JP7829903B2 - Modified fibroin - Google Patents

Description

This invention relates to modified fibroin. More specifically, it relates to modified fibroin in which the content of amino acid residues having free hydroxyl groups is reduced.

Fibroin is a type of fibrous protein. Fibroin contains a high proportion (up to 90%) of amino acid residues with small side chains, such as glycine, alanine, serine, and tyrosine residues. Known examples of fibroin include the proteins that make up the silks produced by insects and spiders (silk protein, hornet silk protein, spider silk protein, etc.).

Silk protein possesses excellent mechanical, hygroscopic, and deodorizing properties, making it a widely used material for clothing. Furthermore, silk thread is an immunotolerant natural fiber with high biocompatibility, and is therefore also used in applications such as surgical sutures.

Various compositions containing modified fibroin have also been manufactured. For example, known compositions containing modified fibroin include spider silk protein films (e.g., Patent Document 1) and protein fibers (e.g., Patent Document 2).

International Publication No. 2014/103799 International Publication No. 2013/065651

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 acid such as formic acid remaining on or inside the protein as a catalyst, releasing the carboxylic acid, which then causes unpleasant odors. This invention aims to solve these problems newly discovered by the inventors.

In other words, the present invention aims to provide a modified fibroin in which the formation of ester bonds upon contact with carboxylic acids 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 upon contact with carboxylic acids such as formic acid.

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

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,
A modified fibroin in which the number of amino acid residues containing free hydroxyl groups after modification is reduced by at least 20% compared to the number of amino acid residues containing free hydroxyl groups before modification.
[2]
The modified fibroin according to [1], wherein the content of amino acid residues having free hydroxyl groups is reduced by at least 6% compared to the pre-modification product.
[3]
Formula 1: [(A) _n motif - REP] _m , or Formula 2: [(A) _n motif - REP] _m - (A) _n motif, comprising a domain sequence
Modified fibroin having a free hydroxyl group amino acid residue content of 22% or less.
[In formulas 1 and 2, (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues in the (A) _n motif is 80% or more of the total number of amino acid residues. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer from 10 to 300. Multiple (A) _n 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.]
[4]
A nucleic acid encoding a modified fibroin as described in any of [1] to [3].
[5]
A nucleic acid that hybridizes under stringent conditions with the complementary strand of the nucleic acid described in [4] 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 formulas 1 and 2, (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues in the (A) _n motif is 80% or more of the total number of amino acid residues. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer from 10 to 300. Multiple (A) _n 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]
A nucleic acid having 90% or more sequence identity with the nucleic acid described in [4], and encoding 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 formulas 1 and 2, (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues in the (A) _n motif is 80% or more of the total number of amino acid residues. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer from 10 to 300. Multiple (A) _n 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 according to [10], 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.
[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 artificially 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].
[21]
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.
[22]
The manufacturing method according to [20], further comprising the step of preparing a modified fibroin solution containing the modified fibroin and a carboxylic acid.
[23]
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 fibroin protein before modification.
[24]
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% based on the content of amino acid residues having free hydroxyl groups in the fibroin protein before modification.
[25]
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.

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.

This graph shows the results of infrared absorption spectrum measurements of films formed with modified fibroin.

The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

[Modified fibroin]
The modified fibroin according to the present invention is a protein containing a domain sequence represented by formula 1: [(A) _n motif - REP] _m , or formula 2: [(A) _n motif - REP] _m - (A) _n motif. The modified fibroin may have an additional amino acid sequence (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 sequence and C-terminal sequence are not limited to these, but are typically regions that do not have repeating amino acid motifs characteristic of fibroin, and consist of about 100 amino acid residues.

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 a protein containing a domain sequence represented by formula 1: [(A) _n motif - REP] _m , or formula 2: [(A) _n motif - REP] _m - (A) _n motif.

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

Examples of insects that produce fibroin include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyratorum, Pilosa cynthia ricinii, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea. Examples include silk proteins produced by silkworms such as *Assama*, and hornet silk proteins excreted by the larvae of the hornet (*Vespa simillima xanthoptera*).

