JP7308507B2 - Method for producing and analyzing recombinant structural protein, and method for producing protein compact - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing and analyzing a recombinant structural protein having a desired molecular weight, a method for producing a protein compact, a diluent for a sparingly soluble protein solution, and a sparingly soluble protein diluent.

Methods for purifying structural proteins (for example, spider silk proteins) include a method using a metal hydroxide such as sodium hydroxide (Patent Document 1) and formic acid or propionic acid for a suspension of host cells. (Patent Document 2), and a method including adding and dissolving an aprotic polar solvent such as dimethyl sulfoxide to the host and separating insoluble matter to obtain a solution (Patent Document 3) have been reported.

Japanese translation of PCT publication No. 2013-523665 Japanese Patent Publication No. 2004-503204 WO2014/103847

In the methods described in Patent Documents 1 and 2, structural proteins with different molecular weights that are present together with the target structural protein (for example, proteins whose translation into proteins has stopped midway, proteins that have been decomposed in the host, etc.) are removed. Therefore, there is a problem that structural proteins having different molecular weights remain as impurities in the isolated structural protein of interest. Further, in the method using an organic acid, there is a problem that the structural proteins that can be isolated are limited because structural proteins that are not resistant to acids are easily degraded.

On the other hand, the method described in Patent Document 3 can purify the target structural protein to about 70%. However, the target structural proteins are limited to hydrophilic recombinant proteins with a hydropathic index (HI) of 0 or less, and structural proteins with different molecular weights still remain, so extremely high purity is required. There is a problem that it is difficult to apply it as it is to fields such as pharmaceuticals and medical supplies.

An object of the present invention is to provide a method for producing a recombinant structural protein having a desired molecular weight, by which the desired structural protein can be obtained with high purity.

As a result of intensive studies, the present inventors prepared a recombinant structural protein solution in which the target recombinant structural protein was dissolved using a dissolving solvent capable of dissolving the target recombinant structural protein, and further containing water. While suppressing gelation of the solution by diluting the solution with a mixed solvent, a recombinant structural protein having a desired molecular weight is subjected to molecular weight fractionation with a porous gel and fractionated to obtain an extremely The present inventors have found that the desired structural protein can be purified with a high degree of purity, and have completed the present invention.

That is, the present invention provides, for example, the following inventions.
[1]
(A) preparing a recombinant structural protein solution in which the recombinant structural protein is dissolved in a solvent for dissolution;
(B) diluting the recombinant structural protein solution prepared in step (A) with a mixed solvent containing water;
(C) a step of passing the recombinant structural protein solution diluted in step (B) through a column filled with a porous gel to fractionate the molecular weight;
(D) collecting a fraction containing the recombinant structural protein having the desired molecular weight.
[2]
The production method of [1], wherein the dissolving solvent is at least one solvent selected from the group consisting of formic acid and aprotic polar solvents.
[3]
The mixed solvent contains water and at least one solvent selected from the group consisting of acetonitrile, isopropanol, N-methyl-2-pyrrolidone (NMP), and dimethylsulfoxide (DMSO) [1] or [2 ] manufacturing method.
[4]
The production method according to any one of [1] to [3], wherein in step (A), the recombinant structural protein solution is collected and host cells and/or host cell-derived contaminants are removed or reduced.
[5]
The production method according to any one of [1] to [4], wherein the weight ratio of water in the mixed solvent is 20 to 80%.
[6]
The production method according to any one of [1] to [5], wherein the dissolution solvent and/or the mixed solvent further contain a salt.
[7]
The production method according to any one of [1] to [6], wherein the porous gel is silica gel.
[8]
The production method according to any one of [1] to [7], wherein the recombinant structural protein is a spider silk protein.
[9]
The production method according to any one of [1] to [8], wherein the recombinant structural protein recovered in step (D) has a purity of 90% or more.
[10]
(A) preparing a recombinant structural protein solution in which the recombinant structural protein is dissolved in a solvent for dissolution;
(B) diluting the recombinant structural protein solution prepared in step (A) with a mixed solvent containing water;
(C) a step of passing the recombinant structural protein solution diluted in step (B) through a column filled with a porous gel to fractionate the molecular weight;
(D) collecting the fraction containing the recombinant structural protein having the desired molecular weight;
(E) A method for producing a protein molded product, comprising the step of molding the recombinant structural protein recovered in step (D) to obtain a molded product.
[11]
The method for producing a protein molded product of [10], wherein in step (E), the recombinant structural protein is molded into a protein fiber.
[12]
A diluent for a poorly soluble protein solution containing a mixed solvent containing water, wherein the poorly soluble protein solution is a solution in which a poorly soluble protein is dissolved in a dissolution solvent, and the diluent is a poorly soluble protein A diluent that can dilute the solution while the poorly soluble protein is dissolved.
[13]
The diluent of [12], wherein the dissolving solvent is at least one solvent selected from the group consisting of formic acid and aprotic polar solvents.
[14]
[12] or [13], wherein the mixed solvent contains water and at least one solvent selected from the group consisting of acetonitrile, isopropanol, N-methyl-2-pyrrolidone (NMP), and dimethylsulfoxide (DMSO); ] diluent.
[15]
The diluent of any one of [12]-[14], wherein the sparingly soluble protein is a spider silk protein.
[16]
A poorly soluble protein diluent comprising a poorly soluble protein, a dissolving solvent for dissolving the poorly soluble protein, and the diluent according to any one of [12] to [15].
[17]
(A) preparing a recombinant structural protein solution in which the recombinant structural protein is dissolved in a solvent for dissolution;
(B) diluting the recombinant structural protein solution prepared in step (A) with a mixed solvent containing water;
(C) a step of passing the recombinant structural protein solution diluted in step (B) through a column filled with a porous gel to fractionate the molecular weight;
(D) collecting a fraction containing a recombinant structural protein having a desired molecular weight; and (F) analyzing the collected fraction in step (D) with a detector. Methods for analysis of recombinant structural proteins.
[18]
The analytical method of [17], wherein the detector comprises a mass spectrometer.
[19]
The analytical method of [17] or [18], wherein the recombinant structural protein is a spider silk protein.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for producing a recombinant structural protein having a desired molecular weight, which enables obtaining the target recombinant structural protein with high purity.

According to the method for producing a recombinant structural protein having a desired molecular weight according to the present invention, the purity of the recombinant structural protein obtained is high, and trace impurities such as pharmaceuticals and medical supplies (same structural protein but different molecular weight It can also be used in fields that must be removed even if it is (including cases). In addition, when the obtained recombinant structural protein is, for example, a spider silk protein, it can be used for production such as spinning and film formation, and the recombinant structural protein designed to have a predetermined molecular weight (for example, spider silk It can also be usefully utilized when analyzing the properties of protein).

1 is a schematic diagram showing an example of a fibroin domain sequence. FIG. 1 is a schematic diagram showing an example of a fibroin domain sequence. FIG. 1 is a schematic diagram showing an example of a fibroin domain sequence. FIG. FIG. 4 is a photograph showing the results of analysis of the purified protein powder obtained in Reference Example 1 by polyacrylamide gel electrophoresis (SDS-PAGE).

