Modified fibroin

Abstract

To provide modified fibroin having reduced formation of ester bond by contact with carboxylic acid such as formic acid.SOLUTION: Provided is a modified fibroin in which the amino acid sequence is modified by substitution, deletion, insertion and / or addition of one or more amino acid residues, the number of amino acid residues having a free hydroxyl group after modification being reduced by at least 20% based on the number of amino acid residues having a free hydroxyl group before modification.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to modified fibroin. More specifically, the present invention relates to modified fibroin with a reduced content of amino acid residues having free hydroxyl groups.

Background Art

[0002] Fibroin is a kind of fibrous protein. Fibroin contains a high proportion of up to 90% of amino acid residues with small side chains such as glycine residues, alanine residues, serine residues, tyrosine residues and the like. As fibroin, proteins (silk proteins, hornet silk proteins, spider silk proteins) that constitute the silk produced by insects and spiders are known.

[0003] Silk protein has excellent mechanical properties, moisture absorption properties and deodorizing properties, and is a material widely used as a clothing raw material. Also, silk thread is an immunotolerant natural fiber and has high biocompatibility, so it is also used for applications such as surgical sutures.

[0004] Compositions containing modified fibroin have also been variously produced. For example, as an example of a composition containing modified fibroin, a spider silk protein film (for example, Patent Document 1), protein fibers and the like are known (for example, Patent Document 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] In the production of compositions containing modified fibroin, carboxylic acids such as formic acid may be used (for example, as a solvent) in the process of preparing protein solutions (e.g., dope solutions). The inventors have found that compositions containing modified fibroin produced using carboxylic acids such as formic acid have the problem of emitting an unpleasant odor when left exposed to the air. The inventors have also found that in compositions containing modified fibroin produced using carboxylic acids such as formic acid, ester groups are formed by a dehydration condensation reaction between hydroxyl groups in the protein and the carboxylic acid. In compositions containing modified fibroin obtained in this way, hydrolysis of the ester groups attached to the protein may proceed using trace amounts of carboxylic acids such as formic acid remaining on or inside the protein as a catalyst, and the carboxylic acid may be released, which causes unpleasant odors. The present invention aims to solve these problems newly discovered by the inventors.

[0007] In other words, the present invention aims to provide a modified fibroin in which the formation of ester bonds by contact with a carboxylic acid such as formic acid is reduced. The present invention also aims to provide a method for suppressing the formation of ester bonds in modified fibroin by contact with a carboxylic acid such as formic acid. [Means for solving the problem]

[0008] The inventors have found that the above objective can be achieved by reducing the content of amino acid residues having free hydroxyl groups in the amino acid sequence of modified fibroin. The present invention is based on this finding.

[0009] This invention relates, for example, to the following inventions. [1] A modified fibroin obtained by altering the amino acid sequence by substituting, deleting, inserting and / or adding one or more amino acid residues, Modified fibroin in which the number of amino acid residues having a free hydroxyl group after modification is at least 20% less than the number of amino acid residues having a free hydroxyl group before modification. [2] The modified fibroin according to [1], wherein the content rate of amino acid residues having a free hydroxyl group is at least 6% less than that before modification. [3] Formula 1: [(A) n Motif-REP] m or Formula 2: [(A) n Motif-REP] m -(A) n containing a domain sequence represented by a motif, Modified fibroin in which the content rate of amino acid residues having a free hydroxyl group is 22% or less. [In Formula 1 and Formula 2, (A) n Motif represents an amino acid sequence composed 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 composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. Multiple (A) n Motifs may have the same amino acid sequence as each other or different amino acid sequences. Multiple REPs may have the same amino acid sequence as each other or different amino acid sequences.] [4] A nucleic acid encoding the modified fibroin according to any one of [1] to [3]. [5] [4] A nucleic acid that hybridizes with the complementary strand of the nucleic acid described in [4] under stringent conditions and encodes a modified fibroin containing a domain sequence represented by Formula 1: [(A) n Motif-REP] m or Formula 2: [(A) n Motif-REP] m -(A) n Motif. [In Formula 1 and Formula 2, (A) n Motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and (A)n The number of alanine residues in the motif is 80% or more of the total number of amino acid residues. REP indicates an amino acid sequence consisting of 10 to 200 amino acid residues. m is an integer between 10 and 300. Multiple such sequences exist (A). n The motifs may have the same amino acid sequence or different amino acid sequences. Multiple REPs may have the same amino acid sequence or different amino acid sequences. [6] [4] The nucleic acid has sequence identity of 90% or more, and formula 1:[(A) n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n A nucleic acid encoding modified fibroin containing a domain sequence represented by a motif. [In equations 1 and 2, (A) n The motif shows an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) n The number of alanine residues in the motif is 80% or more of the total number of amino acid residues. REP indicates an amino acid sequence consisting of 10 to 200 amino acid residues. m is an integer between 10 and 300. Multiple such sequences exist (A). n The motifs may have the same amino acid sequence or different amino acid sequences. Multiple REPs may have the same amino acid sequence or different amino acid sequences. [7] An expression vector having a nucleic acid sequence described in any of [4] to [6] and one or more regulatory sequences operably linked to the nucleic acid sequence. [8] The expression vector described in [7] is a plasmid vector or a viral vector. [9] A host transformed with the expression vector described in [7] or [8].

[10] The host described in [9] is a prokaryote.

[11] The host described in

[10] is a microorganism belonging to a genus selected from the group consisting of Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas.

[12] The host according to claim 9, which is a eukaryote.

[13] The host according to

[12] , wherein the eukaryote is a yeast, filamentous fungus, or insect cell.

[14] An artificial modified fibroin composition comprising the modified fibroin described in any of [1] to [3].

[15] The artificially modified fibroin composition described in

[14] is a protein powder.

[16] The doping solution is the artificially modified fibroin composition described in

[14] .

[17] The synthetic modified fibroin composition described in

[14] , which is a fiber.

[18] A film, the artificially modified fibroin composition described in

[14] .

[19] A method for producing modified fibroin, The process includes expressing a modified fibroin in a host transformed with an expression vector having a nucleic acid sequence encoding a 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 described in any of [1] to [3].

[20] A method for producing an artificial modified fibroin composition containing modified fibroin, This includes the process of preparing modified fibroin. A method for producing the modified fibroin, wherein the modified fibroin is the modified fibroin described in any of [1] to [3]. [twenty one] The method for producing the modified fibroin according to

[19] or

[20] , further comprising the step of contacting the modified fibroin with a carboxylic acid. [twenty two] The manufacturing method according to

[20] , further comprising the step of preparing a modified fibroin solution containing the modified fibroin and the carboxylic acid. [twenty three] A method for reducing the formation of ester bonds between modified fibroin and carboxylic acid, The process includes modifying the amino acid sequence of a fibroin protein by substituting, deleting, inserting and / or adding one or more amino acid residues. A method wherein the number of amino acid residues containing free hydroxyl groups in the modified fibroin is reduced by at least 20% compared to the number of amino acid residues containing free hydroxyl groups in the original fibroin protein. [twenty four] The method according to

[23] , wherein the content of amino acid residues having free hydroxyl groups in the modified fibroin after modification is reduced by at least 6% compared to the content of amino acid residues having free hydroxyl groups in the fibroin protein before modification. [twenty five] Contains the modified fibroin described in any of [1] to [3], Products selected from the group consisting of fibers, threads, films, foams, granules, nanofibrils, gels, and resins. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a modified fibroin in which the formation of ester bonds by contact with carboxylic acids such as formic acid is reduced. [Brief explanation of the drawing]

[0011] [Figure 1] This graph shows the results of infrared absorption spectrum measurements of films formed with modified fibroin. [Modes for carrying out the invention]

[0012] The embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[0013] [Modified fibroin] The modified fibroin according to the present invention is of formula 1:[(A) n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n This is a protein containing a domain sequence represented by a motif. Modified fibroin may have additional amino acid sequences (N-terminal sequence and C-terminal sequence) added to either the N-terminal or C-terminal side, or both, of the domain sequence. The N-terminal and C-terminal sequences are, but are not limited to, regions that do not have repeating amino acid motifs characteristic of fibroin, and consist of about 100 amino acid residues.

