Method for producing a protein molded body, method for producing a protein solution, and method for producing a protein

Abstract

The present invention relates to a method for producing a protein shaped body, which includes the steps of dissolving a protein in a solvent containing formic acid at a temperature of 40°C or higher and lower than 80°C to obtain a protein solution, and molding the protein shaped body using the protein solution.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a protein-formed article, a method for producing a protein solution, and a method for producing protein. [Background technology]

[0002] Conventionally, molded articles using protein materials as polymer materials have been known, such as fibers, films, and porous materials (for example, Patent Documents 1-3). In the case of such protein molded articles, for example, if they are fibers, there may be a need for fibers with superior physical properties such as strength, depending on the intended use. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Patent No. 5540166 [Patent Document 2] Patent No. 5678283 [Patent Document 3] Patent No. 5796147 [Overview of the project] [Problems that the invention aims to solve]

[0004] The object of the present invention is to provide a method for producing protein-molded articles that can be easily manufactured, thereby improving the physical properties of the protein-molded articles. [Means for solving the problem]

[0005] This invention relates, for example, to the following inventions. [1] A method for producing a protein molded article, comprising the steps of: dissolving a protein in a solvent containing formic acid at a temperature of 40°C or higher and less than 80°C to obtain a protein solution; and forming a protein molded article using the protein solution. [2] The method for producing a protein molded article according to [1], wherein the protein molded article is a protein fiber. [3] A method for producing a protein molded article according to [1] or [2], wherein the protein is a structural protein. [4] A method for producing a protein-formed article according to [3], wherein the structural protein is spider silk fibroin. [5] A method for producing a protein solution, comprising the step of dissolving a protein in a solvent containing formic acid at a temperature of 40°C or higher and less than 80°C to obtain a protein solution. [6] The method for producing the protein described in [5], wherein the protein is a structural protein. [7] The method for producing the structural protein described in [6], wherein the structural protein is spider silk fibroin. [8] A method for producing a protein, comprising the steps of: dissolving the target protein and impurities in a solvent containing formic acid at a temperature of 40°C or higher and less than 80°C to obtain a protein solution containing the target protein; and treating the protein solution with a poor solvent for the target protein to aggregate the target protein and obtain the target protein as aggregates. [Effects of the Invention]

[0006] According to the present invention, it is possible to provide a method for producing protein-molded articles with improved physical properties in a simple manner.

[0007] According to the manufacturing method of the present invention, protein fibers with particularly improved strength and elongation among physical properties, protein films that are thinner while maintaining strength, and protein porous materials with low apparent density can be produced more easily. [Brief explanation of the drawing]

[0008] [Figure 1] This is a schematic diagram showing an example of a fibroin domain sequence. [Figure 2] This is a schematic diagram showing an example of a fibroin domain sequence. [Figure 3] This is a schematic diagram showing an example of a fibroin domain sequence. [Figure 4] This graph shows the relationship between the heating temperature and viscosity of the prepared dope solution. [Figure 5]This graph shows the results of evaluating the physical properties of the manufactured protein fibers. [Figure 6] This graph shows the results of evaluating the physical properties of the manufactured protein fibers. [Figure 7] This graph shows the GPC measurement results of the manufactured protein fibers. [Figure 8] This is a photograph showing a protein solution prepared using wet bacterial cells containing spider silk fibroin PRT799. [Figure 9] This figure shows the results of SDS-PAGE of proteins purified from wet bacterial cells containing spider silk fibroin PRT799. [Figure 10] This is a photograph showing a protein solution prepared using dried bacterial cells containing spider silk fibroin PRT799. [Figure 11] This figure shows the results of SDS-PAGE of proteins purified from dried bacterial cells containing spider silk fibroin PRT799. [Figure 12] This figure shows the results of SDS-PAGE of proteins purified from dried bacterial cells containing spider silk fibroin PRT799. [Figure 13] This is a photograph showing a protein solution prepared using dried bacterial cells containing spider silk fibroin PRT918. [Figure 14] This figure shows the results of SDS-PAGE of proteins purified from dried bacterial cells containing spider silk fibroin PRT918. [Modes for carrying out the invention]

[0009] Embodiments of the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[0010] [Method for manufacturing protein-molded bodies] The method for producing a protein molded article according to this embodiment includes the steps of: dissolving a protein in a solvent containing formic acid at a temperature of 40°C or higher and less than 80°C to obtain a protein solution (dissolution step); and molding a protein molded article using the protein solution (molding step).

[0011] The protein molded article manufacturing method of this embodiment allows for the simple production of protein molded articles with improved physical properties. The protein solution obtained in the dissolution step has suppressed gelation and is suitable as a doping solution when molding protein fibers.

[0012] The reason why a protein molded article with improved physical properties can be obtained by the manufacturing method of this embodiment is not clear, but the inventors speculate as follows: First, by dissolving the protein in a solvent containing formic acid at a temperature of 40°C to less than 80°C, a portion of the protein decomposes and the amount of low molecular weight increases. Contrary to expectations, these low molecular weights contribute to strength and other properties, resulting in a protein molded article with improved physical properties.

[0013] (protein) The type of protein is not particularly limited; for example, it may be a structural protein. A structural protein refers to a protein that forms a biological structure or a protein derived from such a structure. That is, a structural protein may be a naturally occurring structural protein, or it may be a modified protein obtained by modifying a part of the amino acid sequence (for example, 10% or less of the amino acid sequence) based on the amino acid sequence of a naturally occurring structural protein.

[0014] Examples of structural proteins include fibroin, collagen, lecithin, elastin, and keratin, as well as proteins derived therefrom. The fibroin may be one or more selected from the group consisting of, for example, silk fibroin, spider silk fibroin, and hornet silk fibroin. The structural protein may be silk fibroin, spider silk fibroin, or a combination thereof.

[0015] The fibroin according to this embodiment includes a naturally-derived fibroin and a modified fibroin. As used herein, "naturally-derived fibroin" means a fibroin having the same amino acid sequence as a naturally-derived fibroin, and "modified fibroin" means a fibroin having an amino acid sequence different from that of a naturally-derived fibroin.

[0016] The fibroin according to this embodiment is preferably spider silk fibroin. Spider silk fibroin includes natural spider silk fibroin and modified fibroin derived from natural spider silk fibroin. Examples of natural spider silk fibroin include spider silk proteins produced by spiders.

[0017] The fibroin according to this embodiment may be, for example, 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 fibroin according to this embodiment may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically regions that do not have repeats of amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

[0018] As used herein, "domain sequence" means an amino acid sequence that gives rise to a crystal region specific to fibroin (typically corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically corresponding to the REP of the amino acid sequence), and is represented by Formula 1: [(A) n motif-REP] m , or Formula 2: [(A) n motif-REP] m -(A) n motif. Here, (A) nThe motif shows an amino acid sequence mainly consisting of alanine residues, with the number of amino acid residues ranging from 2 to 27. (A) n The number of amino acid residues in the motif may be an integer between 2 and 20, 4 and 27, 4 and 20, 8 and 20, 10 and 20, 4 and 16, 8 and 16, or 10 and 16. Also, (A) n The ratio of alanine residues to the total number of amino acid residues in a motif should be 40% or more, but may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning composed solely of alanine residues). Multiple alanine residues are present in the domain sequence (A). n The motif may consist of at least seven alanine residues. REP indicates an amino acid sequence consisting of 2 to 200 amino acid residues. REP may also be an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer between 2 and 300, or between 10 and 300. Multiple motifs 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.

[0019] Examples of naturally derived fibroin include, for example, formula 1:[(A) n Motif-REP] m , or formula 2:[(A) n Motif-REP] m -(A) n Proteins containing domain sequences represented by motifs can be cited. Specific examples of naturally occurring fibroin include, for instance, fibroin produced by insects or spiders.

[0020] Examples of fibroins produced by insects include the silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, as well as the hornet silk protein excreted by the larvae of the hornet (Vespa simillima xanthoptera).

[0021] A more specific example of fibroin produced by insects is, for example, silkworm fibroin L chain (GenBank accession number M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

[0022] 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).

[0023] More specific examples of spider silk proteins produced by arachnids 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 ampullate 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)).

[0024] 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.

[0025] (Modified fibroin) Modified fibroin may be, for example, a modified fibroin whose amino acid sequence is based on the amino acid sequence of naturally occurring fibroin (for example, a modified fibroin whose amino acid sequence is altered by modifying the gene sequence of cloned naturally occurring fibroin), or it may be artificially designed and synthesized without relying on naturally occurring fibroin (for example, a modified fibroin having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid that codes for a designed amino acid sequence).

[0026] Modified fibroin can be obtained, for example, by modifying the amino acid sequence of cloned naturally occurring fibroin, such as by substituting, deleting, inserting, and / or adding one or more amino acid residues. The 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 according to methods described in literature such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0027] The modified fibroin may be, for example, a modified fibroin derived from silk protein produced by silkworms, or a modified fibroin derived from spider silk protein produced by spiders.

[0028] Specific examples of modified fibroin include modified fibroin derived from the large spindle bookmark filament protein produced in the large bottle gland of spiders (first modified fibroin), modified fibroin with reduced glycine residue content (second modified fibroin), (A) n Modified fibroin with reduced motif content (third modified fibroin), glycine residue content, and (A) n Examples include modified fibroin with reduced motif content (fourth modified fibroin), modified fibroin having a domain sequence containing a region with locally high hydrophobicity (fifth modified fibroin), and modified fibroin having a domain sequence with reduced glutamine residue content (sixth modified fibroin).

[0029] As a modified fibroin (first modified fibroin) derived from the large spindle bookmark filament protein produced in the large bottle gland of spiders, Formula 1:[(A) n Motif-REP] m Examples include proteins containing the domain sequence represented by . In formula 1, n is preferably an integer from 3 to 20, more preferably an integer from 4 to 20, even more preferably an integer from 8 to 20, even more preferably an integer from 10 to 20, even more preferably an integer from 4 to 16, particularly preferably an integer from 8 to 16, and most preferably an integer from 10 to 16. In formula 1, the number of amino acid residues constituting REP in the first modified fibroin is preferably 10 to 200 residues, more preferably 10 to 150 residues, even more preferably 20 to 100 residues, and even more preferably 20 to 75 residues. In formula 1, the first modified fibroin is represented by . n Motif-REP] mThe total number of glycine residues, serine residues, and alanine residues contained in the amino acid sequence represented by [the given formula] is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more, of the total number of amino acid residues.