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

Examples of fibroin produced by spiders include those belonging to the genus Araneus (such as the garden orb-weaver spider, the red orb-weaver spider, the blue orb-weaver spider, and the bean orb-weaver spider), those belonging to the genus Neoscona (such as the mountain orb-weaver spider, the house orb-weaver spider, the silver orb-weaver spider, and the Satsuma orb-weaver spider), those belonging to the genus Pronus (such as the false orb-weaver spider), those belonging to the genus Cyrtarachne (such as the bird-dropping spider and the large bird-dropping spider), and those belonging to the genus Gaster (such as the spiny spider and the small spiny spider). Spiders belonging to the genus Acantha, spiders belonging to the genus Ordgarius such as the bean-tailed bell spider and the six-legged bell spider, spiders belonging to the genus Argiope such as the golden orb-weaver, the small golden orb-weaver, and the long golden orb-weaver, spiders belonging to the genus Arachnura such as the white-tailed cane spider, spiders belonging to the genus Acusila such as the bell spider, spiders belonging to the genus Cytophora such as the horn spider, and the broad-winged horn spider, spiders belonging to the genus Poltys such as the horn spider Spider silk protein produced by spiders belonging to the genus Cyclosa, such as the orb-weaver spider, the four-legged orb-weaver spider, the round orb-weaver spider, and the black orb-weaver spider, and spiders belonging to the genus Chorizopes, such as the Japanese orb-weaver spider, and spiders belonging to the genus Tetragnatha, such as the long-legged spider, the broad-legged huntsman spider, and the scaly long-legged spider, spiders belonging to the genus Leucauuge, such as the large white orb-weaver spider, the medium white orb-weaver spider, and the golden orb-weaver spider, etc. Examples include spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Nephila, spiders belonging to the genus Menosira (including golden orb-weaver spiders), spiders belonging to the genus Dyschiriognatha (including long-legged spiders), spiders belonging to the genus Latrodectus (including black widow spiders, redback spiders, gray widow spiders, and thirteen-spotted widow spiders), and spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include MaSp (MaSp1 and MaSp2), ADF (ADF3 and ADF4) and other dragline proteins, and MiSp (MiSp1 and MiSp2).

More specific examples of fibroins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession number AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), and major angute spidroin. 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila 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 *Crientata* (GenBank accession number ABR37278.1 (amino acid sequence)).

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

"Modified fibroin" may be any fibroin having the amino acid sequence specified in this invention, even if it is based on naturally derived fibroin with its amino acid sequence modified (for example, by modifying the gene sequence of cloned naturally derived fibroin), or it may be an artificially designed amino acid sequence without relying on naturally derived fibroin (for example, a nucleic acid encoding a designed amino acid sequence is chemically synthesized to obtain the desired 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.

In this specification, "domain sequence" refers to an amino acid sequence that gives rise to a crystalline region (typically corresponding to the (A) _n motif of an amino acid sequence) and an amorphous region (typically corresponding to the REP of an amino acid sequence) characteristic of fibroin, and means an amino acid sequence represented by formula 1: [(A) _n motif - REP] _m , or formula 2: [(A) _n motif - REP] _m - (A) _n motif. Here, the (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues is 80% or more of the total number of amino acid residues in the (A) _n motif. The REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer from 10 to 300. m is preferably an integer from 20 to 300, and more preferably an integer from 30 to 300. Multiple (A) _n motifs may be the same amino acid sequence or different amino acid sequences. Multiple REPs may have the same amino acid sequence or different amino acid sequences.

(A) The _n motif may have an alanine residue count of 80% or more of the total number of amino acid residues in the (A) _n motif, but it is preferable that it be 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning it is composed only of alanine residues). Of the multiple (A) _n motifs present in the domain sequence, it is preferable that at least seven are composed only of alanine residues. Being composed only of alanine residues means that the (A) _n motif has an amino acid sequence represented as (Ala) _k (Ala represents an alanine residue, and k represents an integer from 4 to 27, preferably an integer from 4 to 20, more preferably an integer from 4 to 16).

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 the number 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.

Examples of amino acid residues containing 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.

The modified fibroin according to this embodiment only needs to have a number of amino acid residues containing free hydroxyl groups that is at least 20% lower than the number of amino acid residues containing free hydroxyl groups before modification (i.e., an OH reduction rate of 20% or more). This reduces the formation of ester bonds when in contact with carboxylic acids such as formic acid. Furthermore, it reduces or makes the generation of off-odors less likely.