[0014] In this specification, "modified fibroin" means fibroin whose amino acid sequence differs from that of naturally occurring fibroin. In this specification, "naturally occurring fibroin" means fibroin whose amino acid sequence is identical to that of fibroin produced by naturally occurring insects or spiders. Naturally occurring fibroin is also defined by formula 1:[(A) n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n It is a protein that contains a domain sequence represented by a motif.

[0015] Examples of naturally occurring fibroin include fibroin produced by insects or spiders.

[0016] Examples of fibroins produced by insects include the silk protein 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, as well as the hornet silk protein excreted by the larvae of the hornet (Vespa simillima xanthoptera).

[0017] A more specific example of fibroin produced by insects is silkworm fibroin L chain (GenBank accession number M76430 (nucleotide sequence), AAA27840.1 (amino acid sequence)).

[0018] Examples of fibroin produced by spiders include those belonging to the genus Araneus, such as the garden orb-weaver spider, red orb-weaver spider, blue orb-weaver spider, and bean orb-weaver spider; those belonging to the genus Neoscona, such as the mountain orb-weaver spider, house orb-weaver spider, and Satsuma orb-weaver spider; those belonging to the genus Pronus, such as the small orb-weaver spider; those belonging to the genus Cyrtarachne, such as the bird-dropping spider and the large bird-dropping spider; spiny spiders and Spiders belonging to the genus Gasteracantha, such as the spiny spider, spiders belonging to the genus Ordgarius, such as the bean spider and the six-legged spiny spider, spiders belonging to the genus Argiope, such as the golden orb-weaver spider, the small golden orb-weaver spider and the long golden orb-weaver spider, spiders belonging to the genus Arachnura, such as the white-tailed cane spider, spiders belonging to the genus Acusilas, such as the bell spider, the golden cane spider and the broad-winged bell spider (C Spider silk protein produced by spiders belonging to the genus Polytrichum (Poltys), such as Polytrichum commune, such as Corbicula japonica, such as Corbicula japonica, such as Corbicula japonica, such as Corbicula japonica and Corbicula japonica, and spiders belonging to the genus Chorizopes, such as Corbicula japonica, as well as spiders belonging to the genus Tetragnatha, such as Polytrichum commune, such as Polytrichum japonica, such as Heteropoda venatoria, and such as Polytrichum commune, and the large white spider Spiders belonging to the genus Leucauge, such as the medium-sized white orb-weaver spider and the small white orb-weaver spider; spiders belonging to the genus Nephila, such as the golden orb-weaver spider and the large golden orb-weaver spider; spiders belonging to the genus Menosira, such as the golden orb-weaver spider; spiders belonging to the genus Dyschiriognatha, such as the small long-legged spider; spiders belonging to the genus Latrodectus, such as the black widow spider, the redback spider, the gray widow spider and the thirteen-spotted widow spider.Examples 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), ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

[0019] More specific examples of fibroins produced by spiders include, for example, fibroin-3 (adf-3) [from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank accession number AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), major angu11ate spidroin 1 [from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [Nephila [Derived from clavata] (GenBank accession number AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [Derived from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank accession number 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 Examples include [cruentata] (GenBank accession number ABR37278.1 (amino acid sequence)).

[0020] A more specific example of naturally derived fibroin is the fibroin whose sequence information is registered in NCBI GenBank. For example, this can be confirmed by extracting sequences from the sequence information registered in NCBI GenBank that include INV as a DIVISION, and whose DEFINITION contains keywords such as spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein," as well as sequences with a specific product string in the CDS or a specific string in the TISSUE TYPE of the SOURCE.

[0021] "Modified fibroin" may be any fibroin having the amino acid sequence specified in this invention, even if it is a modified fibroin based on naturally derived fibroin (for example, a modified fibroin with a modified amino acid sequence by altering the gene sequence of cloned naturally derived fibroin), or an artificially designed fibroin with an amino acid sequence that does not rely on naturally derived fibroin (for example, a fibroin having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding a designed amino acid sequence). Furthermore, a modified fibroin with a modified amino acid sequence is also included in modified fibroin if its amino acid sequence differs from that of naturally derived fibroin.

[0022] In this specification, "domain sequence" refers to a crystalline region specific to fibroin (typically, (A) of an amino acid sequence). n This corresponds to a motif.) and an amino acid sequence that produces an amorphous region (typically corresponding to a REP in an amino acid sequence), and formula 1:[(A) n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n This refers to an amino acid sequence represented as a motif. Here, (A) n The motif shows 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 indicates an amino acid sequence consisting of 10 to 200 amino acid residues. m indicates an integer from 10 to 300. It is preferable that m be an integer from 20 to 300, and more preferably an integer from 30 to 300. Multiple (A) exist. n The motifs may have the same amino acid sequence or different amino acid sequences. Multiple REPs may have the same amino acid sequence or different amino acid sequences.

[0023] (A) n The motif is (A) n The number of alanine residues relative to the total number of amino acid residues in the motif should be 80% or more, but preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning composed solely of alanine residues). Multiple alanine residues are present in the domain sequence (A). n The motif is preferably composed of at least seven alanine residues. Composed of only alanine residues means (A) n The motif is (Ala) k This means having an amino acid sequence represented by (Ala represents an alanine residue, and k represents an integer from 4 to 27, preferably from 4 to 20, more preferably from 4 to 16).

[0024] One embodiment of the modified fibroin is a modified fibroin whose amino acid sequence has been modified by substituting, deleting, inserting and / or adding one or more amino acid residues, wherein the OH reduction rate is at least 20%. Here, the OH reduction rate represents the ratio of amino acid residues having free hydroxyl groups in the modified fibroin after modification, based on the number of amino acid residues having free hydroxyl groups in the fibroin protein before modification, and is calculated by the following formula. OH reduction rate (%) = {1 - (Number of amino acid residues with free hydroxyl groups in the modified fibroin after modification / Number of amino acid residues with free hydroxyl groups in the original fibroin protein)} × 100 Note that the original fibroin protein contains both naturally occurring fibroin and modified fibroin.

[0025] Examples of amino acid residues having a free hydroxyl group include serine residues (S), threonine residues (T), and tyrosine residues (Y), which have a free hydroxyl group in their side chain.

[0026] The modified fibroin according to this embodiment only needs to have a number of amino acid residues having free hydroxyl groups that is reduced by at least 20% compared to the number of amino acid residues having free hydroxyl groups before modification (i.e., the OH reduction rate is 20% or more). This reduces the formation of ester bonds when in contact with carboxylic acids such as formic acid. In addition, the generation of off-odors is reduced or made less likely to occur.

[0027] The OH reduction rate of the modified fibroin according to this embodiment may be 25% or more, 35% or more, 45% or more, 55% or more, 65% or more, 75% or more, 85% or more, 95% or more, or even 100%. This makes the effects of the present invention even more pronounced.

[0028] In this embodiment, the modified fibroin preferably has a free hydroxyl group amino acid residue content that is at least 6%, more preferably at least 6.5%, and even more preferably at least 7%, compared to the free hydroxyl group amino acid residue content in the original fibroin protein. This allows the effects of the present invention to be even more pronounced.

[0029] In this specification, "content of amino acid residues having a free hydroxyl group" is defined as formula 1:[(A) n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n In fibroin containing a domain sequence represented by a motif, x is the total number of amino acid residues having a free hydroxyl group, and y is the total number of amino acid residues in fibroin. The value is calculated as x / y × 100%.

