JP2021119742A - Method for producing protein formed body and target protein formed body - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of easily producing a protein molded product having suppressed gloss. A method for producing a protein molded product, comprising a step of forming a molded product of the target protein using a protein solution containing a target protein, a void-forming substance and a solvent. [Selection diagram] None

Description

The present invention relates to a method for producing a protein molded product and a target protein molded product.

Conventionally, fibers, films, porous bodies and the like have been known as molded products using a protein material as a polymer material (see, for example, Patent Documents 1 to 3). Such a protein molded product may be endowed with various functions depending on the purpose of use.

Japanese Patent No. 5540166 Japanese Patent No. 5678283 Japanese Patent No. 5796147

For example, in the case of protein fibers, an oil agent or the like may be applied to impart gloss or the like to the molded fibers. On the other hand, from the viewpoint of imparting a high-class feeling, a protein molded product having suppressed gloss (particularly protein fiber) has also been required. However, little has been known so far as a method for producing a protein molded product having suppressed gloss.

An object of the present invention is to provide a method capable of easily producing a protein molded product having suppressed gloss.

The present inventors have found that a protein molded product having suppressed gloss can be obtained by using a protein solution containing a target protein, a void-forming substance and a solvent. The present invention is based on this novel finding.

The present invention provides, for example, the following inventions.
[1]
A method for producing a protein molded product, comprising a step of forming a molded product of the target protein using a protein solution containing the target protein, a void-forming substance, and a solvent.
[2]
The method for producing a protein molded product according to [1], wherein the molded product is a fiber.
[3]
The method for producing a protein molded product according to [1] or [2], wherein the void-forming substance is at least one selected from the group consisting of proteins other than the target protein, lipids, sugars, nucleic acids and minerals.
[4]
The method for producing a protein molded product according to any one of [1] to [3], wherein the content of the void-forming substance in the protein solution is 3 parts by mass or more with respect to 100 parts by mass of the target protein.
[5]
The production of the protein molded product according to any one of [1] to [4], wherein the content volume of the void in the protein molded product is 3% by volume or more with respect to 100% by volume of the target protein in the protein molded product. Method.
[6]
The method for producing a protein molded product according to any one of [1] to [5], wherein the target protein is a structural protein.
[7]
The method for producing a protein molded product according to [6], wherein the structural protein is spider silk fibroin.
[8]
A target protein molded product containing a target protein and a void-forming substance, and the content of the void-forming substance is more than 1% by mass with respect to 100% by mass of the target protein.
[9]
A protein molded product containing the target protein and containing more than 1% by volume of voids with respect to 100% by volume of the target protein.
[10]
The target protein molded product has a birefringence of less than 95% when the birefringence of the target protein molded product containing no void is 100%.
[11]
The protein molded product according to any one of [8] to [10], wherein the target protein is a structural protein.
[12]
The protein molded article according to [11], wherein the structural protein is spider silk fibroin.

According to the present invention, it is possible to provide a method capable of easily producing a protein molded product having suppressed gloss.

It is a schematic diagram which shows an example of the domain sequence of fibroin. It is a schematic diagram which shows an example of the domain sequence of fibroin. It is a schematic diagram which shows an example of the domain sequence of fibroin. It is an SEM photograph of a protein fiber in an example. It is an SEM photograph of a protein fiber in an example. It is an SEM photograph of a protein fiber in an example. It is an SEM photograph of a protein fiber in an example. It is an SEM photograph of a protein fiber in an example. It is an SEM photograph of a protein fiber in an example. It is a photograph which shows the evaluation result of the gloss of the protein fiber in an Example.

Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[Manufacturing method of protein molded product]
In the method for producing a protein molded product according to the present embodiment, a protein solution containing a target protein (target protein), a void-forming substance and a solvent is used, and the target protein molded product (hereinafter, also referred to as “target protein molded product”) is used. It is provided with a step of forming (referred to as).

Since the protein solution contains a void-forming substance, the target protein and the void-forming substance are molded together to form a structure derived from the void-forming substance in the molded product. As used herein, a void means a structure derived from this void-forming substance and is present on the surface or inside of a protein molded article. A void-forming substance may be present in the void. Further, the void may be a void from which a part or all of the void-forming substance has been removed.

The gloss of the target protein molded product is suppressed. The target protein molded product has excellent dyeability because its gloss is suppressed. Further, the target protein molded product has improved heat retention.

(protein)
The type of the target protein is not particularly limited, and may be, for example, a structural protein. The structural protein refers to a protein that forms a biological structure or a protein derived from the protein. That is, the structural protein may be a naturally occurring structural protein, and is a modified protein in which a part of the amino acid sequence (for example, 10% or less of the amino acid sequence) is modified depending on the amino acid sequence of the naturally occurring structural protein. May be.

Examples of the structural protein include fibroin, collagen, resilin, elastin and keratin, and proteins derived from these. The fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin. The structural protein may be silk fibroin, spider silk fibroin, or a combination thereof.

The fibroin according to the present embodiment includes naturally occurring fibroin and modified fibroin. As used herein, "naturally occurring fibroin" means fibroin having the same amino acid sequence as naturally occurring fibroin, and "modified fibroin" means fibroin having an amino acid sequence different from that of naturally occurring fibroin. do.

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 the natural spider silk fibroin include spider silk proteins produced by spiders.

The fibroin according to the present embodiment is, for example, a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m − (A) _{n motif.} It may be a protein containing. The fibroin according to the present 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 not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically _{corresponding to (A) n} motif of amino acid sequence) and an amorphous region (typically, REP of amino acid sequence). An amino acid sequence that produces (corresponding.)), _{Which is represented by the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, the (A) _n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2-27. (A) The _number of amino acid residues of the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, (A) _{the ratio of the number of alanine residues to the total number of amino acid residues in the n} motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). A plurality of (A) _n motifs present in the domain sequence may be composed of at least seven alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300 and may be an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences. Specific examples thereof include proteins containing the amino acid sequence shown in SEQ ID NO: 15 (PRT799). The hydropathic index of PRT799 (SEQ ID NO: 15) is −0.80. The value of the hydropathy index is a value calculated according to the method described in International Publication No. 2014/103846.

As naturally derived fibroin, for example, a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif can be used.} Examples include proteins. Specific examples of naturally occurring fibroin include, for example, fibroin produced by insects or spiders.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea perni, tussah, and tussah. ), Silk moth (Samia synthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama) Hornet silk protein can be mentioned.

More specific examples of insect-produced fibroin include, for example, the silk moth fibroin L chain (GenBank accession number M76430 (nucleic acid sequence) and AAA27840.1 (amino acid sequence)).

