, for example, the calcium phosphate method (JP Patent Publication 2-227075) and the lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).

[0115] The recombinant vector according to the present invention preferably further contains a selection marker gene for selecting a transformant. For example, in Escherichia coli, resistance genes against various drugs such as tetracycline, ampicillin, kanamycin, etc. can be used as the selection marker gene. Recessive selection markers capable of complementing gene mutations involved in auxotrophy can also be used. In yeast, resistance genes against geneticin can be used as the selection marker gene, and selection markers such as genes that complement gene mutations involved in auxotrophy, LEU2, URA3, TRP1, HIS3, etc. can also be used. In filamentous fungi, the selection marker genes include niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6, 386-389, (1984)), sC (Gene, 84, 329-334, (1989)), ptrA (BiosciBiotechnol Biochem, 64, 1416-1421, (2000)), pyrG (BiochemBiophys Res Commun, 112, 284-289, (1983)), amdS (Gene, 26, 205-221, (1983)), aureobasidin resistance gene (Mol Gen Genet, 261, 290-296, (1999)), benomyl resistance gene (Proc Natl Acad Sci USA, 83, 4869-4873, (1986)) and hygromycin resistance gene (Gene, 57, 21-26, (1987)), a leucine requirement complementing gene, etc. In addition, when the host is an auxotrophic mutant, a wild-type gene that complements the auxotrophy can also be used as a selection marker gene.

[0116] The host transformed with the expression vector of the present invention can be selected by plaque hybridization, colony hybridization, etc., using a probe that selectively binds to the nucleic acid of the present invention. The probe can be a partial DNA fragment amplified by PCR based on the sequence information of the nucleic acid of the present invention and modified with a radioisotope or digoxigenin.

[0117] [Method of producing modified fibroin] The modified fibroin of the present invention can be produced by a method including a step of expressing the nucleic acid of the present invention by a host transformed with an expression vector of this embodiment. The method of producing modified fibroin of one embodiment includes a step of expressing the nucleic acid by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin of the present invention and one or more regulatory sequences operably linked to the nucleic acid sequence.

[0118] In addition to direct expression, secretory production and fusion protein expression can be performed according to the method described in Molecular Cloning, 2nd Edition. When expressed in yeast, animal cells, or insect cells, the modified fibroin can be obtained as a polypeptide with sugar or a sugar chain added.

[0119] The modified fibroin of the present invention can be produced, for example, by culturing a host transformed with the expression vector of the present invention in a culture medium, producing and accumulating the modified fibroin of the present invention in the culture medium, and collecting it from the culture medium. The method for culturing the host of the present invention in a culture medium can be performed according to a method commonly used for culturing a host.

[0120] When the host according to the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for the host according to the present invention may be either a natural medium or a synthetic medium, so long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host and allows efficient cultivation of the host.

[0121] The carbon source may be any that can be assimilated by the host, and examples of carbon sources that can be used include carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.

[0122] Examples of nitrogen sources that can be used include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria and digests thereof.

[0123] Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0124] Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions, for example, by shaking culture or submerged aeration and stirring culture. The culture temperature is, for example, 15 to 40°C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

[0125] During the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a trp promoter.

[0126] As culture media for insect cells, commonly used TNM-FH medium (manufactured by Pharmingen), Sf-900 II SFM medium (manufactured by Life Technologies), ExCell400, ExCell405 (both manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788 (1962)), etc. can be used.

[0127] Insect cells can be cultured for 1 to 5 days under conditions such as a culture medium pH of 6 to 7 and a culture temperature of 25 to 30° C. Furthermore, antibiotics such as gentamicin may be added to the culture medium during culture, if necessary.

[0128] When the host is a plant cell, the transformed plant cell may be cultured as it is, or may be differentiated into a plant organ and then cultured. As a medium for culturing the plant cell, a commonly used medium such as Murashige and Skoog (MS) medium, White medium, or a medium supplemented with a plant hormone such as auxin or cytokinin can be used.

[0129] The animal cells can be cultured for 3 to 60 days under conditions such as a culture medium pH of 5 to 9 and a culture temperature of 20 to 40° C. Furthermore, antibiotics such as kanamycin and hygromycin may be added to the medium during culture, if necessary.

[0130] Methods for producing modified fibroin using a host transformed with an expression vector according to this embodiment include a method for producing the modified fibroin within a host cell, a method for secreting the modified fibroin outside 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 by changing the host cell used and the structure of the modified fibroin to be produced.