[0030] One embodiment of the modified fibroin is a modified fibroin having a free hydroxyl group amino acid residue content of 22% or less. The free hydroxyl group amino acid residue content may be 20% or less, 18% or less, 16% or less, 14% or less, 12% or less, 10% or less, 8% or less, 6% or less, 4% or less, 2% or less, or 0%. This reduces the formation of ester bonds when in contact with carboxylic acids such as formic acid. It also reduces the generation of off-odors.

[0031] One embodiment of the modified fibroin is a modified fibroin having a serine residue, threonine residue, and tyrosine residue content of 22% or less. The serine residue, threonine residue, and tyrosine residue content may be 20% or less, 18% or less, 16% or less, 14% or less, 12% or less, 10% or less, 8% or less, 6% or less, 4% or less, 2% or less, or 0%. This reduces the formation of ester bonds when in contact with carboxylic acids such as formic acid. It also reduces the generation of off-odors.

[0032] In this specification, "serine residue, threonine residue, and tyrosine residue content" is defined as formula 1:[(A) n Motif-REP]m , or formula 2:[(A) n Motif-REP] m -(A) n In fibroin containing a domain sequence represented by a motif, the value is calculated as (a+b+c) / y × 100%, where a is the total number of serine residues, b is the total number of threonine residues, c is the total number of tyrosine residues, and y is the total number of amino acid residues in fibroin.

[0033] The molecular weight of the modified fibroin according to the present invention is not particularly limited, but may be, for example, 10 kDa or more and 700 kDa or less. The molecular weight of the modified fibroin according to the present invention may be, for example, 20 kDa or more, 30 kDa or more, 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.

[0034] More specific examples of modified fibroin according to the present invention include (i) modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 11, or (ii) modified fibroin comprising 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, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 11.

[0035] The amino acid sequence shown in Sequence ID No. 1 (Met-PRT918) is obtained by substituting GTGA with GPGA, GTGS with GPGS, GLGV with GPGV, GTGI with GPGI, GLY with GPY, and GTS with GPS in the amino acid sequence shown in Sequence ID No. 9 (Met-PRT1083).

[0036] The amino acid sequence shown in SEQ ID NO: 2 (Met-PRT1104) is obtained by substituting most of the serine residues (S) in the amino acid sequence shown in SEQ ID NO: 25 (Met-PRT410) with alanine residues (A) or glycine residues (G). The amino acid sequence shown in SEQ ID NO: 25 (Met-PRT410) is obtained by substituting (A) every two residues from the N-terminus to the C-terminus of the amino acid sequence shown in SEQ ID NO: 26 (Met-PRT380). n The motif is deleted, and furthermore, before the C-terminal sequence, [(A) n This is a sequence with one motif [-REP] inserted. The amino acid sequence shown in SEQ ID NO: 26 (Met-PRT380) is obtained by replacing all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 27 (Met-PRT313), which corresponds to naturally derived fibroin, with GQX.

[0037] The amino acid sequence shown in Sequence ID No. 3 (Met-PRT1105) is obtained by substituting the serine residue (S) with an alanine residue (A) or a glycine residue (G) in the amino acid sequence shown in Sequence ID No. 1 (Met-PRT918).

[0038] The amino acid sequence shown in Sequence ID No. 4 (Met-PRT1103) is obtained by substituting the tyrosine residue (Y) with a phenylalanine residue (F) and substituting most of the serine residues (S) with alanine residues (A) or glycine residues (G) from the amino acid sequence shown in Sequence ID No. 25 (Met-PRT410).

[0039] The amino acid sequence shown in Sequence ID No. 5 (Met-PRT1107) is obtained by substituting the serine residue (S) of the amino acid sequence shown in Sequence ID No. 1 (Met-PRT918) with an alanine residue (A), a valine residue (V), a leucine residue (L), or an isoleucine residue (I).

[0040] The amino acid sequence shown in Sequence ID No. 6 (Met-PRT1146) is the same as the amino acid sequence shown in Sequence ID No. 1 (Met-PRT918), but with the tyrosine residue (Y) deleted and the serine residue (S) replaced with an alanine residue (A) or a glycine residue (G).

[0041] The amino acid sequence shown in Sequence ID No. 7 (Met-PRT1147) is obtained by substituting the tyrosine residue (Y) with a phenylalanine residue (F) and substituting the serine residue (S) with an alanine residue (A) or a glycine residue (G) from the amino acid sequence shown in Sequence ID No. 1 (Met-PRT918).

[0042] The amino acid sequence shown in Sequence ID No. 8 (Met-PRT1148) is obtained by substituting the tyrosine residue (Y) with a leucine residue (L) and the serine residue (S) with an alanine residue (A) or a glycine residue (G) from the amino acid sequence shown in Sequence ID No. 1 (Met-PRT918).

[0043] The amino acid sequence shown in SEQ ID NO: 10 (Met-PRT826) is obtained by substituting the threonine residue (T) with a serine residue (S) from the amino acid sequence shown in SEQ ID NO: 11 (Met-PRT1127), further substituting VF with QQ, and substituting the isoleucine residue (I) with a glutamine residue (Q). The amino acid sequence shown in SEQ ID NO: 10 (Met-PRT826) is also obtained by substituting GTGA with GPGA, GTGS with GPGS, GLGV with GPGV, GTGI with GPGI, GLY with GPY, GTS with GPS, VF with QQ, and substituting the isoleucine residue (I) with a glutamine residue (Q) from the amino acid sequence shown in SEQ ID NO: 9 (Met-PRT1083).

[0044] The amino acid sequence shown in Sequence ID No. 11 (Met-PRT1127) is obtained by substituting GTGA with GPGA, GTGS with GPGS, GLGV with GPGV, GTGI with GPGI, GLY with GPY, GTS with GPS, and then substituting serine residues (S) with threonine residues (T).

[0045] The modified fibroin in (i) may consist of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 11.

[0046] The modified fibroin of (ii) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 11. The modified fibroin of (ii) also contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 11. n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n This protein contains a domain sequence represented by a motif. Preferably, the sequence identity is 95% or higher.

[0047] The modified fibroin of (ii) preferably has a content of amino acid residues having a free hydroxyl group of 22% or less. Furthermore, the modified fibroin of (ii) preferably has a content of serine residues, threonine residues, and tyrosine residues of 22% or less.

[0048] The modified fibroin described above may contain a tag sequence at either the N-terminus or the C-terminus, or both. This enables the isolation, immobilization, detection, and visualization of the modified fibroin.

[0049] Examples of tag sequences include affinity tags that utilize specific affinity (binding affinity) with other molecules. A specific example of an affinity tag is the histidine tag (His tag). The His tag is a short peptide consisting of about 4 to 10 histidine residues, and because it has the property of specifically binding to metal ions such as nickel, it can be used for the isolation of modified fibroin by chelating metal chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24 (an amino acid sequence containing the His tag).

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

[0051] Furthermore, "epitope tags" utilizing antigen-antibody reactions can also be used. By adding an antigenic peptide (epitope) as a tag sequence, antibodies against that epitope can be bound. Examples of epitope tags include HA (hemagglutinin peptide sequence of influenza virus) tags, myc tags, and FLAG tags. By using epitope tags, modified fibroin can be easily purified with high specificity.

[0052] Furthermore, a modified fibroin can be used in which the tag sequence can be cleaved with a specific protease. By treating the protein adsorbed via the tag sequence with a protease, the modified fibroin from which the tag sequence has been cleaved can be recovered.

[0053] More specific examples of modified fibroin containing tag sequences include (iii) modified fibroin containing the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 22, or modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 22.

[0054] The amino acid sequences shown in SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 21, or 22 each have an amino acid sequence obtained by adding the amino acid sequence shown in SEQ ID NOs: 23 or 24 (including the His tag) to the N-terminus of the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 10, or 11, respectively.

[0055] The modified fibroin in (iii) may consist of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22.

[0056] The modified fibroin of (iii) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. The modified fibroin of (iii) also contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) nThis protein contains a domain sequence represented by a motif. Preferably, the sequence identity is 95% or higher.