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 allows the effects of the present invention to be achieved even more significantly.

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% lower than 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.

In this specification, "content of amino acid residues having free hydroxyl groups" is a value calculated by x/y _× 100% when _x is the total number of amino acid residues having free hydroxyl groups and _y is the total number of amino acid residues in fibroin, in a 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.

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 even 0%. This reduces the formation of ester bonds upon contact with carboxylic acids such as formic acid. Furthermore, it reduces the likelihood of off-odors.

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 even 0%. This reduces the formation of ester bonds upon contact with carboxylic acids such as formic acid. Furthermore, it reduces the generation of off-odors.

In this specification, "serine residue, threonine residue, and tyrosine residue content" is a value calculated by (a + b + c) / y × 100% in a 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, 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 the fibroin.

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.

More specific examples of the modified fibroin according to the present invention include (i) 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 (ii) a modified fibroin containing 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.

The amino acid sequence shown in SEQ 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 SEQ ID NO: 9 (Met-PRT1083).

The amino acid sequence shown in SEQ ID NO: 2 (Met-PRT1104) is obtained by replacing 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 deleting every two (A) _n motifs from the amino acid sequence shown in SEQ ID NO: 26 (Met-PRT380) from the N-terminus to the C-terminus, and inserting one [(A) _n motif-REP] before the C-terminal sequence. 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.

The amino acid sequence shown in SEQ ID NO: 3 (Met-PRT1105) is obtained by substituting the serine residue (S) with an alanine residue (A) or a glycine residue (G) from the amino acid sequence shown in SEQ ID NO: 1 (Met-PRT918).

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

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

The amino acid sequence shown in SEQ ID NO: 6 (Met-PRT1146) is the same as the amino acid sequence shown in SEQ 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).

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

The amino acid sequence shown in SEQ 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 SEQ ID NO: 1 (Met-PRT918).

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 isoleucine residue (I) with glutamine residue (Q) from the amino acid sequence shown in SEQ ID NO: 9 (Met-PRT1083).

The amino acid sequence shown in SEQ 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 finally substituting serine residues (S) with threonine residues (T).

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.

The modified fibroin of (ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, 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) is also a protein containing a domain sequence represented by formula 1: [(A) _n motif - REP] _m , or formula 2: [(A) _n motif - REP] _m - (A) _n motif. The above sequence identity is preferably 95% or more.

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

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

As a tag sequence, for example, affinity tags that utilize specific affinity (binding affinity) with other molecules can be cited. A specific example of an affinity tag is the histidine tag (His tag). The His tag is a short peptide consisting of approximately 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 metal chelation 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).

Furthermore, 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 utilized.

Furthermore, "epitope tags" utilizing antigen-antibody reactions can also be used. By attaching 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.

Furthermore, a version in which the tag sequence can be cleaved with a specific protease can also be used. By treating the protein adsorbed via this tag sequence with a protease, the modified fibroin with the tag sequence cleaved can be recovered.

More specific examples of modified fibroin containing tag sequences include (iii) modified fibroin containing the amino acid sequence shown in SEQ ID NOs. 12, 13, 14, 15, 16, 17, 18, 19, 21, or 22, or modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NOs. 12, 13, 14, 15, 16, 17, 18, 19, 21, or 22.

The amino acid sequences indicated by 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 indicated by SEQ ID NOs: 23 or 24 (including the His tag) to the N-terminus of the amino acid sequence indicated by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 10, or 11, respectively.

The modified fibroin of (iii) may consist of the amino acid sequence shown in SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 21, or 22.

The modified fibroin of (iii) contains 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. The modified fibroin of (iii) is also a protein containing a domain sequence represented by formula 1: [(A) _n motif - REP] _m , or formula 2: [(A) _n motif - REP] _m - (A) _n motif. Preferably, the above sequence identity is 95% or more.

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

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 configured depending on the type of host.

[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 (tag sequence) shown in SEQ ID NO: 23 or SEQ ID NO: 24 is attached to either the N-terminus or C-terminus or both of these amino acid sequences.