Examples of fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Spider, spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne), spiders belonging to the genus Trinofundamashi, and spiders belonging to the genus Cyrtarachne. Spiders belonging to (Gasteracantha genus), spiders belonging to the genus Isekigumo (genus Ordgarius) such as Mameitaisekigumo and Mutsutogaysekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc. Spiders belonging to the genus Arachunura, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora ) Spiders, spiders, spiders belonging to the genus Cyclosa, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Tetragnatha, such as Yasagata spider, Harabiroashidaka spider, and Urokoa spider, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as spiders, spiders belonging to the genus Dyschiriognatha, spiders such as spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus (Latrodectus) and spiders belonging to the genus Euprostenops (genus Euprostenops) and other spiders belonging to the family Tetragnathidae Examples include pider silk protein. Examples of the spider silk protein include traction thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleic acid sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleic acid sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank sequence No. AAC4011) (Nucleotide sequence) ), U37520 (nucleic acid sequence)), major amplifier spidroin 1 [derived from Latrodictus protein] (GenBank accession number ABR68856 (nucleic acid sequence), EF595246 (nucleic acid sequence)), dragline silk protein (derived from nuclear protein) Numbers AAL32472 (nucleic acid sequence), AF441245 (nucleic acid sequence)), major aminos protein 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (nucleotide sequence), AJ973155 (nucleic acid sequence)), and major protein (GenBank accession number CAM32249.1 (nucleic acid sequence), AM490169 (nucleic acid sequence)), minor amplify silk protein 1 [Nephila proteins] (GenBank accession number AAC14589.1 (nucleotide sequence)), minor amplifier clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nefilengys cruisetata] (GenBank accession) The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

As a more specific example of naturally occurring fibroin, further, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, among the sequence information registered in NCBI GenBank, among the sequences containing INV as DIVISION, spidroin, complete, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

(Modified fibroin)
The modified fibroin is, for example, one in which the amino acid sequence of naturally-derived fibroin is modified based on the amino acid sequence of naturally-derived fibroin (for example, the amino acid sequence is modified by modifying the gene sequence of cloned naturally-derived fibroin). It may be artificially designed and synthesized regardless of naturally occurring fibroin (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). ..

The modified fibroin is, for example, modifying the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. Can be obtained at. 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 partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Energy, 100, 448 (1983).

The modified fibroin may be, for example, a modified fibroin derived from a silk protein produced by silk moth, or a modified fibroin derived from a spider silk protein produced by spiders.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the large spitting tube bookmarker thread protein produced in the large bottle-shaped gland of spider, and modified fibroin with a reduced content of glycine residues. (Second modified fibroin), (A) _{reduced content of n} motifs Modified fibroin (third modified fibroin), content of glycine residues, and (A) reduced content of _{n motifs} It has a modified fibroin (fourth modified fibroin), a modified fibroin having a domain sequence containing a region having a locally high hydrophobicity index (fifth modified fibroin), and a domain sequence having a reduced content of glutamine residues. Modified fibroin (sixth modified fibroin) can be mentioned.

The modified fibroin (first modified fibroin) derived from the large spider thread tube bookmark thread protein produced in the large bottle-shaped gland of the spider is a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. Examples include proteins containing. In the first modified fibroin, n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, further preferably an integer of 8 to 20, and even more preferably an integer of 10 to 20 in Equation 1. An integer of ~ 16 is even more preferred, an integer of 8-16 is particularly preferred, and an integer of 10-16 is most preferred. In the first modified fibroin, the number of amino acid residues constituting REP in the formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. Is even more preferable, and 20 to 75 residues are even more preferable. In the first modified fibroin, the total number of residues of glycine residue, serine residue and alanine residue contained in the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _{m is the amino acid residue.} It is preferably 40% or more, more preferably 60% or more, and further preferably 70% or more with respect to the total number.

The first modified fibroin contains the unit of the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m , and the C-terminal sequence is the amino acid sequence shown in any of SEQ ID NOs: 1 to 3. Alternatively, it may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 residues of the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is the sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C-terminal of the amino acid sequence shown in No. 1, and the amino acid sequence shown in SEQ ID NO: 3 was 29 residues removed from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It has the same amino acid sequence.

As a more specific example of the first modified fibroin, 90% or more sequence identity with the amino acid sequence shown in (1-i) SEQ ID NO: 4 or the amino acid sequence shown in (1-ii) SEQ ID NO: 4 Examples thereof include modified fibroin containing an amino acid sequence having. The sequence identity is preferably 95% or more.

The amino acid sequence shown by SEQ ID NO: 4 is the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 5) consisting of a starting codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal, and the first to first amino acids. The 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue. The amino acid sequence at the C-terminal of the amino acid sequence shown in SEQ ID NO: 4 is the same as the amino acid sequence shown in SEQ ID NO: 3.

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

The modified fibroin (second modified fibroin) having a reduced content of glycine residue has an amino acid sequence in which the domain sequence thereof is reduced as compared with the naturally occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP replaced with another amino acid residue as compared with naturally occurring fibroin. ..

The second modified fibroin has a domain sequence of GGX and GPGXX in REP as compared with naturally occurring fibroin (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine. In at least one motif sequence selected from), it has an amino acid sequence corresponding to at least one or a plurality of glycine residues in the motif sequence being replaced with another amino acid residue. You may.

In the second modified fibroin, the ratio of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more of the total motif sequence.

The second modified fibroin contains the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and the domain sequence from the (A) _n motif located closest to the C-terminal side from the above domain sequence. The total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal of is z, and the above domain sequence. When the total number of amino acid residues in the sequence excluding the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the above domain sequence is w, z / w is 30% or more. It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The _{number of alanine residues with respect to the total number of amino acid residues in the n} motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed of only alanine residues).

The second modified fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, 6 % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.

The method of calculating z / w will be described in more detail. First, in the fibroin (modified fibroin or naturally-derived fibroin) containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _{m, it is located most C-terminal to the domain sequence (A) n.} The amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50 × 3 = 150. Further, for example, when X (center X) contained in two XGX exists as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used). be). w is the total number of amino acid residues contained in the sequence excluding the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 ( _{excluding the (A) n} motif located most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70% or more. Is even more preferable, and 80% or more is even more preferable. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

The second modified fibroin is, for example, modified from the cloned naturally occurring fibroin gene sequence by substituting at least a part of the base sequence encoding the glycine residue to encode another amino acid residue. Obtainable. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be replaced so that z / w is 50.9% or more. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, in addition to the modification corresponding to the substitution of the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , Insertion and / or modification of the amino acid sequence corresponding to the addition may be performed.

The other amino acid residue described above is not particularly limited as long as it is an amino acid residue other than the glycine residue, but is a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and methionine ( Hydrophobic amino acid residues such as M) residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S) ) Residues, hydrophilic amino acid residues such as lysine (K) residues and glutamate (E) residues are preferred, with valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( The Q) residue is more preferred, and the glutamine (Q) residue is even more preferred.

As a more specific example of the second modified fibroin, (2-i) the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or (2-ii) SEQ ID NO: 6, sequence. Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10, which corresponds to naturally occurring fibroin. _{The amino acid sequence shown in SEQ ID NO: 7 is such that every two (A) n} motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, and further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side _{of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 7, and some glutamine (Q) residues are further added.} It was replaced with a serine (S) residue, and some amino acids on the N-terminal side were deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above four times.

The value of z / w in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence shown in SEQ ID NO: 9 are 58.7%, respectively. It is 70.1%, 66.1% and 70.0%. Further, the values of x / y in the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences shown by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are They are 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.

The modified fibroin of (2-i) may consist of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

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

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography). Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 12 (amino acid sequence including His tag sequence and hinge sequence).

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

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

Further, a tag sequence in which the tag sequence can be separated by a specific protease can also be used. By treating the protein adsorbed via the tag sequence with a protease, the modified fibroin from which the tag sequence has been separated can also be recovered.