[0057] The modified fibroin of (iii) preferably has a content of amino acid residues having a free hydroxyl group of 22% or less. Furthermore, the modified fibroin of (iii) preferably has a content of serine residues, threonine residues, and tyrosine residues of 22% or less.

[0058] The modified fibroin described above 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 host.

[0059] [Nucleic acid] The nucleic acid according to the present invention encodes a modified fibroin according to the present invention. Specific examples of the nucleic acid include a modified fibroin containing the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 11, or a modified fibroin in which the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24 (a tag sequence) is attached to either the N-terminus or C-terminus or both of these amino acid sequences.

[0060] A nucleic acid according to one embodiment hybridizes under stringent conditions with the complementary strand of a nucleic acid encoding the modified fibroin according to the present invention, and also conforms to formula 1:[(A)] n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n This nucleic acid encodes a modified fibroin containing a domain sequence represented by a motif. The modified fibroin encoded by this nucleic acid preferably has a content of 22% or less of amino acid residues having a free hydroxyl group. Furthermore, the modified fibroin encoded by this nucleic acid preferably has a content of 22% or less of serine residues, threonine residues, and tyrosine residues.

[0061] "Stringent conditions" refer to conditions under which so-called specific hybrids are formed, and nonspecific hybrids are not formed. "Stringent conditions" can be low-stringent, medium-stringent, or high-stringent conditions. Low-stringent conditions mean that hybridization occurs only when at least 85% identity exists between the sequences. For example, this could be a condition where 5×SSC containing 0.5% SDS is used and hybridizes at 42°C. Medium-stringent conditions mean that hybridization occurs only when at least 90% identity exists between the sequences. For example, this could be a condition where 5×SSC containing 0.5% SDS is used and hybridizes at 50°C. High-stringent conditions mean that hybridization occurs only when at least 95% identity exists between the sequences. For example, this could be a condition where 5×SSC containing 0.5% SDS is used and hybridizes at 60°C.

[0062] Nucleic acids according to other embodiments have sequence identity of 90% or more with the nucleic acid encoding the modified fibroin according to the present invention, and are of formula 1:[(A) n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n This nucleic acid encodes a modified fibroin containing a domain sequence represented by a motif. The modified fibroin encoded by this nucleic acid preferably has a content of 22% or less of amino acid residues having a free hydroxyl group. Furthermore, the modified fibroin encoded by this nucleic acid preferably has a content of 22% or less of serine residues, threonine residues, and tyrosine residues.

[0063] [Host and expression vector] The expression vector according to the present invention comprises a nucleic acid sequence according to the present invention and one or more regulatory sequences operably linked to the nucleic acid sequence. The regulatory sequences are sequences that control the expression of recombinant proteins in the host (e.g., promoters, enhancers, ribosome-binding sequences, transcription termination sequences, etc.) and can be appropriately selected depending on the type of host. The type of expression vector can be appropriately selected depending on the type of host, such as plasmid vectors, viral vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, etc.

[0064] The host according to the present invention is transformed with the expression vector according to the present invention. Any prokaryotes, as well as eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells, can be suitably used as the host.

[0065] Preferably, the expression vector used is one that can autonomously replicate in host cells or can be incorporated into host chromosomes, and contains a promoter at a position where the nucleic acid according to the present invention can be transcribed.

[0066] When using prokaryotes such as bacteria as hosts, the expression vector according to the present invention is preferably a vector that can autonomously replicate in the prokaryote and also contains a promoter, a ribosome-binding sequence, the nucleic acid according to the present invention, and a transcription termination sequence. It may also contain a gene that controls the promoter.

[0067] Examples of prokaryotes include microorganisms belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas.

[0068] 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, and Escherichia coli. Examples include Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W3110 (ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, etc.

[0069] Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus borsterensis, Brevibacillus centroporus, Brevibacillus formosus, Brevibacillus invocatus, Brevibacillus latillosporus, Brevibacillus limnophilus, Brevibacillus parabrevis, Brevibacillus reuszeri, Brevibacillus thermolvus, Brevibacillus brevis 47 (FERM BP-1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevibacillus brevis 47-5Q (JCM8975), and Brevibacillus chosinensis HPD31 (FERM Examples include BP-1087), Brevibacillus chousinensis HPD31-S (FERM BP-6623), Brevibacillus chousinensis HPD31-OK (FERM BP-4573), and Brevibacillus chousinensis SP3 strain (manufactured by Takara Co., Ltd.).

[0070] 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.

[0071] Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis and Bacillus amyloliquefaciens.

[0072] Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphyllum ATCC15354.

[0073] Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam (Corynebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corynebacterium glutamicum) ATCC14067 ATCC13826, ATCC14067, Brevibacterium immariophilum ATCC14068, Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC13869 ATCC13665, ATCC13869, Brevibacterium roseum ATCC13825, and Brevibacterium saccharoticum (Brevibacterium Examples include *Saccharolyticum* ATCC14066, *Brevibacterium thiogenitalis* ATCC19240, *Brevibacterium album* ATCC15111, and *Brevibacterium selinum* ATCC15112.

[0074] Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC6871, ATCC6872, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, and Corynebacterium acetacidophilum. Examples include Corynebacterium acetoacidophilum ATCC13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanoritycum ATCC21511, Corynebacterium carnae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060, Corynebacterium lylium ATCC15990, Corynebacterium melasecola ATCC17965, Corynebacterium thermoaminogenes AJ12340 (FERMBP-1539), and Corynebacterium hercules ATCC13868.

[0075] Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas fulva, and Pseudomonas sp. D-0110.

[0076] Any method for introducing the expression vector into the host cells can be used as long as it involves introducing DNA into the host cells. For example, methods using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Publication No. 63-248394), or methods described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979) can be used.

[0077] 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).

[0078] Examples of nucleic acid vectors according to the present invention (hereinafter simply referred to as "vectors") include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese Patent Publication No. 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)], and pBluescript II. SK(-) (manufactured by Stratagene), pTrs30 [prepared from Escherichiacoli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), Japanese Patent Publication No. 60-221091], pGKA2 [Escherichia coli IGKA2 (FERM Examples include BP-6798 (prepared from Japanese Patent Publication No. 60-221091), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 (J. Bacteriol., 172, 2392 (1990)), pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), etc.

[0079] When using Escherichia coli as the host, suitable vectors include pUC18, pBluescriptII, pSupex, pET22b, and pCold.

[0080] Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is a known Bacillus subtilis vector, or pHY500 (JP-A-2-31682), pNY700 (JP-A-4-278091), pHY4831 (J. Bacteriol., 1987, 1239-1245), pNU200 (Shigezo Udaka, Journal of the Japan Society for Bioscience, Biotechnology, and Agrochemistry 1987, 61:669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30:75-80), pNU211 (J. Biochem., 1992, 112:488-491), and pNU2 Examples include 11R2L5 (Japanese Patent Publication No. 7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58:525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177:745-749), pHT210 (Japanese Patent Publication No. 6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42:358-363), or pNCO2 (Japanese Patent Publication No. 2002-238569), which is a shuttle vector between Escherichia coli and microorganisms belonging to the genus Brevibacillus.

[0081] The promoter can be any promoter that functions in a host cell. Examples include promoters derived from E. 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 Ptrp x 2 promoter, tac promoter, lacT7 promoter, and let I promoter, can also be used.

[0082] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (e.g., 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 nucleic acids according to the present invention, but it is preferable to place the transcription termination sequence directly below the structural gene.

[0083] Examples of eukaryotic hosts include yeast, filamentous fungi (molds, etc.), and insect cells.

[0084] Examples of yeasts include those belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Jarrowia, 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 include Pichia stipitis, Yarrowia lipolytica, and Hansenula polymorpha.

[0085] When using yeast as the host cell, the expression vector usually preferably includes a replication origin (if amplification in the host is required), a selection marker for vector growth in E. coli, a promoter and terminator for recombinant protein expression in yeast, and a selection marker for yeast.