[0131] For example, when the modified fibroin is produced inside a host cell or on the outer membrane of the host cell, the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Rowe et al. (Proc. Natl. Acad. Sci. USA, 86, 8227 (1989); Genes Develop., 4, 1288 (1990)), or the methods described in JP-A-5-336963 and WO 94 / 23021 can be applied mutatis mutandis to produce the modified fibroin. The modified fibroin can be modified so as to be actively secreted outside the host cell. That is, by using a genetic recombination technique to express a polypeptide containing an active site of the modified fibroin in a form in which a signal peptide has been added, the modified fibroin can be actively secreted outside the host cell.

[0132] The modified fibroin produced by the host transformed with the expression vector according to this embodiment can be isolated and purified by a method commonly used for isolating and purifying proteins. For example, when the modified fibroin is expressed in a dissolved state within the cells, the host cells are collected by centrifugation after the end of the culture, suspended in an aqueous buffer solution, and then disrupted by an ultrasonic homogenizer, French press, Manton Gaulin homogenizer, Dyno Mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified specimen can be obtained by using, alone or in combination, methods commonly used for isolating and purifying proteins, namely, solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, and the like.

[0133] As the above-mentioned chromatography, column chromatography using Phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), or Sephadex G-150 (Pharmacia Biotech) is preferably used.

[0134] In addition, when the modified fibroin is expressed by forming an insoluble body within the cells, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble body of the modified fibroin as a precipitate fraction. The recovered insoluble body of the modified fibroin can be solubilized with a protein denaturant. After this operation, a purified preparation of the modified fibroin can be obtained by the same isolation and purification method as above.

[0135] When the modified fibroin or a derivative in which a sugar chain has been added to the modified fibroin is secreted outside the cells, the modified fibroin or its derivative can be recovered from the culture supernatant. That is, the culture is treated by a technique such as centrifugation to obtain a culture supernatant, and a purified specimen can be obtained from the culture supernatant by the same isolation and purification method as described above.

[0136] [Artificial modified fibroin composition] The artificial modified fibroin composition according to this embodiment contains at least the modified fibroin according to the present invention.

[0137] The content of the modified fibroin in the artificial modified fibroin composition may be 30 to 100 mass %, preferably 35 to 100 mass %, and more preferably 40 to 100 mass %, based on the total amount of the artificial modified fibroin composition.

[0138] The artificial modified fibroin composition according to the present embodiment may further contain other additives depending on the form, application, etc. Examples of additives include plasticizers, leveling agents, crosslinking agents, crystal nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers, and synthetic resins. The content of the additives may be 50 parts by mass or less relative to 100 parts by mass of the total amount of the modified fibroin.

[0139] The artificial modified fibroin composition according to the present embodiment may be in any form, such as a powder, a paste, or a liquid (e.g., a suspension or a solution). The artificial modified fibroin composition according to the present embodiment may be in the form of a raw material composition (e.g., a protein powder or a dope liquid), or in the form of a molded product (e.g., a fiber, a thread, a film, a foam, a granule, a molded product) that contains the artificial modified fibroin composition or that is made of the artificial modified fibroin composition.

[0140] (Dope solution) The artificial modified fibroin composition according to this embodiment may be in the form of a dope solution. The dope solution according to this embodiment contains at least modified fibroin and a solvent. The dope solution according to this embodiment may further contain a dissolution promoter. The dope solution according to this embodiment may also further contain a protein other than the modified fibroin.

[0141] Examples of the solvent include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and aqueous solutions containing urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide, calcium chloride, lithium thiocyanate, etc. These solvents may be used alone or in combination of two or more.

[0142] The content of the modified fibroin in the dope liquid may be 15% by mass or more, 30% by mass or more, 40% by mass or more, or 50% by mass or more based on the total mass of the dope liquid. From the viewpoint of production efficiency of the dope liquid, the content of the modified fibroin may be 70% by mass or less, 65% by mass or less, or 60% by mass or less based on the total mass of the dope liquid.

[0143] Examples of the dissolution promoter include inorganic salts composed of Lewis acids and Lewis bases as shown below. Examples of the Lewis base include oxoacid ions (nitrate ions, perchlorate ions, etc.), metal oxoacid ions (permanganate ions, etc.), halide ions, thiocyanate ions, cyanate ions, etc. Examples of the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, complex ions, etc. Specific examples of inorganic salts composed of Lewis acids and Lewis bases 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, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate; potassium salts such as potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and potassium thiocyanate; sodium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and potassium thiocyanate; zinc salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, zinc perchlorate, and zinc thiocyanate; magnesium salts such as magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium thiocyanate; barium salts such as barium chloride, barium bromide, barium iodide, barium nitrate, barium perchlorate, and barium thiocyanate; and strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate.