[0030] The first modified fibroin is given by formula 1:[(A) n Motif-REP] m The polypeptide may contain units of an amino acid sequence represented by , and its C-terminal sequence is an amino acid sequence shown in any of SEQ ID NOs: 1 to 3, or an amino acid sequence having 90% or more homology to an amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

[0031] The amino acid sequence shown in Sequence ID No. 1 is identical to the amino acid sequence consisting of the C-terminal 50 amino acids of the amino acid sequence of ADF3 (GI: 1263287, NCBI), the amino acid sequence shown in Sequence ID No. 2 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in Sequence ID No. 1, and the amino acid sequence shown in Sequence ID No. 3 is identical to the amino acid sequence obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in Sequence ID No. 1.

[0032] A more specific example of the first modified fibroin is a modified fibroin comprising (1-i) the amino acid sequence shown in SEQ ID NO: 4, or (1-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. Preferably, the sequence identity is 95% or more.

[0033] The amino acid sequence shown in SEQ ID NO: 4 is a modified version of the ADF3 amino acid sequence in which an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, His10 tag, and HRV3C protease (Human rhinovirus 3C protease) recognition site has been added to the N-terminus. The repeat region from positions 1 to 13 has been increased by approximately double, and the translation has been mutated so that it terminates at amino acid residue 1154. The C-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4 is identical to the amino acid sequence shown in SEQ ID NO: 3.

[0034] The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO: 4.

[0035] Modified fibroin with reduced glycine residue content (second modified fibroin) has an amino acid sequence in which the glycine residue content is reduced compared to naturally derived fibroin. The second modified fibroin can be said to have an amino acid sequence equivalent to that in which at least one or more glycine residues in REP are substituted with other amino acid residues compared to naturally derived fibroin.

[0036] The second modified fibroin may have an amino acid sequence in which, compared to naturally occurring fibroin, at least one glycine residue in at least one motif sequence selected from GGX and GPGXX in REP (where G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) is replaced with another amino acid residue.

[0037] In the second modified fibroin, the proportion of motif sequences in which the glycine residue described above is substituted with another amino acid residue may be 10% or more of the total motif sequence.

[0038] The second modified fibroin is given by formula 1:[(A) n Motif-REP] mIt includes a domain sequence represented by the above domain sequence, and the (A) located furthest to the C-terminus n Let z be the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the motif to the C-terminus of the above domain sequence. From the above domain sequence, the (A) located closest to the C-terminus n The amino acid sequence may also have a z / w ratio of 30% or more, 40% or more, 50% or more, or 50.9% or more, when the total number of amino acid residues in the sequence obtained by removing the sequence from the motif to the C-terminus of the above domain sequence is denoted as w. (A) n The number of alanine residues relative to the total number of amino acid residues in the motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning composed solely of alanine residues).

[0039] The second modified fibroin is preferably one in which the content of the XGX amino acid sequence is increased by substituting one glycine residue of the GGX motif with another amino acid residue. The second modified fibroin preferably has a content of GGX amino acid sequences in the domain sequence of 30% or less, more preferably 20% or less, even more preferably 10% or less, even more preferably 6% or less, even more preferably 4% or less, and particularly preferably 2% or less. The content of GGX amino acid sequences in the domain sequence can be calculated in the same way as the method for calculating the content of XGX amino acid sequences (z / w) described below.

[0040] Let's explain the method for calculating z / w in more detail. First, equation 1: [(A) n Motif-REP] m In fibroin (modified fibroin or naturally occurring fibroin) containing a domain sequence represented by (A), the (A) located furthest C-terminal from the domain sequence nFrom the sequence excluding the sequence from the motif to the C-terminus of the domain sequence, amino acid sequences consisting of XGX are extracted from all REPs. The total number of amino acid residues that make up XGX is z. For example, if 50 amino acid sequences consisting of XGX are extracted (no duplicates), z is 50 × 3 = 150. Also, if there is an X (the central X) contained in two XGX sequences, such as in the case of an amino acid sequence consisting of XGXGX, the duplicate is deducted from the calculation (in the case of XGXGX, it is 5 amino acid residues). w is the (A) located furthest towards the C-terminus from the domain sequence. n This is the total number of amino acid residues in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. For example, in the domain sequence shown in Figure 1, w is 4+50+4+100+4+10+4+20+4+30=230 (the one located closest to the C-terminus (A)). n (The motif is excluded.) Next, by dividing z by w, we can calculate z / w(%).

[0041] In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, even more preferably 70% or more, and even more preferably 80% or more. There is no particular upper limit to z / w, but for example it may be 95% or less.

[0042] The second modified fibroin can be obtained, for example, by modifying the gene sequence of cloned naturally occurring fibroin by substituting at least a portion of the base sequence encoding a glycine residue to encode a different amino acid residue. In this case, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or the substitution may be made so that the z / w ratio is 50.9% or more. Alternatively, it can be obtained, for example, by designing an amino acid sequence that satisfies the above embodiment from the amino acid sequence of naturally occurring fibroin and chemically synthesizing the nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification equivalent to substituting a glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, further amino acid sequence modifications equivalent to substituting, deleting, inserting and / or adding one or more amino acid residues may also be performed.

[0043] As for the other amino acid residues mentioned above, there are no particular restrictions as long as they are amino acid residues other than glycine residues, but hydrophobic amino acid residues such as valine (V) residues, leucine (L) residues, isoleucine (I) residues, methionine (M) residues, proline (P) residues, phenylalanine (F) residues and tryptophan (W) residues, and hydrophilic amino acid residues such as glutamine (Q) residues, asparagine (N) residues, serine (S) residues, lysine (K) residues and glutamic acid (E) residues are preferred, valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.

[0044] A more specific example of the second modified fibroin is a modified fibroin that includes (2-i) the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0045] The modified fibroin of (2-i) is described below. The amino acid sequence shown in SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence shown in SEQ ID NO: 10, which corresponds to naturally occurring fibroin, with GQX. The amino acid sequence shown in SEQ ID NO: 7 is obtained by changing every two (A) from the amino acid sequence shown in SEQ ID NO: 6 towards the C-terminus. 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: 8 is the same as the amino acid sequence shown in SEQ ID NO: 7 (A) n Two alanine residues were inserted into the C-terminus of the motif, some glutamine (Q) residues were replaced with serine (S) residues, and some amino acids on the N-terminus were deleted so that the molecular weight would be approximately the same as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a sequence in which the region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 11 (however, a few amino acid residues on the C-terminus of that region are substituted) is repeated four times, with a His tag added to the C-terminus.

[0046] The z / w value for the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values ​​for the amino acid sequences shown in SEQ ID NO: 6, 7, 8, and 9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. Furthermore, the x / y values ​​for the serration ratios (described later) of 1:1.8 to 11.3 for the amino acid sequences shown in SEQ ID NO: 10, 6, 7, 8, and 9 are 15.0%, 15.0%, 93.4%, 92.7%, and 89.3%, respectively.

[0047] The modified fibroin of (2-i) may consist of the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0048] The modified fibroin of (2-ii) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (2-ii) also contains a amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. n Motif-REP] m The protein contains the domain sequence represented by [the specified formula]. Preferably, the sequence identity is 95% or higher.

[0049] The modified fibroin of (2-ii) preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and when z is the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in REP, and w is the total number of amino acid residues in REP in the domain sequence, z / w is preferably 50.9% or more.

[0050] The second modified fibroin 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.

[0051] 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 Sequence ID No. 12 (an amino acid sequence including the His tag sequence and a hinge sequence).

[0052] 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.

[0053] 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.

[0054] 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.

[0055] A more specific example of a second modified fibroin containing a tag sequence is a modified fibroin containing the amino acid sequence shown in (2-iii) SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.

[0056] The amino acid sequences shown in SEQ ID NOs. 16, 17, 13, 11, 14, and 15 are obtained by adding the amino acid sequence shown in SEQ ID NOs. 12 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NOs. 10, 18, 6, 7, 8, and 9, respectively.

[0057] The modified fibroin of (2-iii) may consist of the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.

[0058] The modified fibroin of (2-iv) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (2-iv) also contains a amino acid sequence of formula 1:[(A) n Motif-REP] m The protein contains the domain sequence represented by [the specified formula]. Preferably, the sequence identity is 95% or higher.

[0059] The modified fibroin of (2-iv) preferably has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and when z is the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in REP, and w is the total number of amino acid residues in REP in the domain sequence, it is preferable that z / w is 50.9% or more.

[0060] The second modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

[0061] (A) n Modified fibroin with reduced motif content (third modified fibroin) has a domain sequence that, compared to naturally occurring fibroin, (A) n It has an amino acid sequence with reduced motif content. The domain sequence of the third modified fibroin has at least one or more (A) compared to naturally occurring fibroin. n It can be said that it has an amino acid sequence that corresponds to the deletion of a motif.

[0062] The third modified fibroin is derived from naturally occurring fibroin (A) n It may also have an amino acid sequence that corresponds to the deletion of 10-40% of the motif.

[0063] The third modified fibroin has a domain sequence that, compared to naturally occurring fibroin, has at least 1 to 3 (A) from the N-terminus to the C-terminus. n One (A) for each motif n It may also have an amino acid sequence that corresponds to the deletion of a motif.

[0064] The third modified fibroin had a domain sequence that, compared to naturally occurring fibroin, had at least two consecutive sequences from the N-terminus to the C-terminus (A). n Missing motif, and one (A) n It may also have an amino acid sequence that corresponds to the deletion of a motif being repeated in this order.

[0065] The third modified fibroin has a domain sequence in which at least every two terms from the N-terminus to the C-terminus (A) n It may also have an amino acid sequence that corresponds to the deletion of a motif.