[0086] If the expression vector is a non-integrated vector, it is preferable to further include a self-replicating sequence (ARS). This can improve the stability of the expression vector within cells (Myers, AM, et al. (1986) Gene 45:299-310).

[0087] Examples of vectors that use yeast as a host include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, and pPICZ B.

[0088] The promoter is not limited to those that can be expressed in yeast. Examples include promoters for glycolytic genes such as hexose kinases, 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, and MOX promoter.

[0089] Any method for introducing an expression vector into yeast can be used, such as electroporation (Methods Enzymol., 194, 182 (1990)), spheroplast (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), lithium acetate (J. Bacteriol., 153, 163 (1983)), and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

[0090] Examples of filamentous fungi include those belonging to the genera Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, Penicillium, Myceliophtora, Botryts, Magnaporthe, Mucor, Metarhizium, Monascus, Rhizopus, and Rhizomucoa.

[0091] 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, and Aspergillus parasiticus. Aspergillus parasiticus, Aspergillus ficuum, Aspergillus phoeicus, Aspergillus foetidus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus japonicus, Trichoderma viride, Trichoderma harzianum, Trichoderma reseei, Chrysosporium lucnoens lucknowense), Thermoascus, Sporotrichum, Sporotrichum cellulophilum, Talaromyces, Thielavia terrestris, Thielavia, Neurosporacrassa), Fusarium oxysporus, Fusarium graminearum, Fusarium venenatum, Humicola insolens, Penicillium chrysogenum, Penicillium camemberti, Penicillium canescens, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium perprogenum (Penicillium Mocor purpurogenum), Penicillium roqueforti, Myceliophtaora thermophilum, Mucor ambiguus, Mucor circinelloides, Mucor fragilis, Mucor hiemalis, Mucor inaequisporus, Mucor oblongiellipticus, Mucor racemosus, Mucor recurvus, Mucor saturninus, Mucor subtilissmus, Ogataea polymorpha Examples include *Rhizomucor polymorpha*, *Phanerochaete chrysosporium*, *Rhizomucor miehei*, *Rhizomucor pusillus*, and *Rhizopus arrhizus*.

[0092] When the host is a filamentous fungus, the promoter can be any of the following: genes related to glycolysis, genes related to constitutive expression, enzyme genes related to hydrolysis, etc. Specifically, examples include amyB, glaA, agdA, glaB, TEF1, xynF1tannasegene, No.8AN, gpdA, pgkA, enoA, melO, sodM, catA, catB, etc.

[0093] The introduction of expression vectors into filamentous fungi can be carried out using conventionally known methods. Examples include 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.

[0094] Examples of insect cells include lepidopteran 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.

[0095] Examples of vectors used when insect cells are used as hosts include baculoviruses such as Autographa californica nuclear polyhedrosis virus, a virus that infects moths of the family Croptail (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)).

[0096] When insect cells are used as a host, polypeptides can be expressed by methods 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. Specifically, recombinant gene transfer vectors and baculoviruses are co-introduced into insect cells to obtain recombinant viruses (expression vectors) in the insect cell culture supernatant, and then the recombinant viruses are used to further infect the insect cells and express polypeptides. Examples of gene transfer vectors used in this method include pVL1392, pVL1393, and pBlueBacIII (all manufactured by Invitorogen).

[0097] Methods for co-introducing recombinant gene vectors and baculoviruses into insect cells to prepare recombinant viruses include, for example, the calcium phosphate method (Japanese Patent Publication No. 2-227075) and the lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).

[0098] The recombinant vector according to the present invention preferably further contains a selection marker gene for transformant selection. For example, in Escherichia coli, resistance genes to various drugs such as tetracycline, ampicillin, and kanamycin can be used as the selection marker gene. Recessive selection markers that can complement gene mutations involved in nutritional requirements can also be used. In yeast, a resistance gene to genetisin can be used as the selection marker gene, and genes that complement gene mutations involved in nutritional requirements, as well as selection markers such as LEU2, URA3, TRP1, and HIS3, can also be used. In filamentous fungi, the following genes are used as selection markers: 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 Examples include marker genes selected from the group consisting of Acad Sci USA, 83, 4869-4873, (1986) and the hygromycin resistance gene (Gene, 57, 21-26, (1987)), and leucine requirement complement genes. In addition, if the host is a nutrient requirement mutant, a wild-type gene that complements that nutrient requirement can be used as a selected marker gene.

[0099] The selection of hosts transformed with the expression vector according to the present invention can be performed by plaque hybridization and colony hybridization using a probe that selectively binds to the nucleic acid according to the present invention. As the probe, a partial DNA fragment amplified by PCR based on the nucleic acid sequence information according to the present invention and modified with a radioisotope or digoxigenin can be used.

[0100] [Method for producing modified fibroin] The modified fibroin according to the present invention can be produced by a method that includes the step of expressing the nucleic acid according to the present invention in a host transformed with the expression vector according to the present invention. Expression methods include direct expression, secretory production, fusion protein expression, etc., in accordance with methods described in Molecular Cloning, 2nd Edition. When expressed in yeast, animal cells, or insect cells, the modified fibroin can be obtained as a polypeptide to which sugars or sugar chains have been added.

[0101] The modified fibroin according to the present invention can be produced, for example, by culturing a host transformed with the expression vector according to the present invention in a culture medium, generating and accumulating the modified fibroin according to the present invention in the culture medium, and then collecting it from the culture medium. The method for culturing the host according to the present invention in a culture medium can be carried out according to the method commonly used for culturing hosts.

[0102] When the host according to the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, either a natural culture medium or a synthetic culture medium may be used as the culture medium for the host according to the present invention, as long as it contains a carbon source, nitrogen source, and inorganic salts that the host can utilize and allows for efficient cultivation of the host.

[0103] Any carbon source that the host can utilize is acceptable, and examples 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.

[0104] As nitrogen sources, for example, ammonium salts of inorganic or organic acids such as ammonia, 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 microorganisms and their digests can be used.

[0105] 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.

[0106] Prokaryotes such as Escherichia coli or eukaryotes such as yeast can be cultured under aerobic conditions, such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40°C. The culture time is usually 16 hours to 7 days. It is preferable to maintain the pH of the culture medium at 3.0 to 9.0 during culture. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, and ammonia, etc.

[0107] Furthermore, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during cultivation. When culturing microorganisms transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as needed. For example, when culturing microorganisms transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside may be added to the medium, and when culturing microorganisms transformed with an expression vector using a trp promoter, indoleacrylic acid may be added to the medium.

[0108] Commonly used culture media for insect cells include TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400, ExCell405 (both from JRH Biosciences), and Grace's Insect Medium (Nature, 195, 788 (1962)).

[0109] Insect cells can be cultured for 1 to 5 days under conditions such as a culture medium pH of 6-7 and a culture temperature of 25-30°C. Antibiotics such as gentamicin may also be added to the culture medium during cultivation as needed.

[0110] When the host is a plant cell, the transformed plant cells may be cultured as is, or they may be differentiated into plant organs and then cultured. As a culture medium for these plant cells, commonly used Murashige & Skoog (MS) medium, White medium, or these media to which plant hormones such as auxin and cytokinin have been added can be used.

[0111] 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. Antibiotics such as kanamycin and hygromycin may also be added to the culture medium as needed during the culture process.

[0112] Methods for producing modified fibroin using a host transformed with the expression vector according to the present invention include producing the modified fibroin inside the host cell, secreting it outside the host cell, and producing it on the host cell's outer membrane. These methods can be selected by changing the host cell used and the structure of the modified fibroin produced.

[0113] For example, if the modified fibroin is produced inside or on the host cell membrane, the modified fibroin can be modified to be actively secreted outside the host cell by applying methods such as those of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), Lowe et al. (Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)), or methods described in Japanese Patent Publication No. 5-336963, International Publication No. 94 / 23021, etc. That is, by using genetic recombination techniques to express a polypeptide containing the active site of the modified fibroin with a signal peptide attached, the modified fibroin can be actively secreted outside the host cell.