A nucleic acid according to one embodiment hybridizes under stringent conditions with the complementary strand of the nucleic acid encoding the modified fibroin according to the present invention, and is a nucleic acid encoding a modified fibroin that includes a domain sequence represented by formula 1: [(A) _n motif - REP] _m or formula 2: [(A) _n motif - REP] _m - (A) _n motif. The modified fibroin encoded by the nucleic acid preferably has a content of amino acid residues having free hydroxyl groups of 22% or less. Furthermore, the modified fibroin encoded by the nucleic acid preferably has a content of serine residues, threonine residues, and tyrosine residues of 22% or less.

"Stringent conditions" refer to conditions under which specific hybrids are formed, but non-specific hybrids are not. "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 sequences; for example, using 5×SSC containing 0.5% SDS and hybridizing at 42°C. Medium-stringent conditions mean that hybridization occurs only when at least 90% identity exists between sequences; for example, using 5×SSC containing 0.5% SDS and hybridizing at 50°C. High-stringent conditions mean that hybridization occurs only when at least 95% identity exists between sequences; for example, using 5×SSC containing 0.5% SDS and hybridizing at 60°C.

Nucleic acids according to other embodiments have 90% or more sequence identity with the nucleic acid encoding the modified fibroin according to the present invention, and are nucleic acids encoding a modified fibroin that includes a domain sequence represented by formula 1: [(A) _n motif - REP] _m or formula 2: [(A) _n motif - REP] _m - (A) _n motif. The modified fibroin encoded by the nucleic acid preferably has an amino acid residue content of 22% or less that has a free hydroxyl group. Furthermore, the modified fibroin encoded by the nucleic acid preferably has a serine residue, threonine residue, and tyrosine residue content of 22% or less.

[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.

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.

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

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 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.

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

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), and Escherichia coli No. Examples include 49. Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W3110 (ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, etc.

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 chousinensis 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.).

Examples of microorganisms belonging to the genus Serratia include Serratia liquefacience ATCC14460, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesi, Serratia proteamaculans, Serratia odolifera, Serratia plymuthica, and Serratia rubidae.

Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis and Bacillus amyloricefaciens.

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

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

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 acetoglutamicum* ATCC13870, *Corynebacterium alkanoriticum* ATCC15806, *Corynebacterium carnae* ATCC15991, *Corynebacterium glutamicum* ATCC13020, ATCC13032, ATCC13060, *Corynebacterium lylium* ATCC15990, *Corynebacterium melasecola* ATCC17965, *Corynebacterium thermoaminogenes* AJ12340 (FERMBP-1539), and *Corynebacterium hercules* ATCC13868.

Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, Pseudomonas fluorescein, Pseudomonas brassicacearum, Pseudomonas fluva, and Pseudomonas sp. D-0110.

Any method for introducing the expression vector into the host cells can be used. 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.

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).

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)], and pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli 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 BP-6798, 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.

When using Escherichia coli as the host, suitable vectors include pUC18, pBlueScriptII, pSupex, pET22b, and pCold.

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 Utaka, 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.

The promoter itself is not limited as long as it functions in the 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. Furthermore, artificially designed and modified promoters, such as a promoter with two Ptrp promoters in series (Ptrp x 2), the tac promoter, the lacT7 promoter, and the let I promoter, can also be used.

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.

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

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 Examples include *Pichia utilis*, *Pichia pastoris*, *Pichia angusta*, *Pichia methanolica*, *Pichia polymorpha*, *Pichia stipitis*, *Yarrowia lipolytica*, and *Hansenula polymorpha*.

When using yeast as the host cell, expression vectors typically preferably include 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.

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, A.M., et al. (1986) Gene 45:299-310).

Examples of vectors used with yeast as a host include YEP13 (ATCC37115), YEP24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, and pPICZ B.

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, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP1 promoter, pGAP promoter, pGCW14 promoter, AOX1 promoter, and MOX promoter.

Any method for introducing expression vectors into yeast can be used, including methods for introducing DNA into yeast. Examples include electroporation (Methods Enzymol., 194, 182 (1990)), spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), lithium acetate method (J. Bacteriol., 153, 163 (1983)), and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

Examples of filamentous fungi include the genera Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, and Penicillium. Examples include bacteria belonging to the genera Illium, Myceliophora, Botrytis, Magnaporthe, Mucor, Metalithium, Monascus, Rhizopus, and Rhizomucosa.