[0066] The third modified fibroin is given by formula 1:[(A) n Motif-REP] m It includes a domain sequence represented by [(A) , and from the N-terminus to the C-terminus, two adjacent [(A) n The number of amino acid residues of the REP units in the motif-REP unit is sequentially compared, and when the number of amino acid residues of the REP with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is between 1.8 and 11.3 for two adjacent units [(A) n The amino acid sequence may also have such a ratio of x / y to 20%, 30%, 40%, or 50% or more, where x is the maximum value of the sum of the amino acid residues of the motif-REP units, and y is the total number of amino acid residues in the domain sequence. (A) nThe number of alanine residues relative to the total number of amino acid residues in the motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning composed solely of alanine residues).

[0067] The method for calculating x / y will be explained in more detail with reference to Figure 1. Figure 1 shows the domain sequence obtained by removing the N-terminal and C-terminal sequences from fibroin. The domain sequence is (A) from the N-terminal side (left side). n Motif - First REP (50 amino acid residues) - (A) n Motif - Second REP (100 amino acid residues) - (A) n Motif - Third REP (10 amino acid residues) - (A) n Motif - 4th REP (20 amino acid residues) - (A) n Motif - 5th REP (30 amino acid residues) - (A) n It has an arrangement of motifs.

[0068] Two adjacent [(A) n The [(A)] motif units are selected sequentially from the N-terminus to the C-terminus, ensuring no overlap. n A `motif-REP` unit may exist. Figure 1 shows Pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP and the fourth REP), Pattern 2 (comparison of the first REP and the second REP, and comparison of the fourth REP and the fifth REP), Pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP and the fifth REP), and Pattern 4 (comparison of the first REP and the second REP). Note that other selection methods also exist.

[0069] Next, for each pattern, select two adjacent [(A) nThe number of amino acid residues in each REP within the [motif-REP] unit is compared. The comparison is performed by determining the ratio of the number of amino acid residues of the other REP to the number of amino acid residues of the REP with fewer amino acid residues, with the REP with fewer amino acid residues set to 1. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), with the first REP having fewer amino acid residues set to 1, the ratio of the number of amino acid residues of the second REP is 100 / 50 = 2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), with the fourth REP having fewer amino acid residues set to 1, the ratio of the number of amino acid residues of the fifth REP is 30 / 20 = 1.5.

[0070] In Figure 1, when the amino acid residue count of the one with fewer amino acid residues is set to 1, the ratio of the amino acid residue counts of the other is between 1.8 and 11.3 [(A) n The set of motif-REP units is shown by a solid line. Hereafter, such a ratio will be called the jagged ratio. When the one with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is less than 1.8 or greater than 11.3 [(A) n The set of Motif-REP units is shown with a dashed line.

[0071] In each pattern, the two adjacent [(A) shown by the solid line n Add up the total number of amino acid residues in the motif-REP unit (not just REP, but (A) n The number of amino acid residues in the motif is also included. Then, the sums of these sums are compared, and the sum of the pattern that maximizes this sum (the maximum sum) is defined as x. In the example shown in Figure 1, the sum of pattern 1 is the maximum.

[0072] Next, by dividing x by the total number of amino acid residues in the domain sequence, y, we can calculate x / y(%).

[0073] In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, even more preferably 65% ​​or more, even more preferably 70% or more, even more preferably 75% or more, and particularly preferably 80% or more. There is no particular upper limit to x / y, and for example, it may be 100% or less. When the serration ratio is 1:1.9 to 11.3, x / y is preferably 89.6% or more; when the serration ratio is 1:1.8 to 3.4, x / y is preferably 77.1% or more; when the serration ratio is 1:1.9 to 8.4, x / y is preferably 75.9% or more; and when the serration ratio is 1:1.9 to 4.1, x / y is preferably 64.2% or more.

[0074] A third modified fibroin is present in multiple locations within the domain sequence (A). n If the modified fibroin consists of at least seven alanine residues in the motif, then x / y is preferably 46.4% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and particularly preferably 80% or more. There is no particular upper limit to x / y, as long as it is 100% or less.

[0075] The third modified fibroin is, for example, derived from the gene sequence of cloned naturally occurring fibroin such that the x / y ratio is 64.2% or higher (A) n It can be obtained by deleting one or more sequences that encode the motif. Alternatively, for example, from the amino acid sequence of naturally derived fibroin, one or more (A) sequences can be obtained such that x / y is 64.2% or more. n It can also be obtained by designing an amino acid sequence corresponding to the deletion of a motif and chemically synthesizing the nucleic acid encoding the designed amino acid sequence. In either case, from the amino acid sequence of naturally derived fibroin (A) nIn addition to modifications equivalent to the deletion of a motif, further amino acid sequence modifications equivalent to the substitution, deletion, insertion, and / or addition of one or more amino acid residues may be performed.

[0076] A more specific example of a third modified fibroin is a modified fibroin that includes (3-i) the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0077] (3-i) The modified fibroin will be described. The amino acid sequence shown in Sequence ID No. 18 is derived from the amino acid sequence shown in Sequence ID No. 10, which corresponds to naturally occurring fibroin, with every other amino acid (A) altered from the N-terminus to the C-terminus. n The motif is deleted, and furthermore, before the C-terminal sequence, [(A) n This is a single inserted motif-REP. The amino acid sequence shown in SEQ ID NO: 7 is obtained by replacing all GGX in REP of the amino acid sequence shown in SEQ ID NO: 18 with GQX. The amino acid sequence shown in SEQ ID NO: 8 is obtained by replacing each (A) of the amino acid sequence shown in SEQ ID NO: 7 n Two alanine residues were inserted into the C-terminus of the motif, some glutamine (Q) residues were replaced with serine (S) residues, and some amino acids on the N-terminus were deleted so that the molecular weight would be approximately the same as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a sequence in which the region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 11 (however, a few amino acid residues on the C-terminus of that region are substituted) is repeated four times, with a His tag added to the C-terminus.

[0078] The x / y value for the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) with a jagged ratio of 1:1.8 to 11.3 is 15.0%. The x / y values ​​for the amino acid sequence shown in SEQ ID NO: 18 and SEQ ID NO: 7 are both 93.4%. The x / y value for the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The x / y value for the amino acid sequence shown in SEQ ID NO: 9 is 89.3%. The z / w values ​​for the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.1%, and 70.0%, respectively.

[0079] The modified fibroin of (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0080] The modified fibroin of (3-ii) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (3-ii) also contains a molecule of formula 1:[(A) n Motif-REP] m The protein contains the domain sequence represented by [the specified formula]. Preferably, the sequence identity is 95% or higher.

[0081] The modified fibroin of (3-ii) has more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and has two adjacent [(A) n The number of amino acid residues of the REPs in the motif-REP unit are sequentially compared, and when the number of amino acid residues of the REP with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 (the jagged ratio is 1:1.8 to 11.3) for two adjacent REPs [(A) n When x is the maximum value of the sum of the amino acid residues of the motif-REP units, and y is the total number of amino acid residues in the domain sequence, it is preferable that x / y is 64.2% or more.

[0082] The third modified fibroin may contain the aforementioned tag sequence at either the N-terminus or the C-terminus, or both.

[0083] A more specific example of a third modified fibroin containing a tag sequence is a modified fibroin containing (3-iii) the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.

[0084] The amino acid sequences shown in SEQ ID NOs. 16, 17, 13, 11, 14, and 15 are obtained by adding the amino acid sequence shown in SEQ ID NOs. 12 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NOs. 10, 18, 6, 7, 8, and 9, respectively.

[0085] The modified fibroin of (3-iii) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.

[0086] The modified fibroin of (3-iv) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (3-iv) also contains formula 1:[(A) n Motif-REP] m The protein contains the domain sequence represented by [the specified formula]. Preferably, the sequence identity is 95% or higher.

[0087] The modified fibroin of (3-iv) has more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and has two adjacent [(A) nWhen sequentially comparing the number of amino acid residues of the REP in the [Motif-REP] unit, when the number of amino acid residues of the REP with fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 for two adjacent [(A) n When the maximum value of the total value obtained by adding the number of amino acid residues of the [Motif-REP] unit is x and the total number of amino acid residues of the domain sequence is y, it is preferable that x / y is 64.2% or more.

[0088] The third modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

[0089] The content of glycine residues, and (A) n The modified fibroin with reduced content of [Motif] (the fourth modified fibroin) has a domain sequence that, compared with the naturally derived fibroin, (A) n In addition to the reduced content of [Motif], it has an amino acid sequence with a reduced content of glycine residues. The domain sequence of the fourth modified fibroin, compared with the naturally derived fibroin, has at least one or more (A) n In addition to the deletion of [Motif], it can be said that it has an amino acid sequence corresponding to the substitution of at least one or more glycine residues in the REP with another amino acid residue. That is, the fourth modified fibroin combines the characteristics of the modified fibroin with reduced content of glycine residues (the second modified fibroin) described above and (A) n The modified fibroin having the characteristics of the modified fibroin with reduced content of [Motif] (the third modified fibroin). Specific embodiments and the like are as described for the second modified fibroin and the third modified fibroin.

[0090] A more specific example of the fourth modified fibroin is a modified fibroin that includes (4-i) the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and (4-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The specific form of the modified fibroin containing the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described above.

[0091] A modified fibroin having a domain sequence containing a region with a locally high hydrophobicity index (the fifth modified fibroin) may have an amino acid sequence containing a region with a locally high hydrophobicity index, which corresponds to the substitution of one or more amino acid residues in the REP with amino acid residues with a high hydrophobicity index, and / or the insertion of one or more amino acid residues with a high hydrophobicity index into the REP, compared to naturally derived fibroin.

[0092] Regions with a high local hydrophobicity index are preferably composed of 2 to 4 consecutive amino acid residues.

[0093] The amino acid residues with high hydrophobicity as described above are more preferably amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), and alanine (A).

[0094] The fifth modified fibroin may, in addition to modifications equivalent to the substitution of one or more amino acid residues in the REP with amino acid residues having a high hydrophobicity index, and / or the insertion of one or more amino acid residues having a high hydrophobicity index into the REP, compared to naturally derived fibroin, also have amino acid sequence modifications equivalent to the substitution, deletion, insertion and / or addition of one or more amino acid residues compared to naturally derived fibroin.