[0114] Modified fibroin produced by a host transformed with the expression vector according to the present invention can be isolated and purified by methods commonly used for the isolation and purification of proteins. For example, if the modified fibroin is expressed in a lysed state within cells, after the culture is complete, the host cells are recovered by centrifugation, soaked in an aqueous buffer, and then the host cells are lysed using an ultrasonic disruptor, French press, Manton Gaurine homogenizer, and Dynomil to obtain a cell-free extract. By centrifuging the cell-free extract, the supernatant obtained can be used, individually or in combination, to obtain purified samples from the supernatant obtained by methods commonly used for the isolation and purification of proteins, namely, solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents, 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, and electrophoresis such as isoelectric focusing.

[0115] For the above chromatography, column chromatography using phenyl-toyoparl (Tosoh), DEAE-toyoparl (Tosoh), and Sephadex G-150 (Pharmacia Biotech) is preferably used.

[0116] Furthermore, if the modified fibroin is expressed in the form of an insoluble form within the cell, the host cells are similarly recovered, then lysed and centrifuged to recover the insoluble modified fibroin as a precipitate fraction. The recovered insoluble modified fibroin can be solubilized with a protein denaturant. After this procedure, a purified sample of the modified fibroin can be obtained by the same isolation and purification method as described above.

[0117] If modified fibroin, or a derivative of modified fibroin with added sugar chains, is secreted extracellularly, the modified fibroin or its derivative can be recovered from the culture supernatant. That is, the culture supernatant can be obtained by processing the culture by methods such as centrifugation, and a purified sample can be obtained from the culture supernatant using the same isolation and purification method as described above.

[0118] The modified fibroin according to the present invention has reduced ester bond formation when in contact with carboxylic acids such as formic acid, and as a result, the generation of off-odors is reduced or less likely to occur even when left in the atmosphere. Therefore, the method for producing the modified fibroin according to this embodiment does not have to include a step of contacting the modified fibroin with a carboxylic acid such as formic acid.

[0119] [Artificially modified fibroin composition] The artificially modified fibroin composition according to this embodiment contains at least the modified fibroin according to the present invention.

[0120] The content of modified fibroin in the artificially modified fibroin composition may be 30 to 100% by mass, preferably 35 to 100% by mass, and more preferably 40 to 100% by mass, based on the total amount of the artificially modified fibroin composition.

[0121] The artificially modified fibroin composition according to this embodiment may further contain other additives depending on its form, use, etc. Examples of additives include plasticizers, leveling agents, crosslinking agents, nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers, and synthetic resins. The content of additives may be 50 parts by mass or less per 100 parts by mass of the total amount of modified fibroin.

[0122] The artificially modified fibroin composition according to this embodiment may be in the form of a powder, paste, or liquid (e.g., suspension, solution). In addition, the artificially modified fibroin composition according to this embodiment may be in the form of a raw material composition (e.g., protein powder, dope liquid), or in the form of a molded article (e.g., fiber, yarn, film, foam, granules, molded article) containing the artificially modified fibroin composition or made from the artificially modified fibroin composition.

[0123] (Dope solution) The artificially modified fibroin composition according to this embodiment may be in the form of a dope solution. The dope solution according to this embodiment comprises at least modified fibroin and a solvent. The dope solution according to this embodiment may further contain a dissolution accelerator. The dope solution according to this embodiment may also further contain proteins other than modified fibroin.

[0124] Examples of solvents 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, and lithium thiocyanate. These solvents may be used individually or in combination of two or more.

[0125] The content of modified fibroin in the dope solution 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 solution. From the viewpoint of manufacturing efficiency of the dope solution, the content of 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 solution.

[0126] Examples of dissolution accelerators include inorganic salts composed of Lewis acids and Lewis bases, as shown below. Examples of Lewis bases include oxoacid ions (nitrate ions, perchlorate ions, etc.), metal oxoacid ions (permanganate ions, etc.), halide ions, thiocyanate ions, cyanate ions, etc. Examples of Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions. Specific examples of inorganic salts consisting 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; and sodium chloride. Examples include sodium salts such as sodium bromide, sodium iodide, sodium nitrate, sodium perchlorate, and sodium 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.

[0127] The amount of dissolution accelerator 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, per 100 parts by mass of the total amount of modified fibroin. The amount of dissolution accelerator may be 40 parts by mass or less, 35 parts by mass or less, or 30 parts by mass or less, per 100 parts by mass of the total amount of modified fibroin.

[0128] During the preparation of the dope solution according to this embodiment, the temperature may be heated to 30-90°C. The temperature at which dissolution is possible should be set appropriately depending on the solvent used, the type of modified fibroin, etc. Shaking or stirring may be used to promote dissolution.

[0129] The viscosity of the doping solution according to this embodiment may be set appropriately depending on the intended use of the doping solution. For example, when the doping solution according to this embodiment is used as a spinning stock, its viscosity may be set appropriately depending on the spinning method, for example, to 100 to 15,000 cP (centipoise) at 35°C, 100 to 30,000 cP (centipoise) at 40°C, etc. The viscosity of the spinning stock can be measured using, for example, a product called "EMS Viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd.

[0130] (Protein fiber) The artificially modified fibroin composition according to this embodiment may be in the form of protein fibers. Protein fibers can be obtained, for example, by spinning the doping solution (spinning solution) described above in a manner commonly used for spinning fibroin.

[0131] The spinning method is not particularly limited as long as it is capable of spinning the modified fibroin according to the present invention, and examples include dry spinning, melt spinning, and wet spinning. A preferred spinning method is wet spinning.

[0132] In wet spinning, the doping solution is extruded from the spinneret (nozzle) into a coagulation solution (coagulation solution tank), and the modified fibroin is solidified in the coagulation solution to obtain undrawn yarn in the shape of a thread. The coagulation solution can be any solution from which solvent removal is possible, such as lower alcohols with 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone. Water may be added to the coagulation solution as appropriate. The temperature of the coagulation solution is preferably 0 to 30°C. When using a syringe pump with a nozzle of 0.1 to 0.6 mm in diameter as the spinneret, the extrusion rate 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 solution tank should be long enough to allow efficient solvent removal, for example, 200 to 500 mm. The take-up rate of the undrawn yarn may be, for example, 1 to 20 m / min, and 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. Furthermore, stretching (pre-stretching) may be performed in the coagulation solution. To suppress evaporation of the lower alcohol, the coagulation solution may be kept at a low temperature, and the yarn may be taken up in an unstretched state. The coagulation solution tank may have multiple stages, and stretching may be performed at each stage or at specific stages as needed.

[0133] The undrawn yarn (or pre-drawn yarn) obtained by the above method can be drawn through a drawing process. Drawing methods include wet heat drawing and dry heat drawing.

[0134] Moist heat stretching can be performed in hot water, in a solution of hot water with an organic solvent added, or under steam heating. The temperature can be, for example, 50 to 90°C, with 75 to 85°C being preferred. Moist heat stretching can stretch an unstretched yarn (or pre-stretched yarn) by, for example, 1 to 10 times, with 2 to 8 times being preferred.

[0135] Dry heat stretching can be carried out using an electric tubular furnace, a dry heat plate, etc. The temperature may be, for example, 140°C to 270°C, and preferably 160°C to 230°C. In dry heat stretching, the unstretched yarn (or pre-stretched yarn) can be stretched by, for example, 0.5 to 8 times, and preferably 1 to 4 times.

[0136] Wet heat stretching and dry heat stretching may be performed individually, or they may be performed in multiple stages or in combination. That is, 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 by wet heat stretching, and the third stage stretching by dry heat stretching, and so on, with wet heat stretching and dry heat stretching being performed in an appropriate combination.