[0144] The content of the solubility enhancer may be 1.0 part by mass or more, 5.0 parts by mass or more, 9.0 parts by mass or more, 15 parts by mass or more, or 20.0 parts by mass or more, relative to 100 parts by mass of the total amount of the modified fibroin. The content of the solubility enhancer may be 40 parts by mass or less, 35 parts by mass or less, or 30 parts by mass or less, relative to 100 parts by mass of the total amount of the modified fibroin.

[0145] During the preparation of the dope solution according to the present embodiment, the solution may be heated to 30 to 90° C. The temperature may be appropriately set depending on the solvent used, the type of modified fibroin, etc. The solution may be shaken or stirred to promote dissolution.

[0146] The viscosity of the dope solution according to the present embodiment may be appropriately set depending on the application of the dope solution. For example, when the dope solution according to the present embodiment is used as a spinning stock solution, its viscosity may be appropriately set depending on the spinning method, for example, 100 to 15,000 cP (centipoise) at 35°C, and 100 to 30,000 cP (centipoise) at 40°C, etc. The viscosity of the spinning stock solution can be measured using, for example, an "EMS viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd.

[0147] (Protein Fiber) The artificial modified fibroin composition according to the present embodiment may be in the form of a protein fiber. The protein fiber can be obtained, for example, by spinning the above-mentioned dope solution (spinning solution) by a method commonly used for spinning fibroin.

[0148] The spinning method is not particularly limited as long as it can spin the modified fibroin of the present invention, and examples thereof include dry spinning, melt spinning, wet spinning, etc. A preferred spinning method is wet spinning.

[0149] In wet spinning, the dope solution is extruded from a spinneret (nozzle) into a coagulation liquid (coagulation liquid tank) and the modified fibroin is solidified in the coagulation liquid to obtain an undrawn yarn in the shape of a thread. The coagulation liquid may be any solution capable of removing the solvent, and examples of the coagulation liquid include lower alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, and 2-propanol, and acetone. Water may be added to the coagulation liquid as appropriate. The temperature of the coagulation liquid is preferably 0 to 30°C. When a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the spinneret, the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole, and more preferably 1.4 to 4.0 ml / hour. The length of the coagulation liquid tank may be a length that allows efficient desolvation, and is, for example, 200 to 500 mm. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and is preferably 1 to 3 m / min. The residence time may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes. Also, drawing (pre-drawing) may be performed in the coagulation liquid. In order to suppress evaporation of the lower alcohol, the coagulation liquid may be kept at a low temperature, and the yarn may be taken up in an undrawn state. The coagulation liquid tank may be provided in multiple stages, and drawing may be performed in each stage or in a specific stage as necessary.

[0150] The undrawn yarn (or pre-drawn yarn) obtained by the above method can be made into a drawn yarn through a drawing process. Examples of the drawing method include wet heat drawing and dry heat drawing.

[0151] The wet heat drawing can be carried out in hot water, in a solution of hot water with an organic solvent added, or under steam heating. The temperature may be, for example, 50 to 90° C., and preferably 75 to 85° C. In the wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.

[0152] The hot drawing can be carried out using an electric tubular furnace, a hot plate, etc. The temperature may be, for example, 140° C. to 270° C., and preferably 160° C. to 230° C. In the hot drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.

[0153] The wet heat stretching and the dry heat stretching may be performed alone, or may be performed in multiple stages or in combination. That is, the wet heat stretching and the dry heat stretching may be appropriately combined, for example, the first stage stretching may be performed by wet heat stretching and the second stage stretching by dry heat stretching, or the first stage stretching may be performed by wet heat stretching, the second stage stretching may be performed by wet heat stretching, and the third stage stretching may be performed by dry heat stretching.

[0154] The final draw ratio in the drawing step is, for example, 5 to 20 times, and preferably 6 to 11 times, relative to the undrawn yarn (or pre-drawn yarn).

[0155] After stretching, the protein fiber may be chemically crosslinked between polypeptide molecules in the protein fiber. Examples of functional groups that can be crosslinked include amino groups, carboxyl groups, thiol groups, and hydroxyl groups. For example, The amino group of the lysine side chain can be crosslinked with the carboxyl group of the glutamic acid or aspartic acid side chain through an amide bond by dehydration condensation reaction under vacuum heating or by a dehydration condensation agent such as carbodiimide.