[0095] The fifth modified fibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in the REP of a cloned naturally occurring fibroin gene sequence with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index), and / or by inserting one or more hydrophobic amino acid residues into the REP. Alternatively, it can be obtained, for example, by designing an amino acid sequence equivalent to substituting one or more hydrophilic amino acid residues in the REP of a naturally occurring fibroin gene sequence with hydrophobic amino acid residues, and / or inserting one or more hydrophobic amino acid residues into the REP, and then chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification equivalent to substituting one or more hydrophilic amino acid residues in the REP of a naturally occurring fibroin gene sequence with hydrophobic amino acid residues, and / or inserting one or more hydrophobic amino acid residues into the REP, further amino acid sequence modifications equivalent to substituting, deleting, inserting and / or adding one or more amino acid residues may be performed.

[0096] The fifth modified fibroin is given by formula 1:[(A) n Motif-REP] m The domain sequence represented by (A) is located at the C-terminal end. n In all REPs included in the sequence obtained by removing the sequence from the motif to the C-terminus of the above domain sequence, let p be the total number of amino acid residues in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or higher, and the (A) located closest to the C-terminus. n The amino acid sequence may have a p / q ratio of 6.2% or more, where q is the total number of amino acid residues in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence.

[0097] For the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. 105-132) is used. Specifically, the hydrophobicity index (hydrophathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

[0098]

Table 1

[0099] The calculation method of p / q will be explained in more detail. For the calculation, from the domain sequence represented by formula 1: [(A) n motif-REP] m the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence (hereinafter referred to as “sequence A”) is used. First, for all REPs contained in sequence A, the average value of the hydrophobicity indices of consecutive 4 amino acid residues is calculated. The average value of the hydrophobicity indices is obtained by dividing the sum of the HIs of each amino acid residue contained in the consecutive 4 amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity indices is obtained for all consecutive 4 amino acid residues (each amino acid residue is used 1 to 4 times in the calculation of the average value). Next, a region where the average value of the hydrophobicity indices of consecutive 4 amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of “consecutive 4 amino acid residues where the average value of the hydrophobicity indices is 2.6 or more”, it will be included as 1 amino acid residue in the region. And the total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

[0100] For example, if 20 locations are extracted where "the average hydrophobicity index is 2.6 or higher" (no duplicates), then the region where the average hydrophobicity index of the four consecutive amino acid residues is 2.6 or higher will contain 20 such locations, and p will be 20 × 4 = 80. Also, for example, if two sets of "four consecutive amino acid residues with an average hydrophobicity index of 2.6 or higher" overlap by only one amino acid residue, then the region where the average hydrophobicity index of the four consecutive amino acid residues is 2.6 or higher will contain 7 amino acid residues (p = 2 × 4 - 1 = 7, where "-1" is the deduction for the duplicate). For example, in the domain sequence shown in Figure 2, there are 7 unique sets of "four consecutive amino acid residues with an average hydrophobicity index of 2.6 or higher," so p will be 7 × 4 = 28. Furthermore, for example, in the case of the domain sequence shown in Figure 2, q is 4+50+4+40+4+10+4+20+4+30=170 (located at the end of the C-terminal side (A)). n (Excluding the motif). Next, we can calculate p / q(%) by dividing p by q. In the case of Figure 2, this becomes 28 / 170 = 16.47%.

[0101] In the fifth modified fibroin, the p / q ratio is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, even more preferably 20% or more, and even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

[0102] The fifth modified fibroin can be obtained, for example, by modifying the amino acid sequence of cloned naturally occurring fibroin to include a region with a locally high hydrophobicity index by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in the REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into the REP, so as to satisfy the above p / q condition. Alternatively, it can also be obtained, for example, by designing an amino acid sequence that satisfies the above p / q condition from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to modifications equivalent to substituting one or more amino acid residues in the REP with amino acid residues with a high hydrophobicity index and / or inserting one or more amino acid residues with a high hydrophobicity index into the REP compared to naturally occurring fibroin, further modifications equivalent to substituting, deleting, inserting and / or adding one or more amino acid residues may be performed.

[0103] There are no particular restrictions on the amino acid residues with a high hydrophobicity index, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), and alanine (A) are preferred, and valine (V), leucine (L), and isoleucine (I) are more preferred.

[0104] A specific example of the fifth modified fibroin is a modified fibroin that includes (5-i) the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0105] The modified fibroin (5-i) is described below. The amino acid sequence shown in SEQ ID NO: 22 is the same as that of naturally occurring fibroin (A). nThe amino acid sequence in the motif, which consists of consecutive alanine residues, has been modified by deleting five consecutive alanine residues. The amino acid sequence shown in SEQ ID NO: 19 is obtained by inserting two amino acid sequences (VLI) consisting of three amino acid residues each, at alternating REP positions, into the amino acid sequence shown in SEQ ID NO: 22, and deleting some amino acids at the C-terminus so that the molecular weight is approximately the same as that of the amino acid sequence shown in SEQ ID NO: 22. The amino acid sequence shown in SEQ ID NO: 23 is obtained by modifying the amino acid sequence shown in SEQ ID NO: 22, with each (A) n Two alanine residues are inserted into the C-terminus of the motif, some glutamine (Q) residues are replaced with serine (S) residues, and some amino acids on the C-terminus are deleted so that the molecular weight is approximately the same as that of the amino acid sequence shown in SEQ ID NO: 22. The amino acid sequence shown in SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of 3 amino acid residues at every other REP in the amino acid sequence shown in SEQ ID NO: 23. The amino acid sequence shown in SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) consisting of 3 amino acid residues at every other REP in the amino acid sequence shown in SEQ ID NO: 23.

[0106] The modified fibroin of (5-i) may consist of the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0107] The modified fibroin of (5-ii) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) also contains formula 1:[(A) n Motif-REP] m The protein contains the domain sequence represented by [the specified formula]. Preferably, the sequence identity is 95% or higher.

[0108] The modified fibroin of (5-ii) has more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and the (A) located at the C-terminus nIn all REPs included in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, let p be the total number of amino acid residues in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or higher, and the (A) located closest to the C-terminus. n When the total number of amino acid residues in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence is denoted as q, it is preferable that the p / q ratio is 6.2% or higher.

[0109] The fifth modified fibroin may contain a tag sequence at either the N-terminus or the C-terminus, or both.

[0110] A more specific example of a fifth modified fibroin containing a tag sequence is a modified fibroin containing the amino acid sequence shown in (5-iii)SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or (5-iv)an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0111] The amino acid sequences shown in SEQ ID NOs. 24, 25, and 26 are obtained by adding the amino acid sequence shown in SEQ ID NOs. 12 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, respectively.

[0112] The modified fibroin of (5-iii) may consist of the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0113] The modified fibroin of (5-iv) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. The modified fibroin of (5-iv) also contains formula 1:[(A) n Motif-REP] m The protein contains the domain sequence represented by [the specified formula]. Preferably, the sequence identity is 95% or higher.

[0114] The modified fibroin of (5-iv) has more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, and the most C-terminal (A) n In all REPs included in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, let p be the total number of amino acid residues in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or higher, and the (A) located closest to the C-terminus. n When the total number of amino acid residues in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence is denoted as q, it is preferable that the p / q ratio is 6.2% or higher.

[0115] The fifth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

[0116] Modified fibroin (sixth modified fibroin) having a domain sequence with reduced glutamine residue content has an amino acid sequence with reduced glutamine residue content compared to naturally derived fibroin.

[0117] The sixth modified fibroin preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

[0118] When the sixth modified fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. There is no particular upper limit to the GPGXX motif content; it may be 50% or less, or 30% or less.

[0119] In this specification, the "GPGXX motif content" is a value calculated by the following method. 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 (A) located furthest towards the C-terminus n For all REPs in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, let s be the number obtained by multiplying the total number of GPGXX motifs in that region by 3 (i.e., the total number of G and P in the GPGXX motifs), and the one located closest to the C-terminus (A) n Remove the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, and further (A) n When the total number of amino acid residues in all REPs excluding the motif is denoted as t, the GPGXX motif content is calculated as s / t.

[0120] In calculating the GPGXX motif content, "the one located closest to the C-terminus (A)" n The sequence being targeted is "the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence," which is "the sequence located closest to the C-terminus (A)." n The sequence from the motif to the C-terminus of the domain sequence (the sequence corresponding to REP) may contain sequences that have low correlation with sequences characteristic of fibroin. When m is small (i.e., when the domain sequence is short), this can affect the calculation of the GPGXX motif content, so this is done to eliminate that influence. Note that if the "GPGXX motif" is located at the C-terminus of REP, it will be treated as a "GPGXX motif" even if "XX" is, for example, "AA".

[0121] Figure 3 is a schematic diagram showing the domain sequence of fibroin. The method for calculating the GPGXX motif content will be explained in detail with reference to Figure 3. First, the domain sequence of fibroin shown in Figure 3 ("[(A) n Motif-REP] m -(A) n It is a "motif" type.) In this case, all REPs are "located closest to the C-terminus (A) nSince it is included in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence (the sequence shown as "Region A" in Figure 3), the number of GPGXX motifs to calculate s is 7, and s is 7 × 3 = 21. Similarly, all REPs are "located closest to the C-terminus (A)" n Because it is included in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence (the sequence shown as "Region A" in Figure 3), further from that sequence (A) n The total number of amino acid residues t in all REPs excluding the motif is 50 + 40 + 10 + 20 + 30 = 150. Next, by dividing s by t, we can calculate s / t (%), which in the case of fibroin in Figure 3 is 21 / 150 = 14.0%.

[0122] The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%.

[0123] In this specification, "glutamine residue content" is a value calculated by the following method. 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 (A) located furthest towards the C-terminus n In all REPs contained in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence (corresponding to "Region A" in Figure 3), let u be the total number of glutamine residues contained in that region, and the one located furthest towards the C-terminus (A) n Remove the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, and further (A) n When the total number of amino acid residues in all REPs excluding the motif is denoted as t, the glutamine residue content is calculated as u / t. In calculating the glutamine residue content, the (A) located closest to the C-terminus nThe reason for targeting "the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as the reason mentioned above.