DETAILED DESCRIPTION OF THE INVENTION Embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[Method for Producing Recombinant Structural Protein Having Desired Molecular Weight]
The method for producing a recombinant structural protein having a desired molecular weight according to the present embodiment includes (A) the step of preparing a recombinant structural protein solution in which the recombinant structural protein is dissolved in a dissolution solvent; A) a step of diluting the recombinant structural protein solution prepared in step (C) with a mixed solvent containing water; and (C) passing the recombinant structural protein solution diluted in step (B) through a column filled with a porous gel. and (D) recovering the fraction containing the recombinant structural protein having the desired molecular weight.

[Step (A)]
Step (A) is a step of preparing a recombinant structural protein solution in which a recombinant structural protein is dissolved in a solvent for dissolution.

The recombinant structural protein solution may contain the recombinant structural protein of interest. The recombinant structural protein solution is usually a recombinant structural protein of the same type as the target recombinant structural protein, but with a different molecular weight (e.g., protein whose translation into protein is interrupted, (in the host) disassembled, etc.). The recombinant structural protein solution may contain other contaminants (eg, host cell-derived contaminants).

As the recombinant structural protein to be used as a raw material, for example, host cells expressing the recombinant structural protein of interest or lysate or lysate of the host cells may be used. A solubilized fraction containing the recombinant structural protein of interest obtained from the liquid may be used, or the recombinant structural protein is purified from host cells expressing the recombinant structural protein of interest as described below. Alternatively, crudely purified recombinant structural proteins may be used.

(solvent for dissolution)
The solvent for dissolution can be used without particular limitation as long as it can dissolve the target recombinant structural protein, and may be an organic solvent. Examples of the solvent for dissolution include at least one solvent selected from the group consisting of formic acid and aprotic polar solvents, and formic acid is preferred. The dissolution solvent may further contain a salt.

The aprotic polar solvent is not particularly limited, but preferably has a purity of 90% or more, more preferably 95% or more, from the viewpoint of reducing contaminants. The aprotic polar solvent is preferably an aprotic polar solvent that does not have a cyclic structure. Recombinant structural proteins can be extracted with higher purity using non-cyclic aprotic polar solvents. Aprotic polar solvents having no cyclic structure include dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and the like.

Moreover, the aprotic polar solvent preferably has a dipole moment of 3.0 D or more. The dipole moment is used as an indicator of the strength of the polarity of a molecule, and the greater the dipole moment, the greater the strength of the polarity. Molecules having a dipole moment of 3.0 D or more are generally considered to have a strong polarity, and a solvent having a large polarity is effective in dissolving a polar compound such as a protein. Table 1 below lists the dipole moments of various organic compounds (organic solvents) based on Solvent Handbook (2007) published by Kodansha Scientific. Aprotic polar solvents having a dipole moment of 3.0 D or more include dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl-2 -imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile and the like.

From the viewpoint of high extraction purity of the recombinant structural protein, the aprotic polar solvent preferably has a dipole moment of 3.0 D or more and does not have a cyclic structure. , N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMA).

The dissolution solvent may contain a salt. Containing a salt makes the target recombinant structural protein more soluble and improves the separation performance of size exclusion chromatography, which will be described later, so that a recombinant structural protein of higher purity can be obtained.

Salts contained in the solvent for dissolution include, for example, alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, thiocyanates, inorganic salts such as perchlorates, sodium trifluoroacetate ( _CF3 Organic salts such as COONa) and volatile salts such as ammonium acetate can be mentioned.

Examples of alkali metal halides include potassium bromide, sodium bromide, lithium bromide, potassium chloride, sodium chloride, lithium chloride, sodium fluoride, potassium fluoride, cesium fluoride, potassium iodide, sodium iodide, Lithium iodide etc. can be mentioned. Examples of alkaline earth metal halides include calcium chloride, magnesium chloride, magnesium bromide, calcium bromide, magnesium iodide and calcium iodide. Examples of alkaline earth metal nitrates include calcium nitrate, magnesium nitrate, strontium nitrate and barium nitrate. Examples of thiocyanates include sodium thiocyanate, ammonium thiocyanate, (guanidinium thiocyanate) and the like. Examples of perchlorates include ammonium perchlorate, potassium perchlorate, calcium perchlorate, silver perchlorate, sodium perchlorate and magnesium perchlorate.

One type of these salts may be used alone, or two or more types may be used in combination.

As the salt, alkali metal halides, alkaline earth metal halides and sodium trifluoroacetate are preferable, and lithium chloride, calcium chloride, sodium trifluoroacetate and ammonium acetate are more preferable.

When a salt is added, the optimum amount may be determined according to the type of dissolution solvent used. can. The upper limit of the amount of salt added may be, for example, 0.7 M or less, 0.6 M or less, or 0.5 M or less, and the lower limit of the salt addition amount is 0.05 M or more, 0.1 M or more, or 0 .2M or more.

(recombinant structural protein)
Structural proteins refer to proteins that form biological structures or proteins derived from them. A recombinant structural protein is a structural protein produced by genetic recombination techniques. The recombinant structural protein may be a naturally-occurring structural protein, or a modified structural protein in which a part of the amino acid sequence is modified based on the amino acid sequence of the naturally-occurring structural protein.

Recombinant structural proteins include, for example, any structural proteins that are preferably produced on an industrial scale, and specific examples include structural proteins that can be used for industrial purposes, structural proteins that can be used for medical purposes, and the like. can be done. Specific examples of structural proteins that can be used for industrial or medical purposes include sparingly soluble proteins such as fibroin, collagen, resilin, elastin and keratin, and proteins derived from these. may be Fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin (spider silk protein), and hornet silk fibroin.

The fibroin according to this 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 the present embodiment is preferably spider silk fibroin (spider silk protein). Spider silk fibroin includes natural spider silk fibroin and modified fibroin derived from natural spider silk fibroin. Natural spider silk fibroin includes, for example, spider silk proteins produced by spiders.

The fibroin according to this embodiment has, for example, a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif It may be a protein containing The fibroin according to this embodiment may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but not limited to, regions that do not have repeated amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically corresponding to the REP of the amino acid sequence). ) and is represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif means an array. Here, (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. (A) The number of amino acid residues in _{the n} motif may be an integer of 2-20, 4-27, 4-20, 8-20, 10-20, 4-16, 8-16, or 10-16 . In addition, (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 only of alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed only of alanine residues. REP indicates an amino acid sequence composed of 2-200 amino acid residues. REP may be an amino acid sequence composed of 10-200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

Examples of naturally occurring fibroin include a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. Proteins containing Specific examples of naturally occurring fibroin include fibroin produced by insects or spiders.

Examples of fibroin produced by insects include, for example, Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, Japanese silkworm ( Pilosamia Cynthia ricini ), silk proteins produced by silkworms such as Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, and Vespa simillima xantoptera ) larvae exhale Hornet silk protein.

More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

Examples of fibroin produced by spiders include, for example, spiders belonging to the genus Araneus such as Araneus spiders, Araneus spiders, Red spiders, Green spiders, and Beetle spiders; Spiders belonging to the genus Pronus, spiders belonging to the genus Pronus such as spiders belonging to the Spiders belonging to the genus Gasteracantha (genus Gasteracantha), spiders belonging to the genus Ordgarius (genus Ordgarius) such as the genus Ordina and the spider, spiders belonging to the genus Argiope (genus Argiope) such as the argiope spider, the argiope spider, and the argiope spider, and the like Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas, such as spiders, spiders belonging to the genus Cytophora, such as spiders, spiders, and spiders Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as spiders belonging to the genus Cyclosa, spiders belonging to ), spiders belonging to the genus Cyclosa, and spiders belonging to the genus Chorizopes, such as spiders, and spiders belonging to the genus Chorizopes; Spiders belonging to the genus Tetragnatha such as the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucauge, such as the genus Leucauge, and the genus Nephila Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as golden spiders, spiders belonging to the genus Dyschiriognatha such as the brown spider, widow spiders such as the black widow spider, the redback spider, the gray widow spider and the western widow spider Examples include spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Latrodectus and spiders belonging to the genus Euprosthenops. Spider silk proteins include, for example, MaSp (MaSp1 and MaSp2), dragline proteins such as ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of spider silk proteins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence ), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [Nephila c lavata] (GenBank accession number AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spi droin 2 [Euprosthenops australis] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank Accession No. ABR37278.1 (amino acid sequence), and the like.

More specific examples of naturally occurring fibroin include fibroin whose sequence information is registered in NCBI GenBank. For example, among sequence information registered in NCBI GenBank, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION from among sequences containing INV as DIVISION. It can be confirmed by extracting the specific product character string from the sequence, CDS, and the sequence described with the specific character string from SOURCE to TISSUE TYPE.

Modified fibroin is, for example, one obtained by modifying the amino acid sequence based on the amino acid sequence of naturally occurring fibroin (for example, one obtained by modifying the amino acid sequence by modifying the cloned naturally occurring fibroin gene sequence). Alternatively, it may be one artificially designed and synthesized without relying on naturally occurring fibroin (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding a designed amino acid sequence). .

Modified fibroin is obtained by modifying the amino acid sequence by, for example, substituting, deleting, inserting and/or adding one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. can be obtained with Substitution, deletion, insertion and/or addition of amino acid residues can be performed by methods well known to those skilled in the art such as partial directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

Modified fibroin may be, for example, modified fibroin derived from silk protein produced by silkworms, or modified fibroin derived from spider silk protein produced by spiders.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the dragline silk protein produced in the major pituitary gland of spiders, and modified fibroin with reduced content of glycine residues. (second modified fibroin), (A) modified fibroin with reduced _n -motif content (third modified fibroin), glycine residue content, and (A) _n -motif content reduced A modified fibroin (fourth modified fibroin), a modified fibroin having a domain sequence containing a region with a locally large hydrophobicity index (fifth modified fibroin), and a domain sequence having a reduced content of glutamine residues modified fibroin (sixth modified fibroin);

Modified fibroin (first modified fibroin) derived from the major pituitary dragline protein produced in the major pituitary gland of spiders includes a domain sequence represented by Formula 1: [(A) _n motif-REP] _m Proteins containing In the first modified fibroin, in Formula 1, n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, even more preferably an integer of 10 to 20, 4 Integers from ˜16 are even more preferred, integers from 8 to 16 are particularly preferred, and integers from 10 to 16 are most preferred. In the first modified fibroin, the number of amino acid residues constituting REP in formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, 20 to 100 residues and even more preferably 20 to 75 residues. In the first modified fibroin, the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m is amino acid residue It is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more of the total number.

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

The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of the C-terminal 50 amino acids of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 has the sequence It is identical to the amino acid sequence shown in No. 1 with 20 residues removed from the C-terminus, and the amino acid sequence shown in SEQ ID No. 3 has 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID No. 1. Identical to the amino acid sequence.

As more specific examples of the first modified fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroins comprising amino acid sequences with greater than % sequence identity can be mentioned. Preferably, the sequence identity is 95% or greater.

The amino acid sequence shown by SEQ ID NO: 4 is an amino acid sequence (SEQ ID NO: 5) consisting of an initiation codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus of ADF3. The 13th repeat region was increased to approximately double and mutated so that translation stops at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown by SEQ ID NO:4 is identical to the amino acid sequence shown by SEQ ID NO:3.

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

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

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

In the second modified fibroin, the percentage of the motif sequence in which the glycine residue is substituted with another amino acid residue may be 10% or more of the entire motif sequence.

The second modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the domain sequence, the (A) _n motif located most C-terminal to the domain sequence Let z be the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus of the domain sequence When w is the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence, z/w is 30% or more, It may have an amino acid sequence that is 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative 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. 100% (meaning composed only of alanine residues) is even more preferred.

The second modified fibroin preferably has an increased content of the amino acid sequence consisting of XGX by substituting one glycine residue in the GGX motif with another amino acid residue. 6. In the second modified fibroin, the content of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, even more preferably 10% or less. % or less, even more preferably 4% or less, and particularly preferably 2% or less. 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.

A method for calculating z/w will be described in more detail. First, in a fibroin (modified fibroin or naturally occurring fibroin) containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , the (A) _n located on the most C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminus 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. In addition, for example, when there is an X (central X) contained in two XGX, as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues be). w is the total number of amino acid residues contained in the domain sequence, excluding the sequence from the (A) _n motif positioned most on the C-terminal side to the C-terminus of the domain sequence. For example, for the domain sequence shown in Figure 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the most C-terminal (A) _n motif). Then 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, even more preferably 58.7% or more, and 70% or more. and even more preferably 80% or more. Although the upper limit of z/w is not particularly limited, it may be, for example, 95% or less.

The second modified fibroin is obtained, for example, by substituting at least part of the nucleotide sequence encoding the glycine residue from the cloned naturally-derived fibroin gene sequence so as 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 substituted so that z/w is 50.9% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the alteration corresponding to replacing 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. , insertions and/or additions may be made to the amino acid sequence.

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

More specific examples of the second modified fibroin include (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (2-ii) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Mention may be made of modified fibroin.

The modified fibroin of (2-i) will be explained. The amino acid sequence shown by SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence shown by SEQ ID NO: 10 corresponding to naturally occurring fibroin with GQX. The amino acid sequence shown by SEQ ID NO: 7 is obtained by deleting every two (A) _n motifs from the amino acid sequence shown by SEQ ID NO: 6 from the N-terminal side to the C-terminal side, and furthermore, in front of the C-terminal sequence. [(A) _n motif-REP] is inserted into the . The amino acid sequence shown in SEQ ID NO: 8 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a partial glutamine (Q) residue. It is obtained by substituting serine (S) residues and deleting some amino acids on the C-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and a His tag sequence are added to the C-terminus of a sequence in which is repeated four times.

The z/w value in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z/w values in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are each 58.7%, 70.1%, 66.1% and 70.0%. In addition, the value of x/y in the jagged ratio (described later) of 1:1.8 to 11.3 of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is 15.0%, 15.0%, 93.4%, 92.7% and 89.8% respectively.

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

The modified fibroin (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, 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 Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and XGX contained in REP ( where X represents an amino acid residue other than glycine.), where z is the total number of amino acid residues in the amino acid sequence and w is the total number of amino acid residues of REP in the domain sequence, 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 C-terminus. This enables isolation, immobilization, detection, visualization, and the like of modified fibroin.

Examples of tag sequences include affinity tags that utilize specific affinity with other molecules. A specific example of the affinity tag is a histidine tag (His tag). His-tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has the property of specifically binding to metal ions such as nickel, so isolation of modified fibroin by metal chelating metal chromatography can be used for A specific example of the tag sequence is the amino acid sequence represented by SEQ ID NO: 11 (an amino acid sequence containing a His tag sequence and a hinge sequence).

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

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

Furthermore, a tag sequence that can be cleaved by a specific protease can also be used. The modified fibroin from which the tag sequence has been cut off can also be recovered by treating the protein adsorbed via the tag sequence with protease.

As more specific examples of modified fibroin containing a tag sequence, (2-iii) the amino acid represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) sequence, or (2-iv) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

The amino acid sequences shown in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in SEQ ID NO: 11 (including His tag sequence and hinge sequence) was added to the N-terminus of the amino acid sequence shown.

The modified fibroin of (2-iii) may consist of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(2-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is contained in REP ( where X represents an amino acid residue other than glycine.), where z is the total number of amino acid residues in the amino acid sequence and w is the total number of amino acid residues of REP in the domain sequence, z/w is preferably 50.9% or more.

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

(A) Modified fibroin with reduced _n- motif content (third modified fibroin) has a domain sequence of (A) amino acids with reduced _n -motif content compared to naturally occurring fibroin has an array. The domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to deletion of at least one or more (A) _n motifs compared to the naturally occurring fibroin.

A third modified fibroin may have an amino acid sequence corresponding to 10-40% deletion of the (A) _n motif from the naturally-occurring fibroin.

A third modified fibroin has a domain sequence at least one (A) _n motif for every 1-3 (A) _n motifs from the N-terminal side to the C-terminal side as compared to the naturally-occurring fibroin. may have an amino acid sequence corresponding to the deletion of

The third modified fibroin has at least two consecutive (A) _n motif deletions from the N-terminal side to the C-terminal side and one (A ) may have an amino acid sequence corresponding to deletion of _n motifs repeated in this order.

The third modified fibroin may have an amino acid sequence corresponding to deletion of at least every two (A) _n motifs from the N-terminal side to the C-terminal side of the domain sequence. .

A third modified fibroin comprises a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , with two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -REP] unit sequentially compares the number of amino acid residues of REP, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11. When the maximum value of the total sum of the numbers of amino acid residues of two adjacent [(A) _n motif-REP] units equal to 3 is x, and the total number of amino acid residues of the domain sequence is y Furthermore, 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 relative 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. 100% (meaning composed only of alanine residues) is even more preferred.

A method for calculating x/y will be described in more detail with reference to FIG. FIG. 1 shows the domain sequence of the modified fibroin with the N-terminal and C-terminal sequences removed. The domain sequence is, from the N-terminal side (left side), (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 a sequence called _n motif.

Two adjacent [(A) _n- motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, unselected [(A) _n motif-REP] units may be present. In FIG. 1, pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP and the fourth REP), pattern 2 (comparison of the first REP and the second REP, and comparison of the fourth REP and the fifth REP), pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP and the fifth REP), pattern 4 (comparison of the first REP and A second REP comparison) was shown. Note that 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. Comparison is carried out by determining the ratio of the number of amino acid residues to the number of amino acid residues of the other. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the second REP is The ratio of amino acid residue numbers is 100/50=2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with fewer amino acid residues is set to 1, the fifth REP is 30/20=1.5.

In FIG. 1, a set of [(A) _n motif-REP] units having a ratio of 1.8 to 11.3 for the number of amino acid residues of the other is set to 1 with the smaller number of amino acid residues. It is indicated by a solid line. Such a ratio is hereinafter referred to as a serration ratio. A set of [(A) _n motif-REP] units in which the other amino acid residue number ratio is less than 1.8 or greater than 11.3 when the number of amino acid residues is less than 1 is indicated by a dashed line. Indicated.

In each pattern, the numbers of all amino acid residues of two adjacent [(A) _n motif-REP] units indicated by solid lines are summed up (not only REP but also the number of amino acid residues of (A) _n motifs). be.). Then, the added total values are compared, and the total value (maximum total value) of the pattern with the maximum total value is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the largest.

Then 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, even more preferably 65% or more, and even more preferably 70% or more. Preferably, 75% or more is even more preferable, and 80% or more is particularly preferable. The upper limit of x/y is not particularly limited, and may be, for example, 100% or less. When the serration ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the serration ratio is 1:1.8 to 3.4, x /y is preferably 77.1% or more, and when the serration ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and the serration ratio is 1 : 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 only of alanine residues, x/y is 46.4% or more. is preferably 50% or more, more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and 80% or more It is particularly preferred to have The upper limit of x/y is not particularly limited as long as it is 100% or less.

The third modified fibroin, for example, deletes one or more of the (A) _n motif-encoding sequences from the cloned naturally occurring fibroin gene sequence such that x/y is 64.2% or more. can be obtained by Alternatively, for example, an amino acid sequence corresponding to deletion of one or more (A) _n motifs is designed such that x/y is 64.2% or more 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 described above. In any case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are substituted, deleted, inserted and/or added. Alterations in the amino acid sequence corresponding to what has been done may be made.

More specific examples of the third modified fibroin include (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Mention may be made of modified fibroin.

The modified fibroin of (3-i) will be explained. The amino acid sequence represented by SEQ ID NO: 17 is every two (A) _n The motif was deleted and one [(A) _n motif-REP] was inserted in front of the C-terminal sequence. The amino acid sequence shown by SEQ ID NO: 7 is obtained by substituting all GGX in REP of the amino acid sequence shown by SEQ ID NO: 18 with GQX. The amino acid sequence shown in SEQ ID NO: 8 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a partial glutamine (Q) residue. It is obtained by substituting serine (S) residues and deleting some amino acids on the C-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). A His tag is added to the C-terminus of a sequence consisting of 4 repeats.

The x/y value of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) is 15.0% at the jagged ratio of 1:1.8 to 11.3. The x/y values of the amino acid sequence shown by SEQ ID NO: 18 and the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%. The z/w values in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.0%, respectively. 1% and 70.0%.

The modified fibroin of (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, 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 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 shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and from the N-terminal side to the C-terminal side , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Amino acid residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (Giza ratio is 1:1.8 to 11.3) It is preferable that x/y is 64.2% or more, where x is the maximum sum of the cardinal numbers and y is the total number of amino acid residues in the domain sequence.

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

As more specific examples of modified fibroin containing a tag sequence, (3-iii) the amino acid represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) sequence, or (3-iv) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 have SEQ ID NO: 11 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in (including His tag sequence and hinge sequence) is added.

The modified fibroin of (3-iii) may consist of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

(3-iv) modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(3-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Let x be the maximum value of the total sum of the amino acid residue numbers of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3. , x/y is preferably 64.2% or more, where y is the total number of amino acid residues in the domain sequence.

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

A modified fibroin with a reduced content of glycine residues and a reduced content of (A) _n motifs (fourth modified fibroin) has a domain sequence with (A) _n motif in addition to having a reduced content of glycine residues. The domain sequence of the fourth modified fibroin is that at least one or more (A) _n motifs are deleted, and at least one or more glycine residues in REP are deleted compared to the naturally occurring fibroin. It can be said to have an amino acid sequence corresponding to substitution with another amino acid residue. That is, the fourth modified fibroin consists of the above-described modified fibroin with reduced glycine residue content (second modified fibroin) and (A) modified fibroin with reduced _n motif content (third It is a modified fibroin that has the characteristics of modified fibroin. Specific aspects and the like are as described in the second modified fibroin and the third modified fibroin.

More specific examples of the fourth modified fibroin include (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410 ), the amino acid sequence shown in SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in . Specific embodiments of the modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

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

A region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

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

A fifth modified fibroin is obtained by replacing one or more amino acid residues in REP with amino acid residues having a higher hydrophobicity index than in naturally occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, one or more amino acid residues are substituted, deleted, inserted and / or added as compared to naturally occurring fibroin There may be alterations in the amino acid sequence corresponding to what has been done.

For the fifth modified fibroin, for example, one or more hydrophilic amino acid residues (eg, amino acid residues with a negative hydrophobicity index) in REP from the cloned naturally occurring fibroin gene sequence are replaced with hydrophobic amino acid residues. by substituting a group (for example, an amino acid residue with a positive hydrophobicity index) and/or by inserting one or more hydrophobic amino acid residues into REP. Further, for example, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acid residues in 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 any case, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acids in REP In addition to modifications corresponding to residue insertions, amino acid sequence modifications corresponding to substitutions, deletions, insertions and/or additions of one or more amino acid residues may also be made.

The fifth modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence Let p be the total number of amino acid residues contained in a region where the average value of the hydrophobic index of four consecutive amino acid residues is 2.6 or more in all REPs contained in the sequences excluding the sequences from the domain sequence, p/q is 6, where q is the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. It may have an amino acid sequence that is greater than or equal to .2%.

Hydropathy indexes of amino acid residues are known (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 157). 105-132). Specifically, the hydropathic index (hereinafter also referred to as “HI”) of each amino acid is as shown in Table 2 below.

A method for calculating p/q will be described in more detail. For the calculation, the sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence (hereinafter referred to as “sequence A”) is used. First, in all REPs contained in sequence A, the average value of the hydrophobic index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the sum of 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 four consecutive amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region is identified in which the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more. Even if a certain amino acid residue corresponds to multiple "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

For example, if 20 “continuous 4 amino acid residues with an average hydrophobic index of 2.6 or more” are extracted (no overlap), the average hydrophobic index of 4 consecutive amino acid residues is 2 A region that is 0.6 or greater will contain 20 consecutive 4-amino acid residues (no overlap), and p is 20×4=80. Further, for example, when two "consecutive 4 amino acid residues having an average hydrophobicity index of 2.6 or more" exist overlapping by one amino acid residue, the hydrophobic index of the consecutive 4 amino acid residues A region with an average of 2.6 or more contains 7 amino acid residues (p=2×4−1=7, where “−1” is duplicate subtraction). For example, in the case of the domain sequence shown in FIG. 2, there are seven non-overlapping “continuous 4 amino acid residues with an average hydrophobicity index of 2.6 or more”, so p is 7 × 4 = 28. 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 (not including the (A) _n motif present at the end of the C-terminal side). Then 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, even more preferably 10% or more, and 20% or more. Even more preferably, it is still more preferably 30% or more. Although the upper limit of p/q is not particularly limited, it may be, for example, 45% or less.

For the fifth modified fibroin, for example, the amino acid sequence of the cloned naturally occurring fibroin is modified so as to satisfy the p/q conditions described above, so that one or more hydrophilic amino acid residues in REP (eg, hydrophobicity index). negative amino acid residue) with a hydrophobic amino acid residue (e.g., an amino acid residue with a positive hydrophobicity index), and/or inserting one or more hydrophobic amino acid residues in REP can be obtained by locally modifying an amino acid sequence containing a region with a large hydrophobicity index. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above p/q conditions from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more amino acid residues in REP have been replaced with amino acid residues having a higher hydrophobicity index compared to naturally-occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, further modification corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues may be performed. .

Amino acid residues with a large hydrophobicity index are not particularly limited, but areoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.

As more specific examples of the fifth modified fibroin, (5-i) an amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665), or SEQ ID NO: 21 (Met-PRT666); or (5-ii) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified fibroin of (5-i) will be explained. The amino acid sequence shown by SEQ ID NO: 19 is an amino acid sequence consisting of 3 amino acid residues every other REP except for the terminal domain sequence on the C-terminal side with respect to the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410). The sequence (VLI) was inserted at two locations, and further, some glutamine (Q) residues were substituted with serine (S) residues, and some amino acids on the C-terminal side were deleted. The amino acid sequence shown in SEQ ID NO: 8 is the amino acid sequence shown in SEQ ID NO: 7, with two alanine residues inserted on the C-terminal side of each (A) _n motif, and a partial glutamine (Q) residue. A group is replaced with a serine (S) residue, and some amino acids on the C-terminal side are deleted. The amino acid sequence represented by SEQ ID NO: 20 is the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) in which an amino acid sequence (VLI) consisting of three amino acid residues is inserted at every other REP. be. The amino acid sequence shown by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 8.

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

The modified fibroin (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by 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 shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , 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 C-terminus.

More specific examples of modified fibroin containing a tag sequence include (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665), or SEQ ID NO: 24 (PRT666), or (5-iv ) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are added to the N-terminals of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, where the amino acid sequence shown in SEQ ID NO: 11 (His tag sequence and hinge sequence) are added.

The modified fibroin of (5-iii) may consist of the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

The modified fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by 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 shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

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

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

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

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

As used herein, the "GPGXX motif content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total by three (that is, the total number of G and P in the GPGXX motif) is defined as s, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is taken from the domain sequence. The GPGXX motif content rate is calculated as s/t, where t is the total number of amino acid residues in all REPs excluding (A) _n motifs.

In the calculation of the GPGXX motif content rate, the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is "the most C-terminal side (A) The sequence from _{the n} motif to the C-terminus of the domain sequence” (the sequence corresponding to REP) may include sequences that are poorly correlated with sequences characteristic of fibroin, and m is small In other words, when the domain sequence is short, it affects the calculation result of the GPGXX motif content rate, so this effect is to be eliminated. When a "GPGXX motif" is located at the C-terminus of REP, it is treated as a "GPGXX motif" even if "XX" is, for example, "AA".

FIG. 3 is a schematic diagram showing the domain sequence of fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. First, in the fibroin domain sequence (“[(A) _n motif-REP] _m -(A) _n motif” type) shown in FIG. A) GPGXX for calculating s because it is included in the "sequence obtained by removing the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence" (sequence shown as "region A" in FIG. 3) The number of motifs is 7, and s is 7×3=21. Similarly, all REPs are "sequences obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" (the sequence shown as "region A" in FIG. ), the total number of amino acid residues t of all REPs further 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% for fibroin in FIG.

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

As used herein, "glutamine residue content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), all contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence (sequence corresponding to “region A” in FIG. 3) REP, the total number of glutamine residues contained in the region is u, the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and (A) _n The glutamine residue content is calculated as u/t, where t is the total number of amino acid residues in all REPs excluding motifs. In the calculation of the glutamine residue content, the target is the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because of the reasons described above. It is the same.

A sixth modified fibroin corresponds to a domain sequence that lacks one or more glutamine residues in REP or replaces them with other amino acid residues as compared to the naturally-occurring fibroin. It may have an amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than a glutamine residue, but preferably an amino acid residue with a higher hydrophobicity index than that of a glutamine residue. Hydrophobicity indices of amino acid residues are shown in Table 2.

As shown in Table 2, amino acid residues having a higher hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H); can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

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

As used herein, the "hydrophobicity of REP" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring fibroin), all contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence (sequence corresponding to “region A” in FIG. 3) In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is v, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v/t, where t is the total number of amino acid residues in all REPs excluding _n motifs. In calculating the hydrophobicity of REP, the reason for targeting "the sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.

A sixth modified fibroin has a domain sequence lacking one or more glutamine residues in REP and/or one or more glutamine residues in REP compared to naturally-occurring fibroin In addition to the modification corresponding to the substitution of another amino acid residue, there may be modifications of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. .

A sixth modified fibroin is obtained, for example, by deleting one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence, and/or by deleting one or more glutamine residues in REP. can be obtained by substituting the amino acid residue of Also, for example, deletion of one or more glutamine residues in REP from the amino acid sequence of naturally occurring fibroin, and/or substitution of one or more glutamine residues in REP with other amino acid residues It can also be obtained by designing an amino acid sequence corresponding to , and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

More specific examples of the sixth modified fibroin include (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or sequence Modified fibroin comprising the amino acid sequence represented by number 42 (Met-PRT1028), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42, and a modified fibroin comprising an amino acid sequence having 90% or more sequence identity.

The modified fibroin of (6-i) will be explained. The amino acid sequence shown by SEQ ID NO:25 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:7 (Met-PRT410) with VL. The amino acid sequence shown by SEQ ID NO: 26 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with TS, and replacing the remaining Q with A. The amino acid sequence shown by SEQ ID NO: 27 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with VL, and replacing the remaining Q with I. The amino acid sequence shown by SEQ ID NO:28 is obtained by substituting VI for all QQs in the amino acid sequence shown by SEQ ID NO:7 and L for the remaining Qs. The amino acid sequence shown by SEQ ID NO: 29 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with VF, and replacing the remaining Q with I.

The amino acid sequence shown by SEQ ID NO:30 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:8 (Met-PRT525) with VL. The amino acid sequence shown by SEQ ID NO: 31 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 8 with VL, and replacing the remaining Q with I.

The amino acid sequence represented by SEQ ID NO: 32 consists of 20 domain sequence regions present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) that are repeated twice, and all QQ in the sequence are replaced with VF, And the remaining Q is replaced with I.

The amino acid sequence shown by SEQ ID NO: 41 (Met-PRT917) is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with LI and the remaining Q with V. The amino acid sequence shown by SEQ ID NO:42 (Met-PRT1028) is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:7 with IF, and replacing the remaining Q with T.

SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all glutamine residues The group content is below 9% (Table 3).

The modified fibroin of (6-i) is represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 It may consist of the amino acid sequence shown.

The modified fibroin of (6-ii) is represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 Amino acid sequences having 90% or greater sequence identity with the indicated amino acid sequences are included. The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin (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 C-terminus. This enables isolation, immobilization, detection, visualization, and the like of modified fibroin.

More specific examples of the sixth modified fibroin containing a tag sequence include (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), Modified fibroin comprising an amino acid sequence represented by SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028) , or (6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the sequence can be mentioned.

The amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are SEQ ID NO: 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 at the N-terminus of SEQ ID NO: 11 The amino acid sequence (including the His-tag sequence and hinge sequence) is added. Since the tag sequence was only added to the N-terminus, there was no change in the glutamine residue content, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 4).

The modified fibroin of (6-iii) is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It may consist of the amino acid sequence shown.

The modified fibroin of (6-iv) is represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 Amino acid sequences having 90% or greater sequence identity with the indicated amino acid sequences are included. The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

The modified fibroin has at least two or more of 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. It may be a modified fibroin having the characteristics of

Modified fibroin may be hydrophilic modified fibroin or hydrophobic modified fibroin. As used herein, the term "hydrophobic modified fibroin" refers to a value obtained by obtaining the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin, and then dividing the sum by the total number of amino acid residues. (Average HI) greater than 0 is a modified fibroin. The hydrophobicity index is as shown in Table 1. Further, "modified hydrophilic fibroin" is modified fibroin having an average HI of 0 or less. Hydrophilic modified fibroin is preferable as the modified fibroin from the viewpoint of excellent combustion resistance.

Examples of hydrophobically modified fibroin include amino acid sequences 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, SEQ ID NO: 33, or SEQ ID NO: 43, SEQ ID NO: 35, A modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44 is included.

Examples of modified hydrophilic fibroin include the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, and SEQ ID NO: 14. or the amino acid sequence represented by SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, the amino acid represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15 A modified fibroin comprising the amino acid sequence shown in the sequence SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 is included.

As a collagen-derived structural protein, for example, a protein comprising a domain sequence represented by Formula 3: [REP2] _p (here, in Formula 3, p represents an integer of 5 to 300. REP2 is Gly-X- represents an amino acid sequence composed of Y, where X and Y represent any amino acid residue other than Gly.A plurality of REP2s present may have the same amino acid sequence or different amino acid sequences.) can. Specifically, a protein comprising the amino acid sequence shown in SEQ ID NO:39 can be mentioned. The amino acid sequence represented by SEQ ID NO: 39 corresponds to the repeat portion and motif of the partial sequence of human collagen type 4 obtained from the NCBI database (NCBI GenBank accession number: CAA56335.1, GI: 3702452). The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 12 was added to the N-terminus of the amino acid sequence from the 301st to 540th residues.

As a resilin-derived structural protein, for example, a protein comprising a domain sequence represented by Formula 4: [REP3] _q (here, q represents an integer of 4 to 300 in Formula 4. REP3 is Ser-JJ -Tyr-Gly-U-Pro, J represents an arbitrary amino acid residue, preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr, U is an arbitrary is 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 comprising the amino acid sequence shown in SEQ ID NO:40 can be mentioned. The amino acid sequence represented by SEQ ID NO: 40 is obtained by replacing Thr at the 87th residue with Ser and Asn at the 95th residue in the amino acid sequence of resilin (NCBI GenBank accession number NP 611157, Gl: 24654243). The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 12 is added to the N-terminus of the amino acid sequence from the 19th residue to the 321st residue of the sequence with Asp substituted.

Structural proteins derived from elastin include, for example, proteins having amino acid sequences of NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine), and the like. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO:41 can be mentioned. The amino acid sequence shown by SEQ ID NO: 41 is the amino acid sequence shown by SEQ ID NO: 12 (tag sequence and hinge sequence) are added.

Structural proteins derived from keratin include, for example, type I keratin of Capra hircus. Specifically, a protein comprising the amino acid sequence represented by SEQ ID NO: 42 (amino acid sequence of accession number ACY30466 in GenBank of NCBI) can be mentioned.

(Recombinant expression of structural proteins)
A recombinant structural protein can be produced by, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the structural protein and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expression.

A method for producing a nucleic acid encoding a structural protein is not particularly limited. For example, using a gene encoding a structural protein such as natural fibroin, a method of amplifying and cloning by polymerase chain reaction (PCR) or the like, and modifying it by genetic engineering techniques as necessary, or chemically synthesizing The nucleic acid can be produced by the method described above. The method for chemically synthesizing the 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, etc., with AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.). A gene can be chemically synthesized by a method of linking automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and/or confirmation of the recombinant structural protein, a nucleic acid encoding a structural protein consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence. may be synthesized.

A regulatory sequence is a sequence that controls the expression of a recombinant structural protein in a host (eg, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected according to the type of host. As a promoter, an inducible promoter that functions in the host cell and can induce the expression of the recombinant structural protein may be used. An inducible promoter is a promoter whose transcription can be controlled by physical factors such as the presence of an inducer (expression inducer), the absence of a repressor molecule, or an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected from plasmid vectors, virus vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, etc., depending on the type of host. As the expression vector, those capable of autonomous replication in host cells or capable of integration into the chromosome of the host and containing a promoter at a position at which the nucleic acid encoding the recombinant structural protein can be transcribed are preferably used. .

As hosts, both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammonium philum. Microorganisms belonging to the genus Brevibacterium include, for example, Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

When prokaryotes are used as hosts, examples of vectors for introducing nucleic acids encoding structural proteins include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569) and the like.

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

When a eukaryotic host is used, examples of vectors for introducing nucleic acids encoding structural proteins include YEp13 (ATCC37115) and YEp24 (ATCC37051). Any method for introducing DNA into the host cell can be used as the method for introducing the expression vector into the host cell. 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.

As a method for expressing a 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, Second Edition. .

A recombinant structural 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 a method commonly used for culturing the host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the host so that the host can be efficiently cultured. Either a natural medium or a synthetic medium may be used as long as the medium is used.

Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism. Examples include carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch and starch hydrolysates, acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used. Nitrogen sources include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolysates, soybean meal and soybean meal hydrolysates, various fermented cells and their digests can be used. Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

Prokaryotes such as E. coli or eukaryotes such as yeast can be cultured under aerobic conditions such as shaking culture or deep aeration stir culture. The culture temperature is, for example, 15-40°C. Culture time is usually 16 hours to 7 days. The pH of the culture medium during cultivation is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and 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 necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using a lac promoter, and indole acrylic when culturing a microorganism transformed with an expression vector using a trp promoter. Acids and the like may be added to the medium.

(Method for Collecting Solubilized Fraction Containing Recombinant Structural Protein)
The expressed recombinant structural protein can be recovered as a solubilized fraction. Collection as a solubilized fraction enables the removal or reduction of host cells and/or host cell-derived contaminants, and is therefore preferably performed before step (A).

The method for collecting the solubilized fraction is, for example, when the recombinant structural protein is expressed in solution in the host cell, the host cell is first disrupted by physical or chemical treatment to disrupt the host cell. get the liquid Examples of physical treatment include ultrasonic treatment and crushing treatment with a homogenizer, and examples of chemical treatment include a method of treatment with a solvent that dissolves the target recombinant structural protein but does not dissolve host cells. HFIP etc. are mentioned as a solvent. Then, a solubilized fraction containing the recombinant structural protein of interest is recovered from the host cell lysate. Methods for recovering the solubilized fraction containing the recombinant structural protein of interest include common methods such as centrifugation and filtration through filters such as drum filters and press filters. In the case of filter filtration, a method using a Teflon filter, a method using a filter aid such as celite or diatomaceous earth and a pre-coating agent, etc. can be used to more efficiently recover the soluble fraction containing the target recombinant structural protein. can.

In addition, when the recombinant structural protein forms an insoluble form in the cells and is expressed, the host cells are similarly disrupted by physical or chemical treatment, and then centrifuged to form a precipitate fraction. An insoluble form of the recombinant structural protein is recovered. The recovered insoluble form of the recombinant structural protein can be solubilized with a protein denaturant. After said manipulation, a solubilized fraction containing the recombinant structural protein can be obtained by a method similar to that described above. When the recombinant structural protein is extracellularly secreted, a solubilized fraction containing the recombinant structural protein can be recovered from the culture supernatant. That is, a solubilized fraction containing the recombinant structural protein can be obtained by treating the culture by centrifugation, filtration through a drum filter, a press filter, or the like. In the case of filter filtration, a method using a Teflon filter, a method using a filter aid such as celite or diatomaceous earth and a pre-coating agent, etc. can more efficiently collect the soluble fraction containing the recombinant structural protein.

(Crude purification of recombinant structural protein)
From the soluble fraction containing the recombinant structural protein, methods commonly used for protein isolation and purification, namely, solvent extraction, salting out with ammonium sulfate, desalting, precipitation with an organic solvent, diethylaminoethyl (DEAE )-Sepharose, anion exchange chromatography using resins such as DIAION HPA-75 (manufactured by Mitsubishi Kasei), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), butyl Hydrophobic chromatography using resins such as Sepharose and Phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing alone or in combination can be used to obtain crude preparations of recombinant structural proteins.

(Method for preparing recombinant structural protein solution)
The recombinant structural protein solution is not particularly limited as long as it is a solution in which the recombinant structural protein is dissolved in a solvent for dissolution. For example, it can be obtained by dissolving a crude preparation of the recombinant structural protein described above in a solvent for dissolution, or a soluble fraction containing the recombinant structural protein described above can be obtained as it is, or by replacing the solvent with a solvent for dissolution. It can be obtained by adding a solvent, or by dissolving host cells expressing the recombinant structural protein (or host cell lysate or lysate, etc.) in a dissolution solvent. After dissolving the recombinant structural protein by adding a solvent for dissolution to the host cells used as the recombinant structural protein or the lysate or lysate of the host cells, the recombinant structural protein solution is collected as a solubilized fraction. Both preferably remove or reduce host cells and/or host cell-derived contaminants. The method for collecting the solubilized fraction is as described above.

The conditions for dissolving the recombinant structural protein in the dissolving solvent can be appropriately set according to the type of dissolving solvent, the type and concentration of salt to be added, the type of recombinant structural protein, and the like. In general, the recombinant structural protein can be dissolved in the dissolution solvent by appropriately setting the dissolution conditions.

The dissolution temperature is preferably such that the recombinant structural protein dissolves, but the contaminants derived from the host cell expressing the recombinant structural protein do not dissolve, and the temperature is maintained for a predetermined period of time. The temperature for dissolution may be determined according to the type of dissolution solvent, the type and concentration of salt to be added, and the type of recombinant structural protein. can be mentioned. For example, the upper temperature limit for dissolution may be 100°C, 90°C, 80°C, or 70°C, and the lower temperature limit for dissolution may be 30°C, 40°C, and 50°C. The time for dissolution is not particularly limited as long as the recombinant structural protein is sufficiently dissolved and contaminants are less dissolved, but considering industrial production, it is preferably 10 to 120 minutes. 10 to 60 minutes is more preferable, and 10 to 30 minutes is even more preferable.

When the recombinant structural protein is fibroin, collagen, resilin, elastin, keratin, and proteins derived from these, for example, the following conditions can be mentioned. The amount of the dissolution solvent to be added to the host expressing the structural protein is preferably 100 to 300 times the weight (wt) of the recombinant structural protein as a ratio of solvent (vol)/recombinant structural protein weight (wt). , more preferably 150 to 250 times, and even more preferably 175 to 225 times. As the salt to be added to the solvent for dissolution, lithium chloride, calcium chloride and sodium trifluoroacetate are preferable, and sodium trifluoroacetate is more preferable. In addition, the concentration when adding a salt is preferably more than 0 M and 1.0 M or less, more preferably more than 0 M and 0.6 M or less, further preferably more than 0 M and 0.5 M or less, based on the total amount of the dissolving solvent, and more than 0 M. It may be 0.01M or less.

When the recombinant structural protein is fibroin (including modified fibroin), for example, the following conditions can be mentioned. The amount of the dissolving solvent to be added to the host cells expressing the recombinant structural protein is 100 as a dissolving solvent (vol)/recombinant structural protein weight (wt) ratio per recombinant structural protein weight (wt). ~300 times is preferred, 150 to 250 times is more preferred, and 175 to 225 times is even more preferred. As the salt to be added to the solvent for dissolution, lithium chloride, calcium chloride and sodium trifluoroacetate are preferable, and sodium trifluoroacetate is more preferable. In addition, the concentration when adding a salt is preferably more than 0 M and 1.0 M or less, more preferably more than 0 M and 0.6 M or less, further preferably more than 0 M and 0.5 M or less, based on the total amount of the dissolving solvent, and more than 0 M. It may be 0.01M or less. Temperature conditions include temperatures of 30 to 100° C. and 40 to 60° C. using the above solvents. For example, the upper temperature limit for dissolution may be 100°C, 90°C, 80°C, or 70°C, and the lower temperature limit for dissolution may be 30°C, 40°C, and 50°C. The time for dissolution is, for example, preferably 10 to 120 minutes, more preferably 10 to 60 minutes, even more preferably 10 to 30 minutes.

If the recombinant structural protein solution is obtained by dissolving the recombinant structural protein-expressing host cells (or host cell lysate or lysate, etc.) in a dissolution solvent, the recombinant structural protein solution is collected. In addition, insolubles may be separated to remove or reduce host cells and/or host cell-derived contaminants. Recovery of the structural protein solution and separation of the insoluble matter are not particularly limited as long as the solution and the sediment (aggregate) can be separated. Separation by filtration and/or centrifugation is preferred for ease of handling. Separation by filtration can be performed using, for example, filter paper, a filter membrane, or the like. Conditions for centrifugation are not particularly limited. For example, it can be carried out at room temperature (20±5° C.) at 8000×g to 15000×g for 5 to 20 minutes. The separation of the insoluble matter may be performed twice or more.

[Step (B)]
Step (B) is a step of diluting the recombinant structural protein solution prepared in step (A) with a mixed solvent containing water.

A mixed solvent (hereinafter also referred to as a “mixed solvent for dilution”) is composed of two or more solvents containing water. Water is not particularly limited, but pure water, distilled water, ultrapure water, or the like is preferably used from the viewpoint of reducing contaminants. Examples of water include water-soluble buffer solutions, acidic aqueous solutions, basic aqueous solutions, and the like. Acidic aqueous solutions include acidic aqueous solutions such as formic acid, acetic acid and dilute hydrochloric acid, and basic aqueous solutions include basic aqueous solutions such as sodium hydroxide aqueous solution and calcium hydroxide aqueous solution.

As the water-soluble buffer, for example, a phosphate buffer with a pH of 3 to 9, a Tris buffer with a pH of 3 to 9, or the like can be used. The concentration of the phosphate buffer is, for example, 10 to 200 mM. The concentration of the Tris-based buffer solution is, for example, 10 to 100 mM. For example, a 50 mM Tris buffer of pH 3-9 can be used as the Tris-based buffer. Further, for example, a 50 mM sodium phosphate buffer of pH 3 to 9 can be used as the phosphate buffer.

As the solvent other than water, any organic solvent that can be mixed with water can be used, and known solvents can be used. Specific examples include at least one solvent selected from the group consisting of acetonitrile, isopropanol, N-methyl-2-pyrrolidone (NMP), and dimethylsulfoxide (DMSO). The mixed solvent for dilution is preferably a mixed solvent of water and acetonitrile from the viewpoint of obtaining a recombinant structural protein with high purity and convenience.

From the viewpoint of high purity of the recombinant structural protein, the weight ratio of water in the mixed solvent is preferably 90% or less, more preferably 85% or less, and further preferably 80% or less. It is preferably 70% or less, and particularly preferably 70% or less. In addition, from the viewpoint of preventing aggregation of the recombinant structural protein, the weight ratio of water in the mixed solvent is preferably 10% or more, more preferably 20% or more, and further preferably 30% or more. It is preferably 40% or more, and particularly preferably 40% or more. The weight ratio of water in the mixed solvent may be 20 to 80%, 30 to 70%, 30 to 60%, 40 to 80%, or 40 to 60%. When the mixed solvent contains water and isopropanol, the proportion of water may be 30 to 60% by weight. Moreover, when the mixed solvent contains water and acetonitrile, the ratio of water may be 40 to 80% by weight.

By adding a mixed solvent for dilution to the recombinant structural protein solution and diluting it, most of the contaminant proteins other than the recombinant structural protein are aggregated, the aggregated insoluble matter is separated and removed, and the recombinant structural protein is contained. Collect the protein solution (supernatant).

The amount of the mixed solvent for dilution to be added is not particularly limited as long as proteins other than the recombinant structural protein (hereinafter also referred to as other proteins) can be aggregated. From the viewpoint of uniform dilution, the volume ratio of the recombinant structural protein solution is preferably 0.5 times or more, more preferably 0.75 times or more, and still more preferably 1 time or more. be. In addition, from the viewpoint of preventing aggregation of the recombinant structural protein, it is preferably 10-fold or less, more preferably 6-fold or less, and still more preferably 4-fold or less.

The mixed solvent may further contain a salt. The salt that can be contained in the mixed solvent can be the same as the salt that can be contained in the solvent for dissolution, such as the above-described alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, thiocyanates. , inorganic salts such as perchlorate, organic salts such as sodium trifluoroacetate (CF ₃ COONa), and volatile salts such as ammonium acetate. One type of these salts may be used alone, or two or more types may be used in combination. Preferred salts are alkali metal halides, alkaline earth metal halides, sodium trifluoroacetate and ammonium acetate, more preferably lithium chloride, calcium chloride, sodium trifluoroacetate and ammonium acetate.

When the salt is added, the optimum amount may be determined according to the type of solvent other than water contained in the mixed solvent. Salt can be added. The upper limit of the amount of salt added may be, for example, 0.7 M or less, 0.6 M or less, or 0.5 M or less, and the lower limit of the salt addition amount is 0.05 M or more, 0.1 M or more, or 0 .2M or more.

Separation of insoluble matter is not particularly limited as long as sediments (aggregates) can be separated. Separation by filtration and/or centrifugation is preferred for ease of handling. Separation by filtration can be performed using, for example, filter paper, a filter membrane, or the like. Conditions for centrifugation are not particularly limited. For example, it can be carried out at room temperature (20±5° C.) at 8000×g to 15000×g for 5 to 20 minutes. The separation of the insoluble matter may be performed twice or more.

In the production method of the present embodiment, before or after step (A), the host cells and/or host cell-derived contaminants are removed or reduced from the culture containing the host cells expressing the recombinant structural protein. It may be further provided. Thereby, the operations in steps (A) and (B) can proceed smoothly. Removal or reduction of contaminants can be carried out according to the method described above (Method for Collecting Solubilized Fraction Containing Recombinant Structural Protein).

[Step (C)]
Step (C) is a step of passing the recombinant structural protein solution diluted in step (B) through a column filled with porous gel to fractionate the molecular weight.

The porous gel is not particularly limited, and examples thereof include porous materials based on polystyrene, hydrogel, silica gel, and the like. Silica gel is preferred as the porous gel. By filling a column with these porous gels and passing the recombinant structural protein solution through the column, the components contained in the recombinant structural protein solution can be fractionated according to their molecular weights.

As for the material and shape of the column, those commonly used can be used without particular limitation as long as the components contained in the recombinant structural protein solution can be fractionated according to their molecular weights. As the column packed with porous gel, a size exclusion chromatography column packed with silica gel is preferred, from agar in which a fraction having a specific molecular weight is eluted. This makes it possible to automate the molecular weight fractionation in step (C) and the collection of fractions in step (D) using a chromatographic apparatus. The chromatographic device