[0156] Crosslinking between polypeptide molecules may be performed using a crosslinking agent such as carbodiimide or glutaraldehyde, or an enzyme such as transglutaminase. Carbodiimide is a compound represented by the general formula R1N=C=NR2 (wherein R1 and R2 each independently represent an organic group containing an alkyl group or a cycloalkyl group having 1 to 6 carbon atoms). Specific examples of carbodiimide include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, diisopropylcarbodiimide (DIC), and the like. Among these, EDC and DIC are preferred because they have a high ability to form amide bonds between polypeptide molecules and are easy to crosslink.

[0157] The crosslinking treatment is preferably carried out by applying a crosslinking agent to the protein fibers and crosslinking the fibers by vacuum heating and drying. The crosslinking agent may be applied to the protein fibers in its pure form, or may be applied to the protein fibers after diluting the crosslinking agent with a lower alcohol having 1 to 5 carbon atoms, a buffer solution, or the like to a concentration of 0.005 to 10% by mass. The crosslinking treatment is preferably carried out at a temperature of 20 to 45°C for 3 to 42 hours. The crosslinking treatment can impart even higher stress (strength) to the protein fibers.

[0158] (Film) The artificial modified fibroin composition according to the present embodiment may be in the form of a film. The film can be obtained, for example, by casting the above-mentioned dope solution onto a substrate surface, followed by drying and / or removing the solvent.

[0159] The viscosity of the dope solution is preferably 15 to 80 cP (centipoise), and more preferably 20 to 70 cP.

[0160] When the dope solution is taken as 100% by mass, the concentration of the modified fibroin according to the present invention is preferably 3 to 50% by mass, more preferably 3.5 to 35% by mass, and even more preferably 4.2 to 15.8% by mass.

[0161] The dope solution may be prepared by heating to 30 to 60° C. To promote dissolution, the solution may be shaken or stirred.

[0162] The substrate may be a resin substrate, a glass substrate, a metal substrate, or the like. The substrate is preferably a resin substrate from the viewpoint of easily peeling off the film after cast molding. The resin substrate may be, for example, a polyethylene terephthalate (PET) film, a fluororesin film such as polytetrafluoroethylene, a polypropylene (PP) film, or a release film having a silicone compound fixed on the surface of these films. The substrate is more preferably a PET film or a release film having a silicone compound fixed on the surface of a PET film, from the viewpoint of being stable against HFIP, DMSO solvent, etc., being capable of stably casting the dope solution, and being capable of easily peeling off the film after molding.

[0163] To explain the specific procedure, first, the dope liquid is cast onto the surface of the substrate, and a film thickness control means such as an applicator, knife coater, or bar coater is used to form a wet film of a predetermined thickness (for example, 1 to 1000 μm after drying and / or desolvation).

[0164] The drying and / or desolvation can be carried out by a dry method or 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 the cast film is immersed in a desolvation liquid (also called a coagulation liquid) to remove the solvent. Examples of the desolvation liquid include water, alcohol liquids such as lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and mixtures of water and alcohol. The temperature of the desolvation liquid (coagulation liquid) is preferably 0 to 90°C.

[0165] The unstretched film after drying and / or desolvation can be uniaxially or biaxially stretched in water. The biaxial stretching may be sequential or simultaneous biaxial stretching. Multi-stage stretching of two or more stages may be performed. The stretching ratio is preferably 1.01 to 6 times, more preferably 1.05 to 4 times, both in the longitudinal and transverse directions. Within this range, the balance between stress and strain is easily achieved. Underwater stretching is preferably performed at a water temperature of 20 to 90°C. The stretched film is preferably heat-set with dry heat at 50 to 200°C for 5 to 600 seconds. This heat setting provides dimensional stability at room temperature. A uniaxially stretched film becomes a uniaxially oriented film, and a biaxially stretched film becomes a biaxially oriented film.

[0166] [Method for producing an artificial modified fibroin composition] The artificial modified fibroin composition of the present invention can be produced by a method including a step of preparing the modified fibroin of the present invention. The method for producing an artificial modified fibroin composition of the present invention may further include a step of preparing a modified fibroin solution (e.g., a dope solution) containing the modified fibroin of the present invention.