[0124] The sixth modified fibroin may have an amino acid sequence in which its domain sequence corresponds to the deletion of one or more glutamine residues in REP, or their substitution with other amino acid residues, compared to naturally occurring fibroin.

[0125] The "other amino acid residues" can be any amino acid residue other than glutamine residues, but it is preferable that they be amino acid residues with a higher hydrophobicity index than glutamine residues. The hydrophobicity indexes of amino acid residues are shown in Table 1.

[0126] As shown in Table 1, amino acid residues with a higher hydrophobicity index than glutamine residues include amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), and alanine (A) are more preferable, and amino acid residues selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) are even more preferable.

[0127] The sixth modified fibroin preferably has a hydrophobicity of -0.8 or higher, more preferably -0.7 or higher, even more preferably 0 or higher, even more preferably 0.3 or higher, and particularly preferably 0.4 or higher. There is no particular upper limit to the hydrophobicity of REP; it may be 1.0 or lower, or 0.7 or lower.

[0128] In this specification, the "hydrophobicity of REP" is a value calculated by the following method. 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 (A) located furthest towards the C-terminus n For all REPs in the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence (corresponding to "Region A" in Figure 3), let v be the sum of the hydrophobicity indices of each amino acid residue in that region, and the one located furthest towards the C-terminus (A) n Remove the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, and further (A) n When the total number of amino acid residues in all REPs excluding the motif is denoted as t, the hydrophobicity of the REP is calculated as v / t. In calculating the hydrophobicity of the REP, the (A) located closest to the C-terminus... n The reason for targeting "the sequence obtained by removing the sequence from the motif to the C-terminus of the domain sequence from the domain sequence" is the same as the reason mentioned above.

[0129] The sixth modified fibroin may have a domain sequence that, compared to naturally occurring fibroin, is modified in a manner equivalent to the deletion of one or more glutamine residues in REP and / or the substitution of one or more glutamine residues in REP with other amino acid residues, as well as further amino acid sequence modifications equivalent to the substitution, deletion, insertion and / or addition of one or more amino acid residues.

[0130] The sixth modified fibroin can be obtained, for example, by deleting one or more glutamine residues in REP from the gene sequence of cloned naturally occurring fibroin, and / or by substituting one or more glutamine residues in REP with other amino acid residues. Alternatively, it can be obtained, for example, by designing an amino acid sequence equivalent to deleting one or more glutamine residues in REP and / or substituting one or more glutamine residues in REP with other amino acid residues from the amino acid sequence of naturally occurring fibroin, and then chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0131] More specific examples of the sixth modified fibroin include (6-i) a modified fibroin containing the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, or (6-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: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0132] (6-i) The modified fibroin is described below.

[0133] The amino acid sequence shown in Sequence ID No. 7 (Met-PRT410) is based on the nucleotide and amino acid sequences of the naturally occurring fibroin Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), (A) n The amino acid sequences in the motif have been modified to improve productivity, such as increasing the number of consecutive alanine residues to five. On the other hand, since Met-PRT410 does not have any modifications to the glutamine residue (Q), the glutamine residue content is about the same as that of naturally derived fibroin.

[0134] The amino acid sequence shown in Sequence ID No. 27 (M_PRT888) is obtained by replacing all QQs in Met-PRT410 (Sequence ID No. 7) with VLs.

[0135] The amino acid sequence shown in Sequence ID No. 28 (M_PRT965) is obtained by replacing all QQs in Met-PRT410 (Sequence ID No. 7) with TS, and replacing the remaining Qs with A.

[0136] The amino acid sequence shown in Sequence ID No. 29 (M_PRT889) is obtained by replacing all QQs in Met-PRT410 (Sequence ID No. 7) with VLs, and replacing the remaining Qs with Is.

[0137] The amino acid sequence shown in Sequence ID No. 30 (M_PRT916) is obtained by replacing all QQs in Met-PRT410 (Sequence ID No. 7) with VIs, and replacing the remaining Qs with Ls.

[0138] The amino acid sequence shown in Sequence ID No. 31 (M_PRT918) is obtained by replacing all QQs in Met-PRT410 (Sequence ID No. 7) with VF, and replacing the remaining Qs with I.

[0139] The amino acid sequence shown in Sequence ID No. 34 (M_PRT525) is obtained by inserting two alanine residues into the region (A5) where alanine residues are consecutive, deleting two C-terminal domain sequences to make the molecular weight approximately the same as that of Met-PRT410, and substituting 13 glutamine residues (Q) with serine residues (S) or proline residues (P).

[0140] The amino acid sequence shown in Sequence ID No. 32 (M_PRT699) is obtained by replacing all QQs in M_PRT525 (Sequence ID No. 34) with VLs.

[0141] The amino acid sequence shown in Sequence ID No. 33 (M_PRT698) is obtained by replacing all QQs in M_PRT525 (Sequence ID No. 34) with VLs, and replacing the remaining Qs with Is.

[0142] The amino acid sequences shown in SEQ ID NOs. 27, 28, 29, 30, 31, 32, and 33 all have a glutamine residue content of 9% or less (Table 2).

[0143] [Table 2]

[0144] The modified fibroin of (6-i) may consist of the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0145] The modified fibroin of (6-ii) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33. The modified fibroin of (6-ii) also contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33. 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.

[0146] The modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Furthermore, the modified fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.

[0147] The sixth modified fibroin 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.

[0148] More specific examples of a sixth modified fibroin containing a tag sequence include (6-iii) a modified fibroin containing the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, or (6-iv) a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

[0149] The amino acid sequences shown in SEQ ID NOs. 35, 36, 37, 38, 39, 40, and 41 are obtained by adding the amino acid sequence shown in SEQ ID NOs. 12 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NOs. 27, 28, 29, 30, 31, 32, and 33, respectively. Because only the tag sequence is added to the N-terminus, there is no change in the glutamine residue content, and the amino acid sequences shown in SEQ ID NOs. 35, 36, 37, 38, 39, 40, and 41 all have a glutamine residue content of 9% or less (Table 3).

[0150] [Table 3]

[0151] The modified fibroin of (6-iii) may consist of the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

[0152] The modified fibroin of (6-iv) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41. The modified fibroin of (6-iv) also contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41. 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.

[0153] The modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. Furthermore, the modified fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.

[0154] The sixth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

[0155] The modified fibroin according to this embodiment may be a modified fibroin that combines at least two of the features of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin.

[0156] As a collagen-derived protein, for example, Equation 3:[REP2] pExamples of proteins containing the domain sequence represented by (wherein formula 3, p is an integer from 5 to 300; REP2 represents an amino acid sequence composed of Gly-XY, where X and Y represent any amino acid residues other than Gly; multiple REP2 sequences may be identical or different) include proteins containing the amino acid sequence shown in sequence number 42. The amino acid sequence shown in sequence number 42 is obtained from the NCBI database as a partial sequence of human collagen type 4 (NCBI GenBank accession number: CAA56335.1, GI: 3702452), with the amino acid sequence shown in sequence number 12 (tag sequence and hinge sequence) added to the N-terminus of the amino acid sequence from residue 301 to residue 540, which corresponds to the repeat portion and motif.

[0157] For example, as a protein derived from resilin, see formula 4:[REP3] q Examples include proteins containing the domain sequence represented by (wherein formula 4, q is an integer from 4 to 300. REP3 represents an amino acid sequence consisting of Ser-JJ-Tyr-Gly-U-Pro. J represents an arbitrary amino acid residue, preferably selected from the group consisting of Asp, Ser, and Thr. U represents an arbitrary amino acid residue, preferably selected from the group consisting of Pro, Ala, Thr, and Ser. Multiple REP4s may have the same amino acid sequence or different amino acid sequences). Specifically, examples include proteins containing the amino acid sequence shown in Sequence ID No. 43. The amino acid sequence shown in Sequence ID No. 43 is obtained by substituting Thr at residue 87 with Ser and Asn at residue 95 with Asp, and then adding the amino acid sequence shown in Sequence ID No. 12 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequence from residue 19 to residue 321 of the amino acid sequence of recilin (NCBI GenBank accession number NP 611157, Gl:24654243).

[0158] Examples of elastin-derived proteins include those with amino acid sequences such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, proteins containing the amino acid sequence shown in SEQ ID NO: 44 can be cited. The amino acid sequence shown in SEQ ID NO: 44 is obtained by adding the amino acid sequence shown in SEQ ID NO: 12 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of NCBI GenBank accession number AAC98395.

[0159] Examples of keratin-derived proteins include type I keratin from Capra hircus. Specifically, proteins containing the amino acid sequence shown in SEQ ID NO: 45 (the amino acid sequence with accession number ACY30466 in NCBI's GenBank) can be cited.

[0160] The structural proteins described above and the modified structural proteins derived from said structural proteins can be used individually or in combination of two or more.

[0161] (Method of producing protein) The protein can be produced, for example, by expressing the nucleic acid in a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence.

[0162] There are no particular restrictions on the method for producing nucleic acids that encode proteins. For example, nucleic acids can be produced by using genes that encode proteins such as natural fibroin, amplifying and cloning them using polymerase chain reaction (PCR), and modifying them as needed using genetic engineering techniques, or by chemical synthesis. There are also no particular restrictions on the chemical synthesis method of nucleic acids. For example, genes can be chemically synthesized by linking oligonucleotides, automatically synthesized using an AKTA oligopilot plus 10 / 100 (GE Healthcare Japan Corporation), based on amino acid sequence information of proteins obtained from the NCBI web database, etc., using PCR. In this case, to facilitate the purification and / or confirmation of the protein, nucleic acids encoding proteins consisting of amino acid sequences in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N-terminus of the above amino acid sequence may be synthesized.