[0137] The final stretch ratio in the stretching process is, for example, 5 to 20 times, and preferably 6 to 11 times, compared to the unstretched yarn (or pre-stretched yarn).

[0138] After stretching, protein fibers may be chemically crosslinked between polypeptide molecules within 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 contained in the polypeptide can be crosslinked with the carboxyl group of the glutamic acid or aspartic acid side chain by dehydration condensation via an amide bond. Crosslinking may be carried out by a dehydration condensation reaction under vacuum heating, or by using a dehydration condensation agent such as carbodiimide.

[0139] Crosslinking between polypeptide molecules may be carried out using crosslinking agents such as carbodiimide and glutaraldehyde, or using enzymes 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 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, and diisopropylcarbodiimide (DIC). Among these, EDC and DIC are preferred because they have high ability to form amide bonds between polypeptide molecules and readily undergo crosslinking reactions.

[0140] The crosslinking treatment is preferably carried out by applying a crosslinking agent to the protein fibers and crosslinking them by vacuum heating and drying. The crosslinking agent may be applied to the protein fibers in pure form, or it may be applied to the protein fibers after being diluted to a concentration of 0.005 to 10% by mass with a lower alcohol having 1 to 5 carbon atoms and a buffer solution. 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.

[0141] (film) The artificially modified fibroin composition according to this embodiment may be in the form of a film. The film can be obtained, for example, by casting the doping solution described above onto a substrate surface, drying, and / or desolvating it.

[0142] The viscosity of the doped solution is preferably 15 to 80 cP (centipoise), and more preferably 20 to 70 cP.

[0143] When the doped solution is considered to be 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.

[0144] The doping solution may be heated to 30-60°C during preparation. Shaking or stirring may be used to promote dissolution.

[0145] The substrate may be a resin substrate, a glass substrate, a metal substrate, etc. From the viewpoint of easily peeling off the film after casting, the substrate is preferably a resin substrate. Examples of resin substrates include polyethylene terephthalate (PET) film, fluororesin films such as polytetrafluoroethylene, polypropylene (PP) film, or release films in which a silicone compound is immobilized on the surface of these films. From the viewpoint of being stable to HFIP, DMSO solvent, etc., enabling stable casting of the doped solution, and allowing easy peeling off the film after molding, the substrate is more preferably a PET film or a release film in which a silicone compound is immobilized on the surface of a PET film.

[0146] To explain the specific procedure, first, the doping solution is cast onto the substrate surface, and a wetted film of a predetermined thickness (for example, 1 to 1000 μm after drying and / or desolvation) is prepared using film thickness control means such as an applicator, knife coater, or bar coater.

[0147] Drying and / or desolvation can be carried out dry or wet. Dry methods include vacuum drying, hot air drying, and air drying. Wet methods include immersing the cast film in a desolvation solution (also called a coagulation solution) to remove the solvent. Examples of desolvation solutions include water, alcohol solutions such as methanol, ethanol, and lower alcohols having 1 to 5 carbon atoms such as 2-propanol, and mixtures of water and alcohol. The temperature of the desolvation solution (coagulation solution) is preferably 0 to 90°C.

[0148] The unstretched film after drying and / or desolvation can be uniaxially or biaxially stretched in water. Biaxial stretching may be sequential or simultaneous. Multi-stage stretching of two or more stages is also possible. The stretching ratio is preferably 1.01 to 6 times, more preferably 1.05 to 4 times, in both the longitudinal and transverse directions. This range makes it easy to balance stress and strain. Stretching in water is preferably carried out at a water temperature of 20 to 90°C. After stretching, the film is preferably heat-set with dry heat at 50 to 200°C for 5 to 600 seconds. This heat-set provides dimensional stability at room temperature. Note that a uniaxially stretched film becomes a uniaxially oriented film, and a biaxially stretched film becomes a biaxially oriented film.

[0149] [Method for producing an artificially modified fibroin composition] The artificially modified fibroin composition according to the present invention can be produced by a method that includes the step of preparing the modified fibroin according to the present invention. The method for producing the artificially modified fibroin composition according to the present invention may further include the step of preparing a modified fibroin solution (e.g., a dope solution) containing the modified fibroin and a carboxylic acid according to the present invention.

[0150] The modified fibroin according to the present invention has reduced ester bond formation when in contact with carboxylic acids such as formic acid, and as a result, the generation of off-odors is reduced or less likely to occur even when left in the atmosphere. Therefore, the method for producing the artificial modified fibroin composition according to this embodiment does not have to include a step of contacting the modified fibroin with a carboxylic acid such as formic acid.

[0151] [Method for reducing the formation of ester bonds between modified fibroin and carboxylic acid] The method for reducing the formation of ester bonds between modified fibroin and carboxylic acid according to this embodiment includes the step of modifying the amino acid sequence of a fibroin protein by substituting, deleting, inserting and / or adding one or more amino acid residues, wherein the number of amino acid residues having free hydroxyl groups in the modified fibroin after modification is reduced by at least 20% compared to the number of amino acid residues having free hydroxyl groups in the fibroin protein before modification.

[0152] Substitution, deletion, insertion, and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art, such as partially specific mutagenesis. Specifically, this can be done in accordance with the methods described in literature such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0153] The modification of the fibroin protein in the method according to this embodiment can be carried out in a preferred manner as described in the embodiment of the modified fibroin.

[0154] 〔product〕 The protein fibers according to the present invention can be applied as fibers (long fibers, short fibers, multifilaments, or monofilaments, etc.) or yarns (spun yarns, twisted yarns, false twisted yarns, processed yarns, blended yarns, etc.) to woven fabrics, knitted fabrics, braided fabrics, nonwoven fabrics, etc. They can also be applied to high-strength applications such as ropes, surgical sutures, flexible fasteners for electrical components, and even bioactive materials for implantation (e.g., artificial ligaments and aortic bands).

[0155] Furthermore, the artificially modified fibroin composition according to 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 their equivalents, which can be manufactured in accordance with the methods described in Japanese Patent Publication No. 2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735, etc. [Examples]

[0156] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0157] [Manufacturing of modified fibroin] (1) Preparation of expression vectors Modified fibroins (PRT918, PRT1105, PRT1107, PRT1083, PRT826, and PRT1127, respectively) having the amino acid sequences shown in SEQ ID NOs. 12, 14, 16, and 20-22 were designed.

[0158] (Example 1) The amino acid sequence shown in Sequence ID No. 12 (PRT918) is obtained by substituting GTGA with GPGA, GTGS with GPGS, GLGV with GPGV, GTGI with GPGI, GLY with GPY, and GTS with GPS in the amino acid sequence shown in Sequence ID No. 20 (PRT1083).

[0159] (Example 2) The amino acid sequence shown in Sequence ID No. 22 (PRT1127) is obtained by substituting GTGA with GPGA, GTGS with GPGS, GLGV with GPGV, GTGI with GPGI, GLY with GPY, GTS with GPS, and then substituting serine residues (S) with threonine residues (T).

[0160] (Example 3) The amino acid sequence shown in Sequence ID No. 14 (PRT1105) is obtained by substituting the serine residue (S) of the amino acid sequence shown in Sequence ID No. 12 (PRT918) with an alanine residue (A) or a glycine residue (G).

[0161] (Example 4) The amino acid sequence shown in Sequence ID No. 16 (PRT1107) is obtained by substituting the serine residue (S) of the amino acid sequence shown in Sequence ID No. 12 (PRT918) with an alanine residue (A), a valine residue (V), a leucine residue (L), or an isoleucine residue (I).

[0162] (Comparative Example 1) The amino acid sequence shown in Sequence ID No. 21 (PRT826) is obtained by substituting the threonine residue (T) with a serine residue (S) from the amino acid sequence shown in Sequence ID No. 22 (PRT1127), further substituting VF with QQ, and substituting the isoleucine residue (I) with a glutamine residue (Q).