[0167] [Method for improving the thermal stability of modified fibroin] According to the present embodiment, a modified fibroin having the formula 1: [(A) n Motif-REP] m Or formula 2: [(A) n Motif-REP] m -(A) n The method for improving the thermal stability of a modified fibroin containing a domain sequence represented by the motif includes at least one (A) n At least one (A) is introduced into the motif or REP by inserting one or more alanine residues or substituting other amino acid residues adjacent to the alanine residue with alanine. n The method includes increasing the number of consecutive alanine residues in the motif. The thermal decomposition temperature (Td) of the modified fibroin is 5°C or higher compared to the unmodified fibroin. The unmodified fibroin includes naturally occurring fibroin and modified fibroin.

[0168] Here, (A) n The motif shows an amino acid sequence mainly consisting of alanine residues, and the number of amino acid residues is 4 to 27. (A) n The number of amino acid residues in the motif may be 4 to 20, 8 to 20, 10 to 20, 4 to 16, 5 to 10, 6 to 12, 6 to 18, 7 to 10, 7 to 14, 8 to 16, or 11 to 16. n The ratio of the number of alanine residues to the total number of amino acid residues in the motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. There are multiple (A) n The motifs may be identical to each other or different from each other in amino acid sequence, and the multiple REPs may be identical to each other or different from each other in amino acid sequence.

[0169] (A) n The motif is (A) n The number of alanine residues relative to the total number of amino acid residues in the motif may be 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that the motif is composed only of alanine residues). n It is preferable that at least seven of the motifs are composed of only alanine residues. n The motif is (Ala) k (Ala represents an alanine residue, and k represents an integer of 4 to 27, preferably an integer of 4 to 20, and more preferably an integer of 4 to 16).

[0170] The method according to the present embodiment includes at least one (A) in the unmodified fibroin. n In the method according to the present embodiment, the number of consecutive alanine residues in the motif is increased. nThe number of consecutive alanine residues in the motif may be increased, and the number of consecutive alanine residues in the motif may be increased to at least 5 (A) in the unmodified fibroin. n The number of consecutive alanine residues in the motif may be increased, and the number of consecutive alanine residues in the motif may be increased to at least six (A) in the unmodified fibroin. n The number of consecutive alanine residues in the motif may be increased, and the number of consecutive alanine residues in the motif may be increased to at least 7 (A) in the unmodified fibroin. n The number of consecutive alanine residues in the motif may be increased, and all (A) in the fibroin before modification may be increased. n The number of consecutive alanine residues in the motif may be increased. This can improve the thermal stability of the modified fibroin. n Increasing the number of consecutive alanine residues in the motif increases the number of consecutive alanine residues in at least one (A) in the unmodified fibroin. n The modification may be achieved by inserting one or more alanine residues into the motif or REP, and / or by substituting other amino acid residues adjacent to the alanine residue with alanine. The fibroin before modification may be naturally occurring fibroin or modified fibroin.

[0171] The method according to the present embodiment includes at least one (A) in the unmodified fibroin. nThe motif or REP may include a modification equivalent to inserting one or more alanine residues into the motif or REP, or substituting another amino acid residue adjacent to the alanine residue with alanine, and further including a modification of the amino acid sequence equivalent to substituting, deleting, inserting and / or adding one or more amino acid residues. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, the method can be performed according to the methods described in literature, such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0172] The method according to the present embodiment includes, for example, (A) determining whether a gene sequence of a cloned naturally occurring fibroin is a gene sequence of a cloned naturally occurring fibroin; n The modified fibroin may include, for example, a mutation corresponding to the insertion of one or more alanine residues into the motif or REP, or the substitution of other amino acid residues adjacent to the alanine residue with alanine. n It can also be obtained by inserting one or more alanine residues into the motif or REP, or by designing an amino acid sequence corresponding to the alanine residue by substituting other amino acid residues adjacent to the alanine residue with alanine, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0173] The modified fibroin according to the method of this embodiment is (A) n It is preferable that the total number of consecutive alanine residues in the motif is 20% or more of the total number of amino acid residues in the entire motif. n The total number of six or more consecutive alanine residues in a motif means the total number of alanine residues when only the number of six or more consecutive alanine residues is counted. Therefore, the number of five or more consecutive alanine residues is not counted. nIn the motif, even if there are six or more consecutive alanine residues due to the presence of other amino acid residues between the alanine residues, the number of the alanine residues is not counted.

[0174] (A) n The total number of 6 or more consecutive alanine residues in the motif may be, for example, 20% or more, 21% or more, 22% or more, or 23% or more of the total number of all amino acid residues. n The total number of consecutive alanine residues in the motif is, for example, 20% or more of the total number of amino acid residues in the entire sequence. It may be 1% or more, 22% or more, or 23% or more.