[0163] Regulatory sequences are sequences that control protein expression 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. As a promoter, an inducible promoter that functions in host cells and can induce protein expression may be used. An inducible promoter is a promoter that can control transcription in the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

[0164] The type of expression vector can be appropriately selected depending on the host type, including plasmid vectors, viral vectors, cosmid vectors, fosmid vectors, and artificial chromosome vectors. Preferably, the expression vector should be one that can autoclav in host cells or be incorporated into host chromosomes, and contains a promoter at a position where protein-coding nucleic acids can be transcribed.

[0165] Any of the following can be suitably used as a host: prokaryotes, as well as eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells.

[0166] Preferred hosts for prokaryotes include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus sachilus. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphyllum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

[0167] When using prokaryotes as hosts, examples of vectors for introducing protein-coding nucleic acids include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (Japanese Patent Publication No. 2002-238569).

[0168] Examples of eukaryotic hosts include yeasts and filamentous fungi (molds, etc.). Examples of yeasts include those belonging to the genera Saccharomyces, Pichia, and Schizosaccharomyces. Examples of filamentous fungi include those belonging to the genera Aspergillus, Penicillium, and Trichoderma.

[0169] When using eukaryotes as hosts, examples of vectors for introducing protein-coding nucleic acids include YEp13 (ATCC37115) and YEp24 (ATCC37051). Any method for introducing the expression vector into the host cells can be used, as long as it is a method for introducing DNA into the host cells. Examples include the calcium ion method [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, spheroplast method, protoplast method, lithium acetate method, and competent method.

[0170] Nucleic acid expression by a host transformed with an expression vector can be performed not only by direct expression, but also by secretion, fusion protein expression, etc., in accordance with the methods described in Molecular Cloning, 2nd Edition.

[0171] The protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, allowing the protein to be produced and accumulated in the culture medium, and then harvesting it from the culture medium. The method for culturing the host in a culture medium can be carried out according to the methods commonly used for culturing hosts.

[0172] When the host 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, 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.

[0173] As a carbon source, any material that the transformed microorganisms 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 a nitrogen source, for example, ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, and other nitrogen-containing compounds can be used, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysates, soybean meal and soybean meal hydrolysates, various fermentation cells, and their digests. As inorganic salts, for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

[0174] 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.

[0175] Furthermore, during cultivation, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed. 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.

[0176] The isolation and purification of the expressed protein can be carried out using commonly used methods. For example, if the protein is expressed in a lysed state within cells, after the culture is complete, the host cells are recovered by centrifugation, suspended 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, 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.

[0177] Furthermore, if the protein is expressed in the form of an insoluble form within the cell, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble protein as a precipitate fraction. The recovered insoluble protein can be solubilized with a protein denaturant. After this procedure, a purified protein sample can be obtained by the same isolation and purification method as described above. If the protein is secreted extracellularly, the protein 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.

[0178] The following describes in detail each step of the method for producing the protein molded article according to this embodiment.

[0179] [Dissolution process] The dissolution step is a process of dissolving the protein in a solvent containing formic acid at a temperature of 40°C to less than 80°C to obtain a protein solution.

[0180] In the dissolution step, the protein to be dissolved (hereinafter also referred to as "target protein") may be a purified protein or a protein from a host cell expressing the protein (recombinant protein). The purified protein may be a protein purified from a host cell expressing the protein. When dissolving a protein from a host cell as the target protein, the host cell is brought into contact with a solvent containing formic acid to dissolve the protein in the formic acid-containing solvent. The host cell may be any cell that expresses the target protein, for example, an intact cell or a cell that has undergone a disruption treatment or other process. It may also be a cell that has already undergone a simple purification process.

[0181] The method for purifying proteins from host cells expressing the protein is not particularly limited, but for example, the method described in Japanese Patent Publication No. 6077570 and Japanese Patent Publication No. 6077569 can be used.

[0182] The solvent containing formic acid may consist solely of formic acid, or it may be a mixed solvent containing formic acid and other solvents. Commercially available formic acid can be used. An example of commercially available formic acid is (manufactured by Wako Pure Chemical Industries, Ltd.). The other solvent may be water.

[0183] In a solvent containing formic acid, the concentration of formic acid may be 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 95% by mass or more, based on the total mass of the solvent. In a solvent containing formic acid, the concentration of formic acid may be 99% by mass or less, 95% by mass or less, 90% by mass or more, based on the total mass of the solvent, 80% by mass or less, 70% by mass or less, or 50% by mass or less.

[0184] The temperature in the dissolution process (heating temperature) is 40°C or higher and less than 80°C, and may be 40°C or higher and 75°C or lower, 50°C or higher and 75°C or lower, or 60°C or higher and 75°C or lower. The heating temperature may be less than 80°C, 75°C or lower, 70°C or lower, 60°C or lower, 50°C or lower, or 40°C or lower, and may be 40°C or higher, 50°C or higher, 60°C or higher, or 65°C or higher. When the heating temperature in the dissolution process is high (40°C or higher), proteins and any impurities that may be contained in the proteins can be further decomposed, thus improving the physical properties of the protein-formed product.

[0185] In the dissolution step, the protein is dissolved in a solvent containing formic acid while maintaining the heating temperature as described above. The holding time at the heating temperature is not particularly limited, but may be 10 minutes or more. Considering industrial production, 10 to 120 minutes is preferred, 10 to 60 minutes is more preferred, and 10 to 30 minutes is even more preferred. The holding time at the heating temperature may be set appropriately under conditions in which the protein is sufficiently dissolved and the dissolution of impurities (substances other than the target protein) is minimal.

[0186] The amount of formic acid-containing solvent added to dissolve the protein is not particularly limited, as long as it is sufficient to dissolve the protein.

[0187] When dissolving purified protein, the amount of solvent containing formic acid added may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times the volume of the solvent containing formic acid to the weight (g) of the protein (dried powder containing protein) (volume (mL) / weight (g)).

[0188] When dissolving proteins in host cells expressing a protein, the amount of formic acid-containing solvent added may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times the weight (g) of the host cell.

[0189] The solvent containing formic acid may also contain inorganic salts. Adding inorganic salts to a solvent containing formic acid can increase the solubility of proteins.

[0190] Inorganic salts that can be added to solvents containing formic acid include alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, thiocyanates, and perchlorates.

[0191] Examples of alkali metal halides include potassium bromide, sodium bromide, lithium bromide, potassium chloride, sodium chloride, lithium chloride, sodium fluoride, potassium fluoride, cesium fluoride, potassium iodide, sodium iodide, and lithium iodide.

[0192] Examples of alkaline earth metal halides include calcium chloride, magnesium chloride, magnesium bromide, calcium bromide, magnesium iodide, and calcium iodide.

[0193] Examples of alkaline earth metal nitrates include calcium nitrate, magnesium nitrate, strontium nitrate, and barium nitrate.

[0194] Examples of thiocyanates include sodium thiocyanate, ammonium thiocyanate, and guanidinium thiocyanate.

[0195] Examples of perchlorates include ammonium perchlorate, potassium perchlorate, calcium perchlorate, silver perchlorate, sodium perchlorate, and magnesium perchlorate.

[0196] These inorganic salts may be used individually or in combination of two or more types.

[0197] Suitable inorganic salts include alkali metal halides and alkaline earth metal halides. Specific examples of suitable inorganic salts include lithium chloride and calcium chloride.

[0198] The amount (content) of the inorganic salt added may be 0.5% by mass or more and 10% by mass or 0.5% by mass or more and 5% by mass, relative to the total mass of the solvent containing formic acid.

[0199] The protein solution may be modified to remove insoluble matter as needed. In other words, the method for producing a protein molded body according to this embodiment may include a step to remove insoluble matter from the protein solution after the dissolution step, if necessary. Common methods for removing insoluble matter from a protein solution include centrifugation, filtration using drum filters, press filters, etc. When using filtration, insoluble matter can be removed more efficiently from the protein solution by using filtration aids such as Celite or diatomaceous earth and precoating agents in combination.

[0200] A protein solution contains protein and a solvent (solvent) containing formic acid that dissolves it. The protein solution may contain impurities that were present with the protein during the dissolution process. The protein solution may also be a molding solution for protein-based products.

[0201] The protein content in the protein solution may be 5% by mass or more and 35% by mass or 5% by mass or more and 50% by mass or less, based on the total volume of the protein solution.

[0202] As an embodiment of the present invention, a method for producing a protein solution is provided, which includes the above-described dissolution step. That is, the method for producing a protein solution of this embodiment includes the step of dissolving a protein in a solvent containing formic acid at a temperature of 40°C or higher and less than 80°C to obtain a protein solution.

[0203] [Molding process] The molding process involves forming a protein-molded body using a protein solution. The shape of the protein-molded body is not particularly limited, but examples include fibers, films, porous materials, and the like.

[0204] It is preferable to adjust the protein concentration and viscosity of the protein solution depending on the protein molded product to be formed.

[0205] There are no particular limitations on the method for adjusting the protein concentration in a protein solution, but examples include increasing the protein concentration by volatilizing the solvent containing formic acid through distillation, using a solution with a high protein concentration in the dissolution process, or reducing the amount of solvent containing formic acid added relative to the amount of protein.

[0206] The viscosity suitable for spinning is generally 10 to 50,000 cP (centipoise), and the viscosity can be measured using, for example, a product called "EMS Viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd. If the viscosity of the protein solution is not within the range of 10 to 10,000 cP (centipoise), the viscosity of the protein solution may be adjusted to a viscosity suitable for spinning. The methods described above can be used to adjust the viscosity. The solvent containing formic acid may also contain the suitable inorganic salts exemplified above.

[0207] If the protein molded product to be formed is a protein fiber, the protein content (concentration) in the protein solution may be adjusted to a concentration and viscosity suitable for spinning, if necessary. The method for adjusting the protein concentration and viscosity is not particularly limited. Examples of spinning methods include wet spinning. When a protein solution adjusted to a concentration and viscosity suitable for spinning is added to a coagulation solution as a doping solution, the protein coagulates. In this case, by adding the protein solution to the coagulation solution as a thread-like liquid, the protein coagulates into a thread-like form, and a thread (undrawn yarn) can be formed. The formation of an undrawn yarn can be carried out, for example, in accordance with the method described in Japanese Patent Publication No. 5584932.