Specific examples of filamentous fungi include Acremonium alabamense, Acremonium cellulolitisus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus oryzae, Aspergillus sake, Aspergillus sojae, and Aspergillus tubigensis. Aspergillus tubigensis, Aspergillus niger, Aspergillus nidulans, Aspergillus parasiticus, Aspergillus ficum, Aspergillus phoeicus, Aspergillus foetidus, Aspergillus flavus, Aspergillus fumigatus fumigatus), Aspergillus japonicus, Trichoderma viride, Trichoderma hazianum, Trichoderma reseei, Chrysosporium lucknowense, Thermoascus, Sporotrichum, Sporotrichum cerulofilm Cellulophilum, Talaromyces, Thielavia terrestris, Thielavia, Neurospora crassa, Fusarium oxysporus, Fusarium graminerum, Fusarium venenatum, Humicola insolens, Penicillium chrysogenum, Penicillium camembertii Penicillium canescens, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium purprogenum, Penicillium roqueforti, Myceliophora thermophyllum, Mucor ambigus Mucor ambiguus, Mucor circinelloides, Mucor fragilis, Mucor hiemalis, Mucor inaequisporus, Mucor oblongiellipticus, Mucor racemosus, Mucor recurvus, Mucor saturninus, Mucor subtilissumius Examples include *subtilissmus*, *Ogataea polymorpha*, *Phanerochaete chrysosporium*, *Rhizomucor miehei*, *Rhizomucor pusillus*, and *Rhizopus arrhizus*.

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.

The introduction of expression vectors into filamentous fungi can be performed 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.

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.

Examples of vectors used when insect cells are used as hosts include baculoviruses such as Autographa californica nuclear polyhedrosis virus (Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992)), which infect moths of the family Croptail.

When insect cells are used as a host, polypeptides can be expressed by methods described, for example, in Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992), Bio/Technology, 6, 47 (1988), etc. Specifically, recombinant gene transfer vectors and baculoviruses can be co-introduced into insect cells to obtain recombinant viruses (expression vectors) in the insect cell culture supernatant. Then, the recombinant viruses can be 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 Invitrogen).

Methods for co-introducing recombinant gene vectors and baculoviruses into insect cells for preparing 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)).

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 are used as selectable marker genes: niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6, 386-389, (1984)), sC (Gene, 84, 329-334, (1989)), ptrA (Biosci Biotechnol Biochem, 64, 1416-1421, (2000)), and pyrG (Biochem Biophys Res Examples include marker genes selected from the group consisting of Commun, 112, 284-289 (1983), amdS (Gene, 26, 205-221 (1983)), aureobasidine resistance gene (Mol Gen Genet, 261, 290-296 (1999)), benomyl resistance gene (Proc Natl Acad Sci USA, 83, 4869-4873 (1986)), and hygromycin resistance gene (Gene, 57, 21-26 (1987)), as well as leucine requirement complement genes. Furthermore, if the host is a nutrient requirement mutant, a wild-type gene complementing that nutrient requirement can also be used as a selected marker gene.

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.

[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 the 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.

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 methods commonly used for host culture.

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.

As a carbon source, any substance that the host can utilize is acceptable. For example, 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 can be used.

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.

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.

The culture of prokaryotes such as E. coli or eukaryotes such as yeast can be carried out 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 between 3.0 and 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.

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, and when culturing microorganisms transformed with an expression vector using a trp promoter, indole-acrylic acid may be added.

Commonly used culture media for insect cells include TNM-FH medium (Pharmaingen), Sf-900 II SFM medium (Life Technologies), ExCell 400, ExCell 405 (both from JRH Biosciences), and Grace's Insect Medium (Nature, 195, 788 (1962)).

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.

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 media containing plant hormones such as auxin and cytokinin can be used.

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.

Methods for producing modified fibroin using a host transformed with the expression vector according to the present invention include methods for producing the modified fibroin inside the host cell, methods for secreting the modified fibroin outside the host cell, and methods for 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.

For example, when 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 modified fibroin with a signal peptide attached, the modified fibroin can be actively secreted outside the host cell.