[0175] The total number of amino acid residues in the modified fibroin according to the present embodiment is preferably 580 or more. The total number of amino acid residues in the entirety may be, for example, 580 or more, 590 or more, 600 or more, 610 or more, or 620 or more.

[0176] In the modified fibroin according to the method of the present embodiment, the hydrophobicity of REP is preferably -1.0 or more, more preferably -0.9 or more, even more preferably -0.8 or more, even more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.2 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of REP, and it may be, for example, 1.0 or less, or 0.7 or less. The hydrophobicity of REP may be, for example, -1.0 or more and 1.0 or less, or 0 or more and 0.7 or less.

[0177] In the method according to the present embodiment, the thermal decomposition temperature (Td) of the modified fibroin may be at least 5° C. higher than that of the unmodified fibroin, and may be, for example, at least 8° C., at least 10° C., or at least 12° C. higher. The unmodified fibroin is preferably naturally derived fibroin, more preferably fibroin derived from insects or spiders, and even more preferably large amplexus spider protein (MaSp) or small amplexus spider protein (MiSp) of spiders.

[0178] Furthermore, in the method according to this embodiment, the glass transition point (Tg) of the modified fibroin may be, for example, 5°C or more, 8°C or more, 10°C or more, or 12°C or more higher than that of the fibroin before modification.

[0179] The modification of the fibroin protein in the method according to this embodiment can be carried out in a manner that results in the preferred embodiment described above as the modified fibroin embodiment.

[0180] [Products] The modified fibroin or artificial modified fibroin composition of the present invention can be used as fibers (long fibers, short fibers, multifilaments, monofilaments, etc.) or yarns (spun yarns, twisted yarns, false twisted yarns, textured yarns, blended yarns, blended yarns, etc.) for applications such as woven fabrics, knitted fabrics, braids, nonwoven fabrics, etc. They can also be used for high-strength applications such as ropes, surgical sutures, flexible fasteners for electrical components, and even bioactive materials for transplants (e.g., artificial ligaments and aortic bands).

[0181] In addition, the modified fibroin or artificial modified fibroin composition of the present invention can be applied not only to fibers and films, but also to foams, granules (spherical or non-spherical, etc.), nanofibrils, gels (hydrogels, etc.), resins and equivalents thereof, which can be produced in accordance with the methods described in JP 2009-505668 A, Japanese Patent No. 5678283, Japanese Patent No. 4638735, etc.

[0182] The article of manufacture according to this embodiment comprises the modified fibroin of the present invention and is selected from the group consisting of fibers, threads, films, foams, granules, nanofibrils, gels and resins. EXAMPLES

[0183] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0184] (1) Synthesis of nucleic acid encoding modified fibroin and construction of expression vector Based on the base sequence and amino acid sequence of natural fibroin, Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), fibroin and modified fibroin having the amino acid sequences shown in SEQ ID NOs: 6 to 12 were designed.

[0185] The amino acid sequence (PRT380) shown in SEQ ID NO: 9 corresponds to each of the naturally occurring fibroins (A) n The number of consecutive alanine residues in the motif was deleted so that it was 5, all GGX in REP was replaced with GQX, and the amino acid sequence shown in sequence number 4 (tag sequence and hinge sequence) was added to the N-terminus.

[0186] The amino acid sequence (PRT525) shown in SEQ ID NO: 10 is composed of every two amino acids (A) from the N-terminus to the C-terminus of the naturally occurring fibroin. n Motif ((A) 5 ) was deleted, and the C-terminal sequence was then deleted, followed by the deletion of [(A) n Insert one motif -REP] and replace all GGX in REP with GQX, and each (A) n Two alanine residues were inserted at the C-terminus of the motif, and some glutamine (Q) residues were replaced with serine (S) residues. Some amino acids were deleted at the C-terminus so that the molecular weight was approximately the same as that of PRT380. The amino acid sequence shown in SEQ ID NO:4 (tag sequence and hinge sequence) was added to the N-terminus.

[0187] The amino acid sequence (PRT1068) shown in SEQ ID NO: 11 is a sequence similar to that shown in SEQ ID NO: 12, but is not limited to the following: n One alanine residue was deleted from the C-terminus of the motif, and Q in REP was replaced with VFI.

[0188] The amino acid sequence (PRT1069) shown in SEQ ID NO:6 is a sequence similar to each of (A) of PRT1068. n A single alanine residue was inserted at the C-terminus of the motif.