[0208] Wet spinning - stretching (a) Wet spinning The coagulation solution can be any solution from which solvent removal is possible. Preferably, the coagulation solution uses a lower alcohol with 1 to 5 carbon atoms, such as methanol, ethanol, or 2-propanol, or acetone. The coagulation solution may also contain water. From the viewpoint of spinning stability, the temperature of the coagulation solution is preferably 5 to 30°C.

[0209] The method of supplying the protein solution as a thread-like liquid is not particularly limited, but one example is to extrude it from a spinning nozzle into the coagulation solution in a desolvation tank. Undrawn yarn is obtained by the coagulation of the protein. The extrusion speed when extruding the protein solution into the coagulation solution can be appropriately set according to the diameter of the nozzle and the viscosity of the protein solution, but for example, in the case of a syringe pump with a nozzle of 0.1 to 0.6 mm in diameter, from the viewpoint of spinning stability, the extrusion speed is preferably 0.2 to 6.0 mL / h per hole, and more preferably 1.4 to 4.0 mL / h per hole. The length of the desolvation tank (coagulation solution tank) containing the coagulation solution is not particularly limited, but for example, the length may be 200 to 500 mm. The take-up speed of the undrawn yarn formed by protein coagulation may be, for example, 1 to 14 m / min, and the residence time may be, for example, 0.01 to 0.15 min. From the viewpoint of desolvation efficiency, the take-up speed of the undrawn yarn is preferably 1 to 3 m / min. The undrawn yarn formed by protein coagulation may be further drawn (pre-drawn) in the coagulation solution, but from the viewpoint of suppressing the evaporation of the lower alcohol used in the coagulation solution, it is preferable to maintain the coagulation solution at a low temperature and withdraw the yarn from the coagulation solution in its undrawn state.

[0210] (b) Stretching The process may also include a step of further drawing the undrawn yarn obtained by the method described above. The drawing may be a single-stage drawing or a multi-stage drawing of two or more stages. Multi-stage drawing allows for the orientation of molecules in multiple stages and increases the total drawing ratio, making it suitable for producing tough fibers.

[0211] If the protein-formed product is a film (protein film), the protein solution may be adjusted to a concentration and viscosity that allows for film formation, if necessary. While there are no particular limitations on the method of forming a film from the protein, one method involves applying the protein solution to a predetermined thickness onto a flat plate resistant to a formic acid-containing solvent to form a coating, and then removing the formic acid-containing solvent from the coating to obtain a film of the predetermined thickness.

[0212] One method for forming a film of a predetermined thickness is the casting method. When forming a film by the casting method, a protein solution is cast onto a flat plate to a thickness of several microns or more using a jig such as a doctor coat or knife coater to form a cast film, and then the solvent is removed by vacuum drying or immersion in a solvent removal bath to obtain a protein film (polypeptide film). The formation of the protein film can be carried out in accordance with the method described in Japanese Patent Publication No. 5678283.

[0213] If the protein-molded body is a porous body (protein porous body), it may be adjusted to a concentration and viscosity that allows for porosity formation, if necessary. The method for forming the protein porous body is not particularly limited. For example, a method may be used in which a porous body is obtained by adding an appropriate amount of foaming agent to a protein solution adjusted to a concentration and viscosity suitable for porosity formation and removing the solvent containing formic acid, or by following the method described in Japanese Patent No. 5796147.

[0214] [Method for producing protein] One embodiment of the present invention provides a method for producing a protein, comprising the steps of: dissolving a target protein and impurities in a solvent containing formic acid at a temperature of 40°C or higher and less than 80°C to obtain a protein solution containing the target protein; and treating the protein solution with a poor solvent for the target protein to aggregate the target protein and obtain the target protein as an aggregate. In the protein production method according to this embodiment, the step of obtaining a protein solution containing the target protein may be carried out under the same conditions as the dissolution step described above. The target protein may be the protein described above.

[0215] According to the protein production method of this embodiment, the target protein to be purified and impurities other than the target protein can be removed from a crude raw material containing the target protein to be purified and other impurities, and the purified target protein can be recovered.

[0216] The target protein and contaminants may be those taken out from a culture containing host cells that have produced the target protein by genetic recombination technology. The target protein and contaminants may be those obtained by subjecting those taken out from a culture containing host cells to treatments such as centrifugation, filter filtration, etc. In other words, the method for producing a protein according to one embodiment may include a step of producing a target protein in host cells in a culture, a step of obtaining a crude raw material containing the target protein and contaminants from the culture, and a step of mixing the crude raw material and a solvent containing formic acid at a temperature of 40°C or higher and lower than 80°C to obtain a protein solution.

[0217] As the poor solvent for the target protein, a solvent in which the target protein is less soluble in the solvent contained in the protein solution is preferable. Examples of the poor solvent for the target protein include aprotic polar solvents and protic polar solvents.

[0218] Examples of the protic polar solvent may include water, methanol, ethanol, 1-propanol, 2-propanol (isopropanol), butanol, tert-butanol, ethylene glycol, propylene glycol, glycerin, etc.

[0219] Examples of the aprotic polar solvent may include ketones and nitriles described later, N-methyl-2-pyrrolidone, dimethyl sulfoxide (DMSO), 1,3-dimethyl-2-imidazolidone (DMI), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), propylene carbonate, hexamethylphosphoramide, N-ethylpyrrolidone, nitrobenzene, furfural, γ-butyrolactone, ethylene sulfite, sulfolane, and ethylene carbonate, but are not limited thereto.

[0220] Examples of the ketones may include acetone, methyl ethyl ketone, methyl butyl ketone, and methyl isobutyl ketone.

[0221] The nitriles may be saturated or unsaturated, but saturated nitriles are preferred. The number of carbon atoms in the nitriles may be 2 to 8, preferably 2 to 6, and more preferably 2 to 4. Specific examples of nitriles include acetonitrile, propionitrile, succinonitrile, butyronitrile, and isobutyronitrile.

[0222] The amount of poor solvent added to the target protein should be determined appropriately according to the target protein, so that the target protein precipitates. Usually, the same amount of poor solvent as the protein solution should be added. The amount added should be adjusted as appropriate depending on the aggregation of the target protein and the presence of impurities.

[0223] The poor solvent for the target protein may be a protic polar solvent, or methanol, from the viewpoint of further improving the purity of the target protein and increasing the recovery rate.

[0224] Common methods for recovering aggregated target proteins as aggregates include centrifugation, drum filtration, and press filtration. When using filter filtration, the use of filter aids such as Celite and diatomaceous earth, as well as precoating agents, can more efficiently recover the target protein as aggregates. [Examples]

[0225] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0226] [(1) Preparation of target protein expression cell lines (recombinant cells)] A nucleic acid encoding spider silk fibroin (PRT775) having the amino acid sequence shown in SEQ ID NO: 46 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. The hydroxyl index and molecular weight of each protein are shown in Table 4.

[0227] [Table 4]

[0228] Similarly to the above, nucleic acids encoding spider silk fibroin (PRT799) having the amino acid sequence shown in SEQ ID NO: 15, and nucleic acids encoding spider silk fibroin (PRT918) having the amino acid sequence shown in SEQ ID NO: 39 were synthesized. An NdeI site was added to the 5' end of these nucleic acids, and an EcoRI site was added downstream of the stop codon.

[0229] The above nucleic acids were each cloned into a cloning vector (pUC118). Subsequently, these nucleic acids were cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector. E. coli BLR(DE3) was transformed with the pET-22b(+) expression vectors containing these recombinant nucleic acids to obtain transformed E. coli (recombinant cells) expressing the target protein.

[0230] [(2) Expression of the target protein] The transformed E. coli described above was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was then mixed with OD in 100 mL of seed culture medium containing ampicillin (Table 5). 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.

[0231] [Table 5]

[0232] OD was added to a jar fermenter containing 500 mL of production medium (Table 6). 600The seed culture solution was added to achieve a ratio 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.

[0233] [Table 6]

[0234] 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 culture was incubated for 20 hours while maintaining the dissolved oxygen concentration in the culture medium at 20% of the dissolved oxygen saturation concentration. 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 the target protein. 20 hours after IPTG addition, the culture medium was centrifuged and wet cells were collected. SDS-PAGE was performed using cells (wet cells) prepared from the culture medium before and after IPTG addition, and it was confirmed that the target protein was expressed as an insoluble form by the appearance of a band of the target protein size that was dependent on IPTG addition. The collected wet cells were dried to obtain dried E. coli cells expressing spider silk fibroin. Through the above procedures, wet and dried bacterial cells expressing spider silk fibroins PRT775, PRT799, and PRT918 were obtained, respectively.

[0235] [(3) Purification of protein (PRT775)] The cells expressing PRT775 recovered 2 hours after adding IPTG 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 disrupted using a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity was achieved. 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, stirred with a stirrer at 60 °C for 30 minutes, and dissolved. After dissolution, dialysis was performed with water using a dialysis tube (Cellulose Tube 36 / 32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, and the moisture was removed using a freeze dryer, and the freeze-dried powdered protein (PRT775) was recovered.

[0236] [(4) Preparation of dope solution] 1. A predetermined amount of formic acid was weighed into a screw tube bottle made of Pyrex (registered trademark) glass. 2. The purified target protein (PRT775) was weighed so that the protein content would be 25% by mass at the time of dissolution, and placed in the screw tube bottle. 3. Samples (1 to 6) containing the target protein and formic acid obtained in 2 were stirred using a stirrer at a predetermined temperature for 3 hours or more (the heating temperatures of Samples 1 to 6 were room temperature (RT, 25 °C), 70 °C, 40 °C, 50 °C, 60 °C, and 80 °C, respectively). 4. Each sample was defoamed with Awatori Rentaro (ARE-500) for 30 minutes or more to obtain a dope solution. 5. Each dope solution obtained in 4 was aliquoted into a test tube for viscosity measurement, and the viscosity was measured. Table 7 shows the evaluation results for dissolution and the viscosity measurement results. Figure 4 shows the viscosity measurement results at each heating temperature. Solubility was evaluated visually. In the dissolution evaluation, "B" indicates insoluble, and "A" indicates dissolved. Viscosity was measured using the "EMS Viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd.

[0237] [Table 7]

[0238] [(5) Formation of protein fibers] Using a known spinning apparatus, each doping solution (protein concentration in the doping solution: 25% by mass) was discharged into a coagulation solution (methanol) using a gear pump. The spinning conditions were as shown below. As a result, protein fibers (fibroin fibers) were obtained as protein molded products. (Spinning conditions) Dope solution temperature: 25℃ Hot roller (HR) temperature: 60℃ Total extension ratio: 4x (Sample 1), 5x (Samples 2-5), 1x (Sample 6)

[0239] [(6) Tensile test of protein fibers] Protein fibers were fixed with adhesive to test paper pieces with a gripping jig distance of 20 mm, and stress (strength) and elongation were measured at a tensile speed of 10 cm / min using an Instron 3342 tensile testing machine under conditions of 20°C and 65% relative humidity. A load cell capacity of 10 N and a clip-type gripping jig were used.

[0240] The results are shown in Figures 5-6 and Table 8. Table 8 shows the values ​​for strength (MPa) and elongation (%) (average values ​​for sample size n=10). Figure 7 shows the GPC measurement results for each sample (1-6). In Figure 7, the solid line (thin) indicates heated samples (70°C), the dashed line indicates heated samples (60°C), the dotted line indicates unheated samples (25°C), the dashed line indicates heated samples (80°C), the solid line (thick) indicates heated samples (40°C), and the dotted line indicates heated samples (50°C).

[0241] [Table 8]

[0242] [Test Example 1: Production of the target protein PRT799 using wet bacterial cells] 500 μL of wet bacterial cells containing spider silk fibroin PRT799 were dispensed and centrifuged at 2,500 g for 10 minutes. The supernatant obtained after centrifugation was removed, and an equal volume of formic acid, or a mixed solvent of formic acid and water (reverse osmosis (RO) water) (aqueous formic acid solution), was added to the removed supernatant. The concentration of formic acid in the aqueous formic acid solution was 75% by mass or 50% by mass relative to the total volume of the aqueous formic acid solution. The sample to which the above solvent was added was heated to 40°C and stirred at 1,500 rpm for 1 hour to obtain a protein solution. Photographs of the obtained protein solutions are shown in Figure 8. Figure 8A shows the protein solution obtained by adding formic acid to wet bacterial cells, Figure 8B shows the protein solution obtained by adding a 75% by mass aqueous formic acid solution to wet bacterial cells, and Figure 8C shows the protein solution obtained by adding a 50% by mass aqueous formic acid solution to wet bacterial cells.

[0243] Each protein solution was centrifuged for 10 minutes at a concentration of 2,500 g. The supernatant obtained after centrifugation was added to 1.5 times the volume of methanol and allowed to stand for 2 hours. After standing for 2 hours, the solutions were centrifuged again for 10 minutes at a concentration of 2,500 g, and the supernatant was removed. The solutions were then washed twice with an equal volume of RO water. After washing, the precipitate was freeze-dried, and the protein-containing powdered sample was recovered.

[0244] The total protein and fibroin content in the obtained samples were measured. Total protein content was measured by the BCA method. Fibroin content was measured using Ni Sepharose. Fibroin purity was defined as the ratio of fibroin content (mg / mL) to total protein content (fibroin content / total protein content × 100). The results for fibroin purity and recovery amount are shown in the table below.

[0245] [Table 9]

[0246] (Analysis by SDS-PAGE) Each sample was analyzed using SDS-PAGE. The analysis results are shown in Lane No. 1 (formic acid), Lane No. 2 (75% by mass formic acid aqueous solution), and Lane No. 3 (50% by mass formic acid aqueous solution) of Figure 9.

[0247] Based on the results obtained from measurements using the BCA method, a sample for SDS-PAGE was prepared for a powder containing spider silk fibroin so that the protein concentration was 10 mg / mL. Mini-PROTEAN® Tetra System

[0248] The TGX® Gels system was set up, and each prepared SDS-PAGE sample was loaded and electrophoresis was performed. After electrophoresis, the samples were analyzed using the Gel DOC® EZ Imager. The results are shown in Figure 9.

[0249] The left image in Figure 9 shows the result after electrophoresis, stained with Oriole® fluorescent gel stain (Bio-Rad), which can stain all proteins. The right image in Figure 1 shows the result after electrophoresis, stained with InVision® His-tagged gel staining reagent (Thermo Fisher Scientific), which reacts to the His-tagged region of PRT799. PRT799 (theoretical molecular weight: 211.4 kDa) was detected as a band near the 250 kDa molecular weight marker.

[0250] [Test Example 2: Production of target protein (PRT799) using dried bacterial cells (1)] 50 mg of dried bacterial cells containing spider silk fibroin PRT799 were mixed with formic acid, or a mixture of formic acid and water (reverse osmosis (RO) water) (aqueous formic acid solution). The concentration of formic acid in the aqueous formic acid solution was 75% by mass or 50% by mass relative to the total volume of the aqueous formic acid solution. The sample to which the above solvent was added was heated to 40°C and stirred at 1,500 rpm for 1 hour to obtain a protein solution. Photographs of the obtained protein solutions are shown in Figure 10. Figure 10A shows the protein solution obtained by adding formic acid to dried bacterial cells, Figure 10B shows the protein solution obtained by adding a 75% by mass aqueous formic acid solution to dried bacterial cells, and Figure 10C shows the protein solution obtained by adding a 50% by mass aqueous formic acid solution to dried bacterial cells.

[0251] Using the obtained protein solution, a powdered sample was obtained by performing the same procedure as in Test Example 1. The total protein and fibroin content in the obtained sample were measured in the same manner as in Test Example 1. The results for fibroin purity and recovery amount are shown in the table below.

[0252] [Table 10]

[0253] (Analysis by SDS-PAGE) The obtained samples containing spider fibroin were analyzed using the same method as described in "Analysis by SDS-PAGE" above. The analysis results are shown in Figure 10, lanes No. 1 (formic acid), No. 2 (75% by mass formic acid aqueous solution), and No. 3 (50% by mass formic acid aqueous solution).

[0254] [Test Example 3: Production of the target protein PRT799 using dried bacterial cells (2)] 10 g of dried bacterial cells containing spider silk fibroin PRT799 were mixed with 100 mL of formic acid and water (reverse osmosis (RO) water) to form a formic acid aqueous solution. The concentration of formic acid in the aqueous solution was 50% by mass relative to the total volume of the aqueous solution. The sample with the above solvent added was heated to 40°C and stirred at 1,500 rpm for 1 hour to obtain a protein solution. 10 g of filter aid was added to the protein solution, and suction filtration was performed using a disposable bottle top filter pre-coated with the filter aid. After suction filtration, the supernatant was added to 1.5 times the volume of methanol and allowed to stand for 2 hours. After standing for 2 hours, the sample was centrifuged for 10 minutes at 2,500 g, and the supernatant was removed and washed twice with an equal volume of RO water. After washing, the precipitate was freeze-dried, and a powdered sample containing protein was recovered.

[0255] (Analysis by SDS-PAGE) The obtained samples were analyzed using the same method as described in "Analysis by SDS-PAGE" above. The analysis results are shown in Figure 12, lane No. 1.

[0256] [Test Example 4: Production of the target protein PRT918 using dried bacterial cells] 50 mg of dried bacterial cells containing spider silk fibroin PRT918 were mixed with formic acid, or a mixture of formic acid and water (reverse osmosis (RO) water) (aqueous formic acid solution). The concentration of formic acid in the aqueous formic acid solution was 75% by mass or 50% by mass relative to the total volume of the aqueous formic acid solution. The sample to which the above solvent was added was heated to 40°C and stirred at 1,500 rpm for 1 hour to obtain a protein solution. Photographs of the obtained protein solutions are shown in Figure 13. Figure 13A shows the protein solution obtained by adding formic acid to dried bacterial cells, Figure 13B shows the protein solution obtained by adding a 75% by mass aqueous formic acid solution to dried bacterial cells, and Figure 13C shows the protein solution obtained by adding a 50% by mass aqueous formic acid solution to dried bacterial cells.

[0257] Each protein solution was centrifuged for 10 minutes at a concentration of 2,500 g. The supernatant obtained after centrifugation was added to twice the volume of RO water and allowed to stand for 2 hours. After standing for 2 hours, the solution was centrifuged again for 10 minutes at a concentration of 2,500 g, the supernatant was removed, and the solution was washed twice with an equal volume of RO water. After washing, the precipitate was freeze-dried, and the protein-containing powdered sample was recovered.

[0258] (Analysis by SDS-PAGE) The obtained samples containing spider fibroin were analyzed using the same method as described in "Analysis by SDS-PAGE" above. The analysis results are shown in Figure 14, lanes No. 1 (formic acid), No. 2 (75% by mass formic acid aqueous solution), and No. 3 (50% by mass formic acid aqueous solution).

Claims

1. A step of dissolving a protein in a solvent containing formic acid at a temperature of 40°C or higher but less than 80°C to obtain a protein solution, Using the aforementioned protein solution, a protein molded body (excluding those molded by electrospinning and protein nanofibers) is produced. The process includes molding ) and, The protein is fibroin, A method for producing a protein-formed article, wherein the concentration of the formic acid is 30% by mass or more, based on the total mass of the solvent.

2. The method for producing a protein molded article according to claim 1, wherein the protein is a modified protein.

3. The aforementioned protein is given by 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, In formula 1 or formula 2, (A) ｎ The motif shows an amino acid sequence consisting of 2 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 40% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer between 2 and 300. There are multiple (A) ｎ The motifs may have the same amino acid sequence or different amino acid sequences. The method for producing a protein molded article according to claim 1 or 2, wherein the multiple REPs may have the same amino acid sequence or different amino acid sequences.

4. A method for producing a protein molded article according to any one of claims 1 to 3, wherein the protein molded article is a protein fiber.

5. A method for producing a protein-formed article according to any one of claims 1 to 4, wherein the protein is spider silk fibroin.

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