[0163] Table 1 shows the serine residue content (S residue content), threonine residue content (T residue content), tyrosine residue content (Y residue content), amino acid residue content with free hydroxyl groups (amino acid residue content with free OH groups), and the reduction rate of the number of amino acid residues with free hydroxyl groups after modification (OH reduction rate) relative to the number of amino acid residues with free hydroxyl groups before modification for modified fibroins having the amino acid sequences shown in Sequence IDs 12, 14, 16 and 20-22. [Table 1]

[0164] A nucleic acid encoding the designed modified fibroin was synthesized. An NdeI site was added to the 5' end of this nucleic acid, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Subsequently, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

[0165] (2) Protein production The obtained expression vector was used to transform E. coli BLR(DE3). The transformed E. coli were cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture medium was then mixed with OD in 100 mL of seed culture medium containing ampicillin (Table 2). 600 The amount was added to make it 0.005. The culture medium temperature was maintained at 30°C, and the OD 600 Flask culture was performed until the concentration reached 5 (approximately 15 hours) to obtain the seed culture medium. [Table 2]

[0166] Adding 500 mL of the seed culture solution to the production medium (Table 3) in a jar fermenter, then adding OD 600 The solution was added to achieve a concentration of 0.05. The culture medium temperature was maintained at 37°C, and the pH was controlled to a constant 6.9 during cultivation. The dissolved oxygen concentration in the culture medium was also maintained at 20% of the dissolved oxygen saturation concentration.

[0167] [Table 3]

[0168] Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g glucose / 1 L, 120 g yeast extract / 1 L) was added at a rate of 1 mL / min. The culture medium temperature was maintained at 37°C, and the pH was controlled to a constant 6.9. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was performed for 20 hours. Subsequently, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce the expression of modified fibroin. 20 hours after IPTG addition, the culture medium was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture medium before and after IPTG addition, and the expression of the target modified fibroin was confirmed by the appearance of a band of the target modified fibroin size that was dependent on IPTG addition.

[0169] (3) Purification of proteins Two hours after adding IPTG, the collected bacterial cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF, and the cells were lysed using a high-pressure homogenizer (GEA Niro Soavi). The lysed cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until it reached high purity. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and dissolved by stirring with a stirrer at 60°C for 30 minutes. After dissolution, dialysis was performed with water using dialysis tubing (cellulose tubing 36 / 32, Sanko Pure Chemical Industries, Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed using a freeze-dryer, and the freeze-dried powder was recovered to obtain modified fibroin (PRT918, PRT1105, PRT1107, PRT1083, PRT826, and PRT1127).

[0170] [Production and evaluation of protein films] (1) Production of protein films The dried powder of the modified fibroin obtained was added to formic acid and heated at 40°C for 1 hour to dissolve it and obtain a dope solution (protein concentration in the dope solution: 26% by mass).

[0171] The obtained doping solution was applied to a glass slide to a thickness of approximately 0.5 mm, and then immersed in acetone and water sequentially (for 15 minutes each) to solidify and wash. After air drying overnight, the film was peeled off the glass slide to obtain the sample. The film thickness was approximately 0.5 to 1.0 mm.

[0172] (2) Evaluation of protein films The degree of formic acid ester formation within the film samples was evaluated by measuring the infrared absorption spectra of the manufactured film samples using the following measuring equipment. Measurement device: Nicolet iS50 FT-IR (Manufacturer: Thermo Fisher Scientific Co., Ltd.)

[0173] The degree of formate ester formation was evaluated by calculating the absorbance ratio P1 / P2. A smaller absorbance ratio P1 / P2 indicates less formate ester formation. P1: 1725cm -1 (Peak height based on the C=O of the ester) P2: 1445cm -1 Peak height (of the peak based on protein amide III)

[0174] The results are shown in Figure 1 and Table 1. As shown in Figure 1, by reducing the content of amino acid residues with free hydroxyl groups (for example, an OH reduction rate of 20% or more), the formic acid ester absorption region (1715-1730 cm²) is reduced. -1 The peak in the ) region was reduced (Examples 1-4). On the other hand, even when an amino acid residue without a free hydroxyl group was substituted, the formic acid ester absorption region (1715-1730 cm) was reduced. -1 No significant changes were observed in the peak (Comparative Example 1).

Claims

1. Formula 1: [(A) ｎ Motif - REP] ｍ , or formula 2: [(A) ｎ Motif - REP] ｍ - (A) ｎ Includes a domain sequence represented by a motif, Modified fibroin having a free hydroxyl group amino acid residue content of 10% or less. [In equations 1 and 2, (A) ｎ The motif shows an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) ｎ The number of alanine residues in the motif is 80% or more of the total number of amino acid residues, and it is an amino acid sequence that produces a crystalline region. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues and is an amino acid sequence that produces an amorphous region. m represents an integer from 10 to 300. Multiple sequences exist (A). ｎ The motifs may have the same amino acid sequence or different amino acid sequences. Multiple REPs may have the same amino acid sequence or different amino acid sequences.

2. A nucleic acid encoding the modified fibroin described in claim 1.

3. An expression vector comprising a nucleic acid sequence according to claim 2 and one or more regulatory sequences operably linked to the nucleic acid sequence.

4. The expression vector according to claim 3, which is a plasmid vector or a viral vector.

5. A host (excluding humans) transformed with the expression vector described in claim 3 or 4.

6. The host according to claim 5, which is a prokaryote.

7. The host according to claim 6, wherein the prokaryote is a microorganism belonging to a genus selected from the group consisting of Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas.

8. The host according to claim 7, which is a eukaryote.

9. The host according to claim 8, wherein the eukaryote is a yeast, a filamentous fungus, or an insect cell.

10. A synthetic modified fibroin composition comprising the modified fibroin described in claim 1.

11. The artificially modified fibroin composition according to claim 10, which is a protein powder.

12. The artificially modified fibroin composition according to claim 10, which is a dope liquid.

13. The artificially modified fibroin composition according to claim 10, which is a fiber.

14. The artificially modified fibroin composition according to claim 10, which is a film.

15. A method for producing modified fibroin, A host (excluding humans) transformed with an expression vector having a nucleic acid sequence encoding a modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. The process includes expressing the nucleic acid by the following steps: A method for producing the modified fibroin, wherein the modified fibroin is the modified fibroin described in claim 1.

16. A method for producing an artificial modified fibroin composition containing modified fibroin, This includes the process of preparing modified fibroin. A method for producing the modified fibroin, wherein the modified fibroin is the modified fibroin described in claim 1.

17. The manufacturing method according to claim 15 or 16, further comprising the step of contacting the modified fibroin with a carboxylic acid.

18. The manufacturing method according to claim 16, further comprising the step of preparing a modified fibroin solution containing the modified fibroin and carboxylic acid.

19. A method for reducing the formation of ester bonds between modified fibroin and carboxylic acid, The process includes modifying the amino acid sequence of a fibroin protein by substituting, deleting, inserting and / or adding one or more amino acid residues. The modified fibroin after modification has the formula 1: [(A) ｎ motif - REP] ｍ , or the formula 2: [(A) ｎ motif - REP] ｍ -(A) ｎ and contains a domain array represented by the motif and has an amino acid residue content having a free hydroxyl group of 10% or less. [In equations 1 and 2, (A) ｎ The motif shows an amino acid sequence consisting of 4 to 27 amino acid residues, and (A) ｎ The number of alanine residues in the motif is 80% or more of the total number of amino acid residues, and it is an amino acid sequence that produces a crystalline region. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues and is an amino acid sequence that produces an amorphous region. m represents an integer from 10 to 300. Multiple sequences exist (A). ｎ The motifs may have the same amino acid sequence or different amino acid sequences. Multiple REPs may have the same amino acid sequence or different amino acid sequences.

20. comprising the modified fibroin described in claim 1, Products selected from the group consisting of fibers, threads, films, foams, granules, nanofibrils, gels, and resins.

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