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 disrupted 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 to obtain purified samples by employing, alone or in combination, methods commonly used for the isolation and purification of proteins, such as 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, chromatographic focusing, and electrophoresis methods such as isoelectric focusing.

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

Furthermore, if the modified fibroin is expressed as an insoluble form within cells, the host cells are similarly recovered, disrupted, 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.

When 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. Specifically, the culture supernatant can be obtained by processing the culture by methods such as centrifugation, and a purified sample can be obtained from this supernatant using the same isolation and purification method as described above.

The modified fibroin according to the present invention has reduced ester bond formation upon 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 exposed to the air. Therefore, the method for producing the modified fibroin according to this embodiment does not require a step of contacting the modified fibroin with a carboxylic acid such as formic acid.

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

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.

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

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

(Dope liquid)
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.

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.

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.

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.

The content of the 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 content of the 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.

During the preparation of the dope solution according to this embodiment, the temperature may be heated to 30-90°C. The appropriate temperature for dissolution should be set as needed, depending on the solvent used, the type of modified fibroin, etc. Shaking or stirring may be used to promote dissolution.

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, or to 100 to 30,000 cP (centipoise) at 40°C. The viscosity of the spinning stock can be measured using, for example, an EMS viscometer manufactured by Kyoto Electronics Manufacturing Co., Ltd.

(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.

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

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 diameter of 0.1 to 0.6 mm as the spinneret, the extrusion speed is preferably 0.2 to 6.0 ml/hour per hole, and more preferably 1.4 to 4.0 ml/hour. The length of the coagulation solution tank should be long enough to allow efficient solvent removal, for example, 200 to 500 mm. The take-up speed 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 the evaporation of the lower alcohol, the coagulation solution may be maintained 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.

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.

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 the unstretched yarn (or pre-stretched yarn) by, for example, 1 to 10 times, with 2 to 8 times being preferred.

Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, etc. The temperature can be, for example, 140°C to 270°C, with 160°C to 230°C being preferred. In dry heat stretching, the unstretched yarn (or pre-stretched yarn) can be stretched, for example, 0.5 to 8 times, and 1 to 4 times is preferred.

Moist heat stretching and dry heat stretching may be performed individually, or in multiple stages or in combination. That is, the first stage stretching may be performed using moist heat stretching and the second stage using dry heat stretching, or the first stage stretching may be performed using moist heat stretching, the second stage using moist heat stretching, and the third stage using dry heat stretching, etc. Moist heat stretching and dry heat stretching can be combined as appropriate.

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).

Protein fibers may be chemically crosslinked between polypeptide molecules within the protein fiber after stretching. 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 in a polypeptide can be crosslinked with the carboxyl group of the glutamic acid or aspartic acid side chain via amide bonding through dehydration condensation. Crosslinking may be achieved by carrying out a dehydration condensation reaction under vacuum heating, or by using a dehydration condensation agent such as carbodiimide.

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 independently represent organic groups 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 amide bond formation ability between polypeptide molecules and readily undergo crosslinking reactions.

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 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. Crosslinking can impart even higher stress (strength) to the protein fibers.

(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.

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

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.

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

The substrate may be a resin substrate, glass substrate, 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 with a silicone compound 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 with a silicone compound immobilized on the surface of a PET film.

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.

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 with 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.

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, both longitudinally and transversely. This range facilitates a good stress-strain balance. Underwater stretching is preferably performed at a water temperature of 20 to 90°C. After stretching, the film is preferably heat-set at 50 to 200°C for 5 to 600 seconds. This heat-set provides dimensional stability at room temperature. Note that uniaxially stretched films become uniaxially oriented films, and biaxially stretched films become biaxially oriented films.

[Method for producing an artificially modified fibroin composition]
The artificially modified fibroin composition according to the present invention can be manufactured by a method that includes the step of preparing the modified fibroin according to the present invention. The method for manufacturing 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.

The modified fibroin according to the present invention has reduced ester bond formation upon 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 exposed to the air. Therefore, the method for producing the artificial modified fibroin composition according to this embodiment does not require a step of contacting the modified fibroin with a carboxylic acid such as formic acid.

[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.

Substitution, deletion, insertion, and/or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial-directed mutagenesis. Specifically, this can be done according to methods described in literature such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

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.