[0189] The amino acid sequence shown in SEQ ID NO: 7 (PRT1070) is a sequence similar to each of (A) of PRT1068. n Two alanine residues were inserted into the C-terminus of the motif.

[0190] Nucleic acids encoding proteins having the designed amino acid sequences shown in SEQ ID NOs: 6 to 12 were synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. These five types of nucleic acids were cloned into a cloning vector (pUC118). The nucleic acids were then excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

[0191] (2) Protein Expression: E. coli BLR(DE3) was transformed with the pET22b(+) expression vector containing nucleic acids encoding proteins having the amino acid sequences shown in SEQ ID NOs: 6 to 12. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture was transferred to 100 mL of seed culture medium (Table 5) containing ampicillin at OD 200. 600 The culture temperature was kept at 30°C, and the OD 600 The flask culture was continued until the number of cells reached 5 (about 15 hours), and a seed culture solution was obtained.

[0192] [Table 5]

[0193] The seed culture solution was transferred to a jar fermenter containing 500 mL of production medium (Table 6) to obtain an OD 600 The transformed E. coli was inoculated with 0.05 NaCl. The temperature of the culture medium was kept at 37°C, and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration.

[0194] [Table 6]

[0195] Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455g / 1L, yeast extract 120g / 1L) was added at a rate of 1mL / min. The culture temperature was kept at 37°C and the culture was controlled to a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours. Then, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1mM to induce expression of the target protein. 20 hours after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the target protein was confirmed by the appearance of a band of the target protein size depending on the addition of IPTG.

[0196] (3) Protein purification: (1) Approximately 50 g of E. coli cells expressing the modified fibroin protein and 300 ml of buffer solution AI (20 mM Tris-HCl, pH 7.4) were added to a centrifuge tube (1,000 ml). The cells were dispersed using a mixer (IKA "T18 Basic Ultra Turrax", level 2), and then centrifuged (11,000 g, 10 minutes, room temperature) using a centrifuge (Kubota "Model 7000"), and the supernatant was discarded.

[0197] (2) 300 ml of Buffer AI and 3 ml of 0.1 M PMSF (dissolved in isopropanol) were added to the precipitate (bacterial cells) obtained by centrifugation, and the mixture was dispersed for 3 minutes using the IKA mixer (level 2).Then, the bacterial cells were repeatedly disrupted three times using a high-pressure homogenizer (GEA Niro Saovi's "Panda Plus 2000").

[0198] (3) 300 mL of buffer B (50 mM Tris-HCL, 100 mM NaCl, pH 7.0) containing 3 w / v% SDS was added to the disrupted cells, and the mixture was thoroughly dispersed using the IKA mixer (level 2) and then stirred for 60 minutes using a shaker (Taitec, 200 rpm, 37°C).The mixture was then centrifuged (11,000 g, 30 minutes, room temperature) using the Kubota centrifuge, and the supernatant was discarded to obtain SDS-washed granules (precipitate).

[0199] (4) The SDS-washed granules were suspended in a DMSO solution containing 1 M lithium chloride to a concentration of 100 mg / mL, and heat-treated at 80° C. for 1 hour. Then, the suspension was centrifuged (11,000 g, 30 minutes, room temperature) using the Kubota centrifuge, and the supernatant was collected.

[0200] (5) Ethanol was prepared in an amount three times the amount of the collected supernatant, and the collected supernatant was added to the ethanol and allowed to stand at room temperature for 1 hour. The mixture was then centrifuged (11,000 g, 30 minutes, room temperature) using the Kubota centrifuge to collect the aggregated proteins. The aggregated proteins were then washed with pure water and collected by centrifugation. This process was repeated three times, after which the moisture was removed using a freeze-dryer to collect the freeze-dried powder.

[0201] (4) Measurement of thermal decomposition temperature (Td) and glass transition temperature (Tg) Measurements were performed using a Shimadzu Corporation differential thermal and thermogravimetric simultaneous analyzer (DTG-60H) according to the manual provided by the manufacturer. 10 mg of protein powder was used for each sample, and 20 mg of alumina powder was used as a reference material. The reference material and sample were placed in separate aluminum containers and covered with an aluminum cover. The samples were heated to a target temperature of 300°C at 10°C / min in a nitrogen atmosphere. Thermal analysis measurement graphs were analyzed using Softwere TA60 (Shimadzu Corporation).

[0202] The thermal decomposition temperature (Td) was measured by tangent point analysis of the differential thermal analysis (DTA) curve, and the glass transition temperature (Tg) was measured by tangent point analysis of the DTA curve.

[0203] (5) Results The characteristics of the amino acid sequences of PRT380 and PRT525 are shown in Table 7. The thermal decomposition temperature and glass transition temperature of PRT380 and PRT525 are compared in Figures 2 and 3, respectively. Compared to PRT380, PRT525 exhibited a higher thermal decomposition temperature and glass transition temperature.

[0204] [Table 7]

[0205] The characteristics of the amino acid sequences of PRT1068, PRT1069 and PRT1070 are shown in Table 8. The results of comparing the thermal decomposition temperatures of PRT1068, PRT1069 and PRT1070 are shown in Figure 4. n There was a tendency for the pyrolysis temperature to increase by increasing the number of consecutive alanine residues in the motif.

[0206] [Table 8]

[0207] Each (A) nThe thermal decomposition temperatures (Td) of PRT525 (SEQ ID NO: 10) and PRT1070 (SEQ ID NO: 7), which have 7 consecutive alanine residues in their motifs, are compared in Figure 5. The hydrophobicity of REP in PRT525 is -1.32, and the hydrophobicity of REP in PRT1070 is 0.21.

[0208] PRT1070 has high hydrophobicity of REP showed a higher thermal decomposition temperature than PRT525.

Claims

1. Formula 1: [(A) ｎ Motif-REP] ｍ Or formula 2: [(A) ｎ Motif-REP] ｍ - (A) ｎ A modified fibroin comprising a domain sequence represented by the motif: The (A) ｎ the total number of consecutive alanine residues in the motif is 22% or more of the total number of amino acid residues in the entire motif, The hydrophobicity of the REP is 0.2 or more; A modified fibroin having a total number of amino acid residues of 620 or more. ｎ The motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) ｎ The number of alanine residues relative to the total number of amino acid residues in the motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. (A) ｎ The motifs may be identical to each other or different from each other in amino acid sequence. The REPs present in multiple locations may be identical to each other or different from each other in amino acid sequence.

2. The amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, The (A) ｎ A modified fibroin, in which the total number of consecutive alanine residues in a motif is 20% or more of the total number of amino acid residues.

3. The modified fibroin according to claim 1 or 2, further comprising a tag sequence at either or both of the N-terminus and C-terminus.

4. The modified fibroin according to claim 3 , wherein the tag sequence comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO:

5.

5. The amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, The (A) ｎ A modified fibroin, in which the total number of consecutive alanine residues in a motif is 20% or more of the total number of amino acid residues.

6. The modified fibroin according to any one of claims 1 to 5, having a thermal decomposition temperature (Td) of 268°C or higher.

7. A nucleic acid encoding the modified fibroin according to any one of claims 1 to 6.

8. A method for producing modified fibroin, comprising the steps of: expressing the nucleic acid by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence; A method for producing the modified fibroin, wherein the modified fibroin is the modified fibroin according to any one of claims 1 to 6.

9. Formula 1: [(A) ｎ Motif-REP] ｍ Or formula 2: [(A) ｎ Motif-REP] ｍ - (A) ｎ A method for improving the thermal stability of a modified fibroin comprising a domain sequence represented by a motif, comprising: At least one (A) in the fibroin before modification ｎ At least one (A) is obtained by inserting one or more alanine residues into the motif or REP, or by substituting other amino acid residues adjacent to the alanine residue with alanine. ｎ increasing the number of consecutive alanine residues in the motif; The method for producing the modified fibroin, wherein the thermal decomposition temperature (Td) of the modified fibroin is 5° C. or higher than that of the unmodified fibroin. ｎ The motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) ｎ The number of alanine residues relative to the total number of amino acid residues in the motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. (A) ｎ The motifs may be identical to each other or different from each other in amino acid sequence. The REPs present in multiple locations may be identical to each other or different from each other in amino acid sequence.

10. The method according to claim 9 , wherein the unmodified fibroin is naturally occurring fibroin.

11. The method of claim 10, wherein the naturally occurring fibroin is fibroin from an insect or arachnid.

12. The method according to claim 11, wherein the naturally occurring fibroin is arachnid major ampullate spider protein (MaSp) or minor ampullate spider protein (MiSp).

13. The modified fibroin according to any one of claims 1 to 6 is contained therein, 1. An article of manufacture selected from the group consisting of fibers, yarns, films, foams, granules, nanofibrils, gels and resins.

Images