As a more specific example of the second modified fibroin comprising the tag sequence, (2-iii) SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or the amino acid sequence set forth in SEQ ID NO: 15 or (2-iv). SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 can be mentioned as a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 15, or SEQ ID NO: 15.

The amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15, are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. And the amino acid sequence shown in SEQ ID NO: 12 (including His tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence shown in SEQ ID NO: 9.

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

The modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15 and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

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

(A) modified fibroin content of _n motifs has been reduced (third modified fibroin), the domain sequence is compared to the naturally occurring fibroin, amino acids with reduced content of (A) _n motif Has a sequence. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of _{at least one or more (A) n motifs as compared with naturally occurring fibroin.}

The third modified fibroin may have an amino acid sequence corresponding to a 10-40% deletion of the _{(A) n motif from naturally occurring fibroin.}

The third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal C-terminal one to three toward the side of the (A) _n motif every one (A) _n motif It may have an amino acid sequence corresponding to the deletion of.

_{The third modified fibroin has a domain sequence of at least two consecutive (A) n-} motif deletions and one (A) from the N-terminal side to the C-terminal side as compared to naturally occurring fibroin. ) It may have an amino acid sequence corresponding to the deletion of the _{n-motif being repeated in this order.}

The third modified fibroin may have an amino acid sequence corresponding to the deletion _{of the (A) n} motif at least every other two domain sequences from the N-terminal side to the C-terminal side. ..

The third modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _{m , and two adjacent [(A) n} motifs from the N-terminal side to the C-terminal side. -REP] When the number of amino acid residues of REP in the unit is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 1. When x is the maximum value of the sum of the number of amino acid residues of two adjacent [(A) _{n motif-REP] units, which is 11.3, and y is the total number of amino acid residues in the domain sequence.} In addition, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The _{number of alanine residues with respect to the total number of amino acid residues in the n} motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed of only alanine residues).

The calculation method of x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from fibroin. From the N-terminal side (left side), the domain sequence consists of (A) _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-Fourth REP (20 amino acid residues)-(A) _n Motif-Fifth REP (30 amino acid residues)-(A) _It has an arrangement called n motifs.

Two adjacent [(A) _n motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, _{there may be a [(A) n} motif-REP] unit that is not selected. In FIG. 1, pattern 1 (comparison between the first REP and the second REP and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. There are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in _{two adjacent [(A) n motif-REP] units selected is compared.} The comparison is performed by obtaining the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparing the 4th REP (20 amino acid residues) and the 5th REP (30 amino acid residues), when the 4th REP having a smaller number of amino acid residues is 1, the 5th REP The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 1, when the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is 1.8 to 11.3 [(A) _n motif-REP] unit set. Shown by solid line. Hereinafter, such a ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 _{. The set of [(A) n} motif-REP] units is indicated by a broken line. Indicated.

In each pattern, add up the total number of amino acid residues of the two adjacent [(A) _n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of _{(A) n motif.} be.). Then, the total values added are compared, and the total value (maximum value of the total value) of the pattern in which the total value is maximized is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.

Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, still more preferably 70% or more. It is preferably 75% or more, even more preferably 80% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be, for example, 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x. / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.

When the third modified fibroin is a modified fibroin in which _{at least 7 of the (A) n} motifs present in the domain sequence are composed of only alanine residues, the x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further more preferable, 70% or more is even more preferable, and 80% or more. It is particularly preferable to have. The upper limit of x / y is not particularly limited and may be 100% or less.

The third modified fibroin, for example, deletes one or more of the sequences encoding the _{(A) n} motif from the cloned naturally occurring fibroin gene sequence so that x / y is 64.2% or more. Can be obtained by Further, for example, an amino acid sequence corresponding to the deletion _{of one or more (A) n} motifs so that x / y is 64.2% or more is designed and designed from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence. _{In each case, in addition to the modification corresponding to the deletion of (A) n} motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. The amino acid sequence corresponding to the above may be modified.

As a more specific example of the third modified fibroin, (3-i) the amino acid sequence shown by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or (3-ii) SEQ ID NO: 18, sequence. Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (3-i) will be described. The amino acid sequence shown in SEQ ID NO: 18 is obtained by deleting _{every other (A) n} motif from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 10, which corresponds to naturally occurring fibroin. Further, one [(A) _n motif-REP] is inserted before the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 7 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 18. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side _{of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 7, and some glutamine (Q) residues are further added.} It was replaced with a serine (S) residue, and some amino acids on the N-terminal side were deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above four times.

The value of x / y in the jagged ratio of 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 15.0%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 18 and the amino acid sequence shown in SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 9 is 89.3%. The values of z / w in 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% and 66. 1% and 70.0%.

The modified fibroin of (3-i) may consist of the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _{The amino acid residue of two adjacent [(A) n} motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3) When the maximum value of the total value obtained by adding the radix is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

The third modified fibroin may contain the tag sequence described above at either or both of the N-terminus and the C-terminus.

As a more specific example of the third modified fibroin containing the tag sequence, the amino acid sequence set forth in (3-iii) SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15 or the (3-iv) sequence. Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15.

The amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15, are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. And the amino acid sequence shown in SEQ ID NO: 12 (including His tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence shown in SEQ ID NO: 9.

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

The modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _Let x be the maximum value of the total value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units in which the ratio of the number of amino acid residues in REP is 1.8 to 11.3. , When the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

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

The modified fibroin (fourth modified fibroin) having a reduced content of glycine residue and (A) _{n motif has a domain sequence of (A) n} motif as compared with naturally occurring fibroin. In addition to having a reduced content of glycine, it has an amino acid sequence with a reduced content of glycine residues. The domain sequence of the fourth modified fibroin lacked at least one or more (A) _n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with another amino acid residue. That is, the fourth modified fibroin includes the above-mentioned modified fibroin having a reduced content of glycine residue (second modified fibroin) and (A) modified fibroin having a reduced content of _{n motif (third).} It is a modified fibroin that also has the characteristics of modified fibroin). Specific aspects and the like are as described in the second modified fibroin and the third modified fibroin.

As a more specific example of the fourth modified fibroin, (4-i) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, (4-ii) SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 9. Specific embodiments of the modified fibroin containing the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 are as described above.

A modified fibroin (fifth modified fibroin) having a domain sequence containing a region having a locally high hydrophobicity index has one or more amino acid residues in the REP as compared to the naturally occurring fibroin. Is replaced with an amino acid residue having a large hydrophobicity index, and / or one or more amino acid residues having a large hydrophobicity index are inserted in the REP. It may have an amino acid sequence containing a region.

The region having a locally large hydrophobicity index is preferably composed of consecutive 2 to 4 amino acid residues.

The amino acid residue having a large hydrophobicity index described above is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). It is more preferably a residue.

In the fifth modified fibroin, one or more amino acid residues in the REP were replaced with amino acid residues having a higher hydrophobicity index as compared with the naturally occurring fibroin, and / or one or more in the REP. In addition to the modification corresponding to the insertion of an amino acid residue with a high hydrophobicity index, one or more amino acid residues were substituted, deleted, inserted and / or added as compared with naturally occurring fibroin. There may be a modification of the amino acid sequence corresponding to the above.

The fifth modified fibroin, for example, leaves one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting for a group (eg, an amino acid residue with a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. Also, for example, one or more hydrophilic amino acid residues in the REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in the REP. It can also be obtained by designing an amino acid sequence corresponding to the insertion of and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acids in the REP. In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The fifth modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _{m , from the (A) n} motif located closest to the C-terminal side to the C-terminal of the above domain sequence. In all REPs contained in the sequence excluding the sequence from the above domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more is defined as p. _{When the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, p / q is 6 It may have an amino acid sequence of .2% or more.

For the hydrophobicity index of amino acid residues, a known index (Hydrotherapy index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic protein, B. 105-132) is used. Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m. (Hereinafter referred to as "sequence A") is used. First, in all the REPs contained in the sequence A, the average value of the hydrophobicity index of consecutive four amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all consecutive 4 amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index", it should be included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Further, the total number of amino acid residues contained in the sequence A is q.

For example, when 20 consecutive "4 consecutive amino acid residues having an average hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of .6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are duplicated by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues is present. A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 2, p is 7 × 4 = because there are seven “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, for example, in the case of the domain sequence shown in FIG. 2, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) _n motif existing at the end of the C-terminal side). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 2, 28/170 = 16.47%.

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

The fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (eg, a hydrophobic index) in the REP so that the amino acid sequence of the cloned naturally occurring fibroin satisfies the above p / q condition. (Amino acid residue with a negative value) is replaced with a hydrophobic amino acid residue (for example, an amino acid residue with a positive hydrophobicity index), and / or one or more hydrophobic amino acid residues are inserted in the REP. By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. It can also be obtained, for example, by designing an amino acid sequence satisfying 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 each case, one or more amino acid residues in the REP were replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring fibroin, and / or one or more amino acid residues in the REP. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. ..

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) Is preferable, and valine (V), leucine (L) and isoleucine (I) are more preferable.

As a specific example of the fifth modified fibroin, (5-i) the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or (5-ii) SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 21 can be mentioned.

The modified fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 22 is an amino _{acid sequence in which alanine residues in the (A) n} motif of naturally occurring fibroin are contiguous, and the number of contiguous alanine residues is five. .. The amino acid sequence shown in SEQ ID NO: 19 is the amino acid sequence shown in SEQ ID NO: 22, in which two amino acid sequences (VLI) consisting of 3 amino acid residues are inserted every other REP, and the amino acid sequence is shown in SEQ ID NO: 22. A part of the amino acid on the C-terminal side is deleted so that the molecular weight of the amino acid sequence is almost the same as that of the amino acid sequence. In the amino acid sequence shown in SEQ ID NO: 23, two alanine residues are inserted on the C-terminal side of _{each (A) n} motif with respect to the amino acid sequence shown in SEQ ID NO: 22, and a part of glutamine (Q) remains. The group was replaced with a serine (S) residue, and some amino acids on the C-terminal side were deleted so as to have substantially the same molecular weight as 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 three amino acid residues every other REP into the amino acid sequence shown in SEQ ID NO: 23. The amino acid sequence shown by SEQ ID NO: 21 is the amino acid sequence shown by SEQ ID NO: 23, in which two amino acid sequences (VLI) consisting of 3 amino acid residues are inserted every other REP.

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

The modified fibroin of (5-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located most on the C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p and the total number of _{amino acid residues contained in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.

As a more specific example of the fifth modified fibroin comprising a tag sequence, the amino acid sequence set forth in (5-iii) SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, or (5-iv) SEQ ID NO: 24, sequence. Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 25 or SEQ ID NO: 26.

The amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 are the amino acid sequences shown in SEQ ID NO: 12 (His tag) at the N-terminal of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including array and hinge array) is added.

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

The modified fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. The modified fibroin of (5-iv) is also a protein containing the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and is located most on the C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p and the total number of _{amino acid residues contained in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

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

Modified fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified fibroin) has an amino acid sequence with a reduced content of glutamine residues as compared to naturally occurring fibroin.

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.

When the sixth modified fibroin contains the GPGXXX motif in the REP, the content of the GPGXXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited and may be 50% or less, or 30% or less.

In the present specification, the "GPGXX motif content" is a value calculated by the following method.
In the fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif, the most C-terminal side. In all REPs included in the sequence excluding the sequence from the located (A) _n motif to the C-terminal of the domain sequence from the domain sequence, the total number of GPGXX motifs contained in the region is tripled (that is, that is). Let s be (corresponding to the total number of G and P in the GPGXX motif), remove the _{sequence from the (A) n} motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence, and further (A) _n motif. The GPGXX motif content is calculated as s / t, where t is the total number of amino acid residues in all REPs excluded.

In the calculation of the GPGXX motif content, "the _{sequence obtained by excluding the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is targeted at "the most C-terminal side". (A) The _{sequence from the n} motif to the C-terminal of the domain sequence (the sequence corresponding to REP) may contain a sequence having a low correlation with the sequence characteristic of fibroin, and m is small. In this case (that is, when the domain sequence is short), it affects the calculation result of the GPGXX motif content, and this effect is eliminated. When the "GPGXX motif" is located at the C-terminal of the REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 3 is a schematic diagram showing a domain sequence of fibroin. The method of calculating the GPGXX motif content will be specifically described with reference to FIG. First, in the fibroin domain sequence shown in FIG. 3 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are “located closest to the C-terminal side (). A) _{GPGXX for calculating s because it is included in the "sequence excluding the sequence from the n} motif to the C-terminal of the domain sequence from the domain sequence" (the sequence shown by "region A" in FIG. 3). The number of motifs is 7, and s is 7 × 3 = 21. _{Similarly, all REPs are "sequences obtained by removing the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (the sequence shown in "Region A" in FIG. 3). ), The total number t of amino acid residues in all REPs excluding the (A) _{n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150.} Next, s / t (%) can be calculated by dividing s by t, which is 21/150 = 14.0% in the case of fibroin in FIG.

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

In the present specification, the "glutamine residue content" is a value calculated by the following method.
In the fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif, the most C-terminal side. (A) _{All REPs included in the sequence obtained by removing the sequence from the n} motif to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "region A" in FIG. 3) are included in that region. _Let u be the total number of glutamine residues, and remove the sequence from the (A) n motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence, and further (A) the amino acid residue of all REPs excluding the _{n motif.} The glutamine residue content is calculated as u / t, where t is the total number of groups. In the calculation of the glutamine residue content, the _{reason why "the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin corresponds to its domain sequence lacking one or more glutamine residues in the REP or substituting for other amino acid residues as compared to naturally occurring fibroin. It may have an amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than the glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

As shown in Table 1, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M). ), Alanin (A), Glycine (G), Threonine (T), Serine (S), Tryptophan (W), Tyrosine (Y), Proline (P) and Histidine (H). Can be done. 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. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are more preferably amino acid residues.

The sixth modified fibroin has a REP hydrophobicity of -0.8 or more, more preferably -0.7 or more, further preferably 0 or more, and 0.3 or more. Is even more preferable, and 0.4 or more is particularly preferable. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

In the present specification, the "hydrophobicity of REP" is a value calculated by the following method.
In the fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif, the most C-terminal side. In all REPs contained in the sequence obtained by excluding the sequence from the located (A) _n motif to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "region A" in FIG. 3), each amino acid in that region. _Let v be the sum of the hydrophobicity indexes of the residues, and remove the sequence from the (A) n motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence, and further (A) all REP excluding the _{n motif.} The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues in. In the calculation of the hydrophobicity of REP, the _{reason for targeting "the sequence obtained by excluding the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in REP. In addition to the modification corresponding to the replacement of one or more amino acid residues with other amino acid residues, there may be further modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. ..

The sixth modified fibroin, for example, deletes one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence and / or removes one or more glutamine residues in REP. It can be obtained by substituting with the amino acid residue of. Also, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to this and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As a more specific example of the sixth modified fibroin, (6-i) the amino acid sequence shown by 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, or the amino acid sequence set forth in (6-ii) 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 and 90% or more of the sequence. A modified fibroin containing an amino acid sequence having the same identity can be mentioned.

The modified fibroin of (6-i) will be described.

The amino acid sequence (Met-PRT410) shown in SEQ ID NO: 7 is based on the nucleotide sequence and amino acid sequence of Nephila clavipes (GenBank accession number: P4684.1, GI: 11744415), which is a naturally occurring fibroin, (A) _n. The amino acid sequence in which the alanine residues are continuous in the motif is modified to improve the productivity, such as increasing the number of consecutive alanine residues to five. On the other hand, since Met-PRT410 has not been modified for the glutamine residue (Q), the glutamine residue content is about the same as the glutamine residue content of naturally occurring fibroin.

The amino acid sequence (M_PRT888) shown in SEQ ID NO: 27 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 7) with VL.

In the amino acid sequence (M_PRT965) shown in SEQ ID NO: 28, all QQs in Met-PRT410 (SEQ ID NO: 7) are replaced with TS, and the remaining Qs are replaced with A.

The amino acid sequence (M_PRT889) shown in SEQ ID NO: 29 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 7) with VL and substituting the remaining Qs with I.

In the amino acid sequence (M_PRT916) shown in SEQ ID NO: 30, all QQs in Met-PRT410 (SEQ ID NO: 7) are replaced with VI, and the remaining Qs are replaced with L.

In the amino acid sequence (M_PRT918) shown in SEQ ID NO: 31, all QQs in Met-PRT410 (SEQ ID NO: 7) are replaced with VF, and the remaining Qs are replaced with I.

_{The amino acid sequence (M_PRT525) shown in SEQ ID NO: 34 has two alanine residues inserted in the region (A 5} ) where the alanine residues are continuous with respect to Met-PRT410 (SEQ ID NO: 7), and the molecular weight of Met-PRT410. Two domain sequences on the C-terminal side were deleted, and 13 glutamine residues (Q) were replaced with serine residues (S) or proline residues (P) so as to be substantially the same as.

The amino acid sequence (M_PRT699) shown in SEQ ID NO: 32 is obtained by substituting all QQs in M_PRT525 (SEQ ID NO: 34) with VL.

The amino acid sequence (M_PRT698) shown in SEQ ID NO: 33 is obtained by substituting all QQs in M_PRT525 (SEQ ID NO: 34) with VL and substituting the remaining Qs with I.

The amino acid sequences 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 and SEQ ID NO: 33 all have a glutamine residue content of 9% or less (Table 2). ).

The modified fibroin of (6-i) may consist of the amino acid sequence represented by 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) has 90% or more sequence identity with the amino acid sequence represented by 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. It contains an amino acid sequence having. The modified fibroin of (6-ii) is also a domain represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

As a more specific example of the sixth modified fibroin containing the tagged sequence, (6-iii) 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. Modified fibroin containing the indicated amino acid sequence, or (6-iv) amino acid sequence and 90 represented by 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. A modified fibroin containing an amino acid sequence having a sequence identity of% or more can be mentioned.

The amino acid sequences shown by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 are SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively. , The amino acid sequence shown in SEQ ID NO: 12 (including His tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33. Since only the tag sequence was added to the N-terminal, there was no change in the glutamine residue content, and SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41. All of the amino acid sequences shown in (Table 3) have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) may consist of the amino acid sequence set forth 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) has 90% or more sequence identity with the amino acid sequence set forth 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. It contains an amino acid sequence having. The modified fibroin of (6-iv) is also a domain represented by _{formula 1: [(A) n} motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

The modified fibroin according to the present embodiment is among the characteristics 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. , May be a modified fibroin having at least two or more features.

As a collagen-derived protein, for example, a protein _{containing a domain sequence represented by the formula 3: [REP2] p} (where, in formula 3, p represents an integer of 5 to 300. REP2 is Gly-XY. Indicates an amino acid sequence composed of, and X and Y indicate arbitrary amino acid residues other than Gly. Multiple REP2s may have the same amino acid sequence or different amino acid sequences.) .. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 42 can be mentioned. The amino acid sequence shown in SEQ ID NO: 42 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (NCBI GenBank accession number: CAA5635.1, GI: 3702452) obtained from the NCBI database. The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 12 is added to the N-terminal of the amino acid sequence from the 301st residue to the 540th residue.

As a protein derived from resilin, for example, a protein _{containing a domain sequence represented by the formula 4: [REP3] q} (where, in formula 4, q represents an integer of 4 to 300. REP3 is Ser-JJ-. The amino acid sequence composed of Tyr-Gly-U-Pro is shown. J indicates an arbitrary amino acid residue, and it is particularly preferable that it is an amino acid residue selected from the group consisting of Asp, Ser and Thr. U is an arbitrary amino acid residue. It indicates an amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser. A plurality of REP4s may have the same amino acid sequence or different amino acid sequences.) Can be mentioned. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 43 can be mentioned. The amino acid sequence shown in SEQ ID NO: 43 replaces Thr at residue 87 with Ser and Asn at residue 95 in the amino acid sequence of recillin (Axion No. NP 61157, Gl: 24654243 of NCBI GenBank). The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 12 is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced with Asp.

Examples of elastin-derived proteins include proteins having amino acid sequences such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 44 can be mentioned. The amino acid sequence shown by SEQ ID NO: 44 is the amino acid sequence shown by SEQ ID NO: 12 at the N-terminal of the amino acid sequence from the 121st residue to the 390th residue of the amino acid sequence of accession number AAC98395 of GenBank of NCBI. And the hinge arrangement) are added.

Examples of the keratin-derived protein include Type I keratin of Capra hilcus. Specifically, a protein containing the amino acid sequence shown by SEQ ID NO: 45 (amino acid sequence of accession number ACY30466 of GenBank of NCBI) can be mentioned.

The above-mentioned structural protein and the modified structural protein derived from the structural protein may be used alone or in combination of two or more.

(Protein manufacturing method)
The protein is expressed, 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. Can be produced.

The method for producing the nucleic acid encoding the protein is not particularly limited. For example, using a gene encoding a protein such as natural fibroin, it is amplified and cloned by polymerase chain reaction (PCR) or the like, and if necessary, modified by a genetic engineering method or chemically synthesized. The nucleic acid can be produced by the method. The chemical synthesis method of nucleic acid is not particularly limited, and for example, based on the amino acid sequence information of the protein obtained from the NCBI web database or the like, AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.) or the like. Genes can be chemically synthesized by ligating automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the protein, a nucleic acid encoding a protein consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N-terminal of the above amino acid sequence is synthesized. May be good.

The regulatory sequence is a sequence that controls the expression of a protein in a host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the protein may be used. An inducible promoter is a promoter that can control transcription by 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.

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, a vector that can be autonomously replicated in a host cell or can be integrated into the chromosome of the host and contains a promoter at a position where a nucleic acid encoding a protein can be transcribed is preferably used.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia marcescens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus satirus and the like. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium, Ammonia Philum and the like. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium and Ammonia Genes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, as a vector into which a nucleic acid encoding a protein is introduced, for example, pBTrp2 (manufactured by Berliner Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238569) and the like can be mentioned.

Eukaryotic hosts include, for example, yeast and filamentous fungi (molds, etc.). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

When a eukaryote is used as a host, examples of the vector into which the nucleic acid encoding the protein is introduced include YEp13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing an expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like can be mentioned.

As a method for expressing nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. ..

The protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host, and the host can be efficiently cultured. If so, either a natural medium or a synthetic medium may be used.

The carbon source may be any assimilated by the transforming microorganisms, for example, glucose, fructose, sucrose, carbohydrates such as molasses, starch and starch hydrolysate containing them, acetic acid, propionic acid and the like. Organic acids and alcohols such as ethanol and propanol can be used. Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof can be used. As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

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

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium during the culture, if necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

The expressed protein can be isolated and purified by a commonly used method. For example, when the protein is expressed in a lysed state in cells, after the culture is completed, the host cells are collected by centrifugation, suspended in an aqueous buffer solution, and then an ultrasonic crusher, a French press, or manton gaulin. Crush the host cells with a homogenizer, dynomil, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolating and purifying a protein, that is, a solvent extraction method, a salting-out method using sulfuric acid, a desalting method, or an organic solvent is used. Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography method, hydrophobic chromatography method using a resin such as butyl Sepharose, phenyl Sepharose, gel filtration method using a molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis, etc. Purified preparations can be obtained by using the above methods alone or in combination.

When the protein is expressed by forming an insoluble matter in the cell, the host cell is similarly recovered, crushed, and centrifuged to recover the insoluble matter of the protein as a precipitate fraction. The insoluble form of the recovered protein can be solubilized with a protein denaturing agent. After the operation, a purified protein preparation can be obtained by the same isolation and purification method as described above. When the protein is secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

(Void forming substance)
The void-forming substance is a substance that forms voids on the surface and / or inside of a protein molded product obtained by using the solution by containing it in a molding solution together with a protein. Examples of the void-forming substance include proteins other than the target protein (other proteins), lipids, sugars, nucleic acids, and minerals (for example, phosphoric acid). The void-forming substance may be one kind of substance alone or a combination of two or more kinds of substances.

When the target protein is a protein derived from a cell expressing a recombinant protein, the other protein refers to a protein other than the target protein intentionally expressed in the cell. In this case, examples of other proteins include enzyme proteins, structural proteins, transport proteins, storage proteins, contractile proteins, defense proteins, regulatory proteins and the like.

The lipid is not particularly limited, and examples thereof include simple lipids, complex lipids, and inducible lipids. Simple lipids are esters of alcohols and fatty acids. The simple lipid is not particularly limited, and examples thereof include acylglycerol, wax, and ceramide. The complex lipid is a lipid containing phosphoric acid, sugar, etc. in the molecule. The complex lipid is not particularly limited, and examples thereof include phospholipids, glycolipids, lipoproteins, and sulfolipids. Induced lipids are compounds derived by hydrolysis from simple lipids and / or complex lipids. The inducible lipid is not particularly limited, and examples thereof include fatty acids, terpenoids, steroids, and carotenoids.

Nucleic acids are ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Examples of ribonucleic acid (RNA) include adenine, uracil, guanine, and cytosine. Examples of deoxyribonucleic acid (DNA) include adenine, thymine, guanine, and cytosine.

Examples of sugars include sucrose, glucose and galactose.

Examples of minerals include phosphoric acid, phosphate, alkali metal (eg, sodium, potassium) hydrochloride or sulfate, alkaline earth metal (eg, calcium) hydrochloride or sulfate sulfate. ..

The void-forming substance may be at least one substance selected from the group consisting of other proteins, sugars, nucleic acids, lipids and minerals.

The content of the void-forming substance in the protein solution is 1 part by mass or more, 2 parts by mass or more, 3 parts by mass or more, 4 parts by mass or more, 5 parts by mass or more, and 10 parts by mass or more with respect to 100 parts by mass of the target protein. , 15 parts by mass or more, 20 parts by mass or more, 30 parts by mass or more, 40 parts by mass or more, 60 parts by mass or more, 80 parts by mass or more or 100 parts by mass or more, 40 parts by mass or less, 50 parts by mass or less, It may be 80 parts by mass or less or 100 parts by mass or less. The content of the void-forming substance in the protein solution is 1 part by mass or more and 100 parts by mass or less, 2 parts by mass or more and 100 parts by mass or less, 3 parts by mass or more and 100 parts by mass or less, 4 parts by mass with respect to 100 parts by mass of the target protein. Parts or more and 100 parts by mass or less, 5 parts by mass or more and 100 parts by mass or less, 10 parts by mass or more and 80 parts by mass or less, 15 parts by mass or more and 80 parts by mass or less, 20 parts by mass or more and 50 parts by mass or less, or 40 parts by mass or more and 50 parts by mass It may be less than or equal to a part.

(solvent)
The protein solution contains a solvent. The solvent may be any solvent that dissolves the target protein. Solvents include, for example, hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and urea, guanidine, sodium dodecylsulfate (SDS), Examples thereof include an aqueous solution containing lithium bromide, calcium chloride, lithium thiocyanate and the like. These solvents may be used alone or in combination of two or more.

(Protein solution)
The protein solution contains a protein of interest, a void-forming substance and a solvent. The protein solution can be obtained by dissolving the target protein and the void-forming substance in a solvent. There is no limitation on the order in which the target protein and the void-forming substance are dissolved, and the target protein may be dissolved in a solvent and then the void-forming substance may be dissolved in the solution. The target protein may be dissolved, or the target protein and the void-forming substance may be dissolved together in a solvent. When preparing the protein solution, the insoluble fraction may be removed by filtration, if necessary.

The content of the target protein in the protein solution may be 1% by mass or more, 10% by mass or more, 15% by mass or more, or 20% by mass or more, and 30% by mass or less, 35% by mass, based on the total amount of the protein solution. % Or less, or 40% by mass or less. The content of the target protein may be 1 to 40% by mass, preferably 10 to 40% by mass, and more preferably 15 to 35% by mass with respect to the total amount of the protein solution.

The protein solution may further contain a dissolution accelerator. Examples of the dissolution accelerator include the following inorganic salts composed of Lewis acid and Lewis base. Examples of the Lewis base include oxoacid ion (nitrate ion, perchlorate ion, etc.), metal oxoacid ion (permanganate ion, etc.), halide ion, thiocyanate ion, cyanate ion, and the like. Examples of the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions. Specific examples of the inorganic salt composed of Lewis acid and Lewis base include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate, calcium chloride, and calcium bromide. , Calcium salts such as calcium iodide, calcium nitrate, calcium perchlorate, and calcium thiocyanate, iron chlorides, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron salts such as iron thiocyanate, Aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate, potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and potassium thiocyanate. Potassium salts, sodium chloride, sodium bromide, sodium iodide, sodium nitrate, sodium perchlorate, and sodium salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, perchloric acid, etc. Zinc and zinc salts such as zinc thiocyanate, magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium salts such as magnesium thiocyanate, barium chloride, barium bromide, barium iodide, Examples thereof include barium salts such as barium nitrate, barium perchlorate, and barium thiocyanate, and strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate.

The content of the dissolution accelerator is 1.0 part by mass or more, 5.0 part by mass or more, 9.0 part by mass or more, 15 part by mass or more, or 20.0 part by mass with respect to 100 parts by mass of the total amount of the target protein. That may be the above. The content of the dissolution accelerator may be 40 parts by mass or less, 35 parts by mass or less, or 30 parts by mass or less with respect to 100 parts by mass of the total amount of the target protein.

When producing the protein solution according to this embodiment, it may be heated to 30 to 90 ° C. The solubilizable temperature may be set in a timely manner according to the solvent used, the target protein, the type of void-forming substance, and the like. It may be shaken and stirred to promote dissolution.

The viscosity of the protein solution according to this embodiment may be appropriately set according to the use of the protein solution and the like. For example, when the protein solution according to the present embodiment is used as a spinning stock solution, its viscosity may be appropriately set according to the spinning method, and is, for example, 100 to 15,000 cP (centipoise) and 100 to 50,000 at 35 ° C. It may be set, and may be set to 100 to 30,000 cP (centipoise) and 100 to 50,000 at 40 ° C. The viscosity of the protein solution can be measured using, for example, the trade name "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

(Target protein molded product)
The shape of the target protein molded product is not particularly limited, and may be, for example, a fiber, a film, a porous body, or the like.

The content of the target protein in the target protein molded product is, for example, 50% by mass or more, 60% by mass or more, 80% by mass or more, 85% by mass or more with respect to 100% by mass (W / W%) of the target protein molded product. , 90% by mass or more, 95% by mass or more, 96% by mass or more, 97% by mass or more, 98% by mass or more, or 99% by mass or more. The content of the target protein in the target protein molded product can be appropriately adjusted by, for example, adjusting the content of the void-forming substance in the protein solution.

When the target protein molded product contains a void-forming substance, the content of the void-forming substance in the target protein molded product is, for example, more than 1% by mass and 2% by mass with respect to 100% by mass (W / W%) of the target protein. % Or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, or 40% by mass or more, 100% by mass or less, 80% by mass. % Or less, 50% by mass or less, or 40% by mass or less. The content of the void-forming substance in the target protein molded product can be adjusted within the above numerical range by, for example, adjusting the content of the void-forming substance in the protein solution.

The method for measuring the content of the void-forming substance in the target protein molded product is not particularly limited, and for example, a method using an electrophoresis apparatus, an ICP emission spectrometer, a spectrophotometer, or the like, an electronic balance (for example, UX6200H manufactured by Shimadzu Corporation). ) Is used to quantify.

The target protein molded product may contain more than 1% by volume of voids with respect to 100% by volume of the target protein. The volume of voids contained in the target protein compact is, for example, more than 1% by volume, 2% by volume or more, 3% by volume or more, 4% by volume or more, and 5 volumes with respect to 100% by volume (V / V%) of the target protein. % Or more, 10% by volume or more, 15% by volume or more, 20% by volume or more, or 40% by volume or more, and 50% by volume or less, or 40% by volume or less.

The method for measuring the volume of voids contained in the target protein molded product is not particularly limited, and examples thereof include an electronic balance, a density meter, and an optical microscope.

The birefringence of the protein article of interest is reduced, for example, by including voids. The birefringence of the target protein molded product is, for example, 95% or less, less than 95%, 90% or less, 85% or less, 80% when the birefringence of the target protein molded product having no void is 100%. Below, it may be 75% or less, 70% or less, 65% or less, 60% or less, or 55% or less, and may be 20% or more, 30% or more, 40% or more, or 50% or more.

The method for measuring the birefringence of the target protein molded product is not particularly limited, and examples thereof include a polarizing microscope. When using a polarizing microscope: BX51 (OLYMPUS), retardation measurement software: WPA-View Ver.2.4.2.9 (Photonic) Lattice, Inc.) can be used to calculate.

When the molded product is a fibrous molded product (protein fiber), the spinning method includes wet spinning and the like. When a protein solution is applied to a coagulation solution as a spinning stock solution, the protein coagulates. At this time, by applying the protein solution to the coagulating liquid as a thread-like liquid, the protein can be coagulated into a thread-like form to form an undrawn thread. The undrawn yarn can be formed, for example, according to the method described in Japanese Patent No. 5584932.

The coagulating solution may be any solution that can be desolvated. The coagulation liquid is preferably a lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, or acetone. The coagulant may contain water. The temperature of the coagulating liquid is preferably 5 to 30 ° C. from the viewpoint of spinning stability.

The method of applying the protein solution as a filamentous liquid is not particularly limited, and examples thereof include a method of extruding the protein solution from a spinning mouthpiece into a coagulating liquid in a solvent-removing tank. Undrawn yarn is obtained by coagulation of the protein. The extrusion speed when extruding the protein solution into the coagulation liquid can be appropriately set according to the diameter of the mouthpiece, the viscosity of the protein solution, etc., but for example, in the case of a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm, spinning From the viewpoint of 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 (coagulant tank) for containing the coagulant is not particularly limited, but may be, for example, 200 to 500 mm. The take-up speed of the undrawn yarn formed by the coagulation of the protein may be, for example, 1 to 14 m / min, and the residence time may be, for example, 0.01 to 0.15 min. The take-up speed of the undrawn yarn is preferably 1 to 3 m / min from the viewpoint of solvent removal efficiency. The undrawn yarn formed by coagulation of the protein may be further stretched (pre-stretched) in the coagulation liquid, but from the viewpoint of suppressing evaporation of the lower alcohol used in the coagulation liquid, the coagulation liquid is kept at a low temperature and is not yet drawn. It is preferable to take it from the coagulating liquid in the state of drawn yarn.

It is also possible to include a step of further drawing the undrawn yarn obtained by the above-mentioned method. The stretching may be one-step stretching or multi-step stretching of two or more steps. When drawn in multiple stages, the molecules can be oriented in multiple stages and the total draw ratio can be increased, which is suitable for producing fibers with high toughness.

When the molded product is a film-shaped molded product (protein film), the method for forming the protein into a film is not particularly limited, and the protein solution is applied to a solvent-resistant flat plate to a predetermined thickness to form a coating film. A method of obtaining a film having a predetermined thickness or the like can be used by forming the film and removing the solvent from the coating film. As the flat plate on which the coating film is formed, a flat plate resistant to a solvent such as a glass plate is used. The thickness of the coating film is not particularly limited, but may be, for example, 1 to 1000 μm. The protein film can be produced, for example, according to the method described in Japanese Patent No. 5678283.

The porous molded body (protein porous body) is not particularly limited as long as the conditions for obtaining the porous body as the molded body are not particularly limited. For example, the method described in Japanese Patent No. 5796147 using the above protein solution. It can be manufactured according to the above.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples.

[Manufacturing of modified fibroin]
[(1) Preparation of expression strain (recombinant cell) of target protein]
A nucleic acid encoding fibroin (spider silk fibroin) (PRT799) having a sequence derived from spider silk having the amino acid sequence shown in SEQ ID NO: 15 was synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The hydropathic index and molecular weight of each protein are as shown in Table 4.

This nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into a protein expression vector pET-22b (+) to obtain an expression vector. Escherichia coli BLR (DE3) was transformed with a pET-22b (+) expression vector containing a nucleic acid encoding PRT799 to obtain transformed Escherichia coli (recombinant cells) expressing the target protein.

[(2) Expression of target protein]
The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 5) containing ampicillin so that OD _{600 was 0.005.} The culture solution temperature was maintained at 30 ° C., _{and flask culture was carried out until the OD 600} reached 5, (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to the jar fermenter to which 500 mL of the production medium (Table 6) was added so that the OD _{600 was 0.05.} The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Moreover, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

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

[(3) Purification of protein]
The cells collected 2 hours after the addition of 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 about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL and 30 at 60 ° C. Stir for minutes with a stirrer to dissolve. 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, water was removed by a freeze-dryer, and the freeze-dried powdered target protein (PRT799) was recovered.

[Preparation of undiluted spinning solution and molded product]
The following materials were prepared as void-forming substances.
・ Lipid (cell extract)
・ Sucrose (reagent / special grade, manufactured by Wako Pure Chemical Industries, Ltd.)
-Phosphoric acid (reagent-99% grade, manufactured by Sigma-Aldrich)
-Host cell powder (sample containing proteins other than the target protein, lipids, sugars and minerals, dried cultured cells)
・ Deoxyribonucleic acid (reagent, deoxyribonucleic acid derived from fish sperm, manufactured by Sigma-Aldrich)

A predetermined amount of formic acid (solvent) was weighed in a screw tube bottle made of Pyrex (registered trademark) glass. The target protein is added to the solvent, weighed to the desired concentration (26% by mass at the time of dissolution), and at the same time, various void-forming substances are added so as to have the content (mass%) shown in Table 7 or Table 8. And put it in a screw tube bottle. Subsequently, DMSO containing the target protein and various void-forming substances was stirred with a stirrer for 1 hour or more under the condition of a temperature of 40 ° C. The obtained solution was defoamed by a centrifuge (conditions: 2000 rpm, 30 minutes or more). As a result, a spinning stock solution (protein solution) containing the target protein and the void-forming substance was prepared. Further, a protein solution for the protein fiber of the comparative example was prepared in the same manner as described above except that the void-containing substance was not added. The viscosity of each protein solution was adjusted to 5000 cp. Viscosity measurement was performed using an EMS viscometer. The content (unit: mass%) shown in Tables 7 to 8 and FIGS. 5 to 8 is the content with respect to 100% by mass of the protein molded product.

The undiluted spinning solution is filtered at 40 ° C. (heater temperature) with a filter having an opening of 1 μm, and then defoamed (defoaming conditions: 30 seconds, 2000 rpm), and then the gear pump is operated from the nozzle at 40 ° C. (heater temperature). It was discharged into the coagulation liquid (methanol). The protein after coagulation was spun under the following spinning conditions to obtain protein fibers (fibroin fibers) of Examples and Comparative Examples shown in Table 7 or Table 8. In the process of spinning the protein after coagulation, washing was carried out using a washing bath (washing bath solution: water) except for Example 5-2.
Spinning conditions (first solidification bath)
Coagulant: Methanol (Temperature: 5 ° C)
(Hot roller)
Temperature: 55 ° C
Total draw ratio: 5.17 times (washing bath)
Washing bath liquid: water (implemented except for Example 5-2)

The measurement results of the birefringence of protein fibers are shown in Tables 7-8. The birefringence is correlated with the voids present in the protein fibers, and it is known that the value of the birefringence decreases when the voids are formed. The birefringence was calculated using a polarizing microscope: BX51 (OLYMPUS) and retardation measurement software: WPA-View Ver.2.4.2.9 (Photonic Lattice, Inc.). The birefringence of the protein compact of each example is the birefringence when the birefringence of the void-forming substance and the protein compact of Comparative Example 1 is 100%.

The gloss evaluation was performed visually. In the gloss evaluation, it was evaluated as "-" when there was gloss, "+" when the gloss was suppressed as compared with Comparative Example 1, and "++" when there was no gloss.

FIGS. 4-9 are micrographs of protein fibers and scanning electron microscope (SEM) photographs of the surface structure (skin layer) and internal structure (cut surface). FIG. 10 is a photograph of protein fibers of Examples and Comparative Examples. In FIG. 10, A is Comparative Example 1 (control), B is Example 1-1 (DNA), C to D are Examples 2-1 and Example 2-2 (lipid), and E to F are carried out, respectively. Examples 3-1 to 3-2 (sucrose) and GH are from Examples 4-1 and 4-2 (phosphoric acid), respectively, and I to J are from Examples 5-1 and 5-2 (dried cells), respectively. A photograph of protein fibers is shown.

As shown in FIGS. 5 to 9, when protein fibers were molded using a spinning stock solution containing a void-forming substance, it was confirmed that voids were formed in the obtained protein fibers.

As shown in Tables 7 to 8 and FIG. 10, the protein fibers obtained by using the spinning stock solution containing the void-forming substance have suppressed gloss as compared with the protein fibers obtained by using the spinning stock solution containing no void-forming substance. Was there. When the birefringence was 95% or more, the protein fibers were glossy. On the other hand, when the birefringence is less than 95% and 75% or more, the gloss of the protein fiber is suppressed (compared to Comparative Example 1), and when the birefringence is less than 75%, the protein fiber is glossy. There wasn't.

Claims (12)

A method for producing a protein molded product, comprising a step of forming a molded product of the target protein using a protein solution containing the target protein, a void-forming substance, and a solvent. The method for producing a protein molded product according to claim 1, wherein the molded product is a fiber. The method for producing a protein molded product according to claim 1 or 2, wherein the void-forming substance is at least one selected from the group consisting of proteins other than the target protein, lipids, sugars, nucleic acids and minerals. The method for producing a protein molded product according to any one of claims 1 to 3, wherein the content of the void-forming substance in the protein solution is 3 parts by mass or more with respect to 100 parts by mass of the target protein. .. The protein molded product according to any one of claims 1 to 4, wherein the content volume of the void in the protein molded product is 3% by volume or more with respect to 100% by volume of the target protein in the protein molding product. Manufacturing method. The method for producing a protein molded product according to any one of claims 1 to 5, wherein the target protein is a structural protein. The method for producing a protein molded product according to claim 6, wherein the structural protein is spider silk fibroin. Contains target proteins and void-forming substances
A target protein molded product in which the content of the void-forming substance is more than 1% by mass with respect to 100% by mass of the target protein. Contains the protein of interest
A target protein molded product containing more than 1% by volume of voids with respect to 100% by volume of the target protein. The target protein molded product has a birefringence of less than 95% when the birefringence of the target protein molded product containing no void is 100%. The target protein molded product according to any one of claims 8 to 10, wherein the target protein is a structural protein. The target protein molded product according to claim 11, wherein the structural protein is spider silk fibroin.

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