〔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 transplantation (e.g., artificial ligaments and aortic bands).

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. These can be manufactured in accordance with the methods described in Japanese Patent Publication No. 2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735, and the like.

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

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

(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).

(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).

(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).

(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).

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

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.

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 using restriction enzymes with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Protein production E. coli BLR(DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 2) containing ampicillin so that the OD ₆₀₀ was 0.005. The culture solution temperature was maintained at 30°C and flask culture was performed until the OD ₆₀₀ reached 5 (approximately 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter containing 500 mL of production medium (Table 3) so that the OD ₆₀₀ was 0.05. 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 also maintained at 20% of the dissolved oxygen saturation concentration.

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 bacterial cells were collected. SDS-PAGE was performed using bacterial 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 desired modified fibroin size that was dependent on IPTG addition.

(3) Protein purification Two hours after adding IPTG, the cells were collected and 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 stirred with a stirrer at 60°C for 30 minutes to dissolve. After dissolution, dialysis was performed with water using dialysis tubes (cellulose tubes 36/32 manufactured by 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).

[Production and evaluation of protein films]
(1) Production of protein film 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).

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 0.5–1.0 mm.

(2) Evaluation of protein films The degree of formate ester formation in the film samples was evaluated by measuring the infrared absorption spectra of the prepared film samples using the following measuring device.
Measurement device: Nicolet iS50 FT-IR (Manufacturer: Thermo Fisher Scientific Co., Ltd.)

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: Peak height of 1725 ^cm⁻¹ (peak based on ester C=O) P2: Peak height of 1445 ^cm⁻¹ (peak based on protein amide III)

The results are shown in Figure 1 and Table 1. As shown in Figure 1, reducing the content of amino acid residues with free hydroxyl groups (for example, an OH reduction rate of 20% or more) reduced the peak in the formic acid ester absorption region (1715-1730 ^cm⁻¹ ) (Examples 1-4). On the other hand, substituting amino acid residues without free hydroxyl groups did not result in a significant change in the peak in the formic acid ester absorption region (1715-1730 ^cm⁻¹ ) (Comparative Example 1).

Claims (20)

Formula 1: [(A) _n motif - REP] _m , or Formula 2: [(A) _n motif - REP] _m - (A) _n motif, comprising a domain sequence
Modified fibroin having a free hydroxyl group amino acid residue content of 10% or less.
[In formulas 1 and 2, (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues in (A) _n motif is 80% or more of the total number of amino acid residues, and 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 (A) _n 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.] A nucleic acid encoding the modified fibroin described in claim 1. 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. The expression vector according to claim 3 , which is a plasmid vector or a viral vector. A host (excluding humans) transformed with the expression vector described in claim 3 or 4 . The host according to claim 5 , which is a prokaryote. 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 . The host according to claim 7 , which is a eukaryote. The host according to claim 8 , wherein the eukaryote is a yeast, a filamentous fungus, or an insect cell. A synthetic modified fibroin composition comprising the modified fibroin described in claim 1. The artificially modified fibroin composition according to claim 10 , which is a protein powder. The artificially modified fibroin composition according to claim 10 , which is a dope liquid. The artificially modified fibroin composition according to claim 10 , which is a fiber. The artificially modified fibroin composition according to claim 10 , which is a film. A method for producing modified fibroin,
The process includes expressing a modified fibroin in a host (excluding humans) transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence.
A method for producing the modified fibroin, wherein the modified fibroin is the modified fibroin described in claim 1. 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. The manufacturing method according to claim 15 or 16 , further comprising the step of contacting the modified fibroin with a carboxylic acid. The manufacturing method according to claim 16 , further comprising the step of preparing a modified fibroin solution containing the modified fibroin and carboxylic acid. 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, contains a domain sequence represented by formula 1: [(A) _n motif - REP] _m or formula 2: [(A) _n motif - REP] _m - (A) _n motif, and the content of amino acid residues having a free hydroxyl group is 10% or less.
[In formulas 1 and 2, (A) _n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues in (A) _n motif is 80% or more of the total number of amino acid residues, and 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 (A) _n 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.] comprising the modified fibroin described in claim 1,
Products selected from the group consisting of fibers, threads, films, foams, granules, nanofibrils, gels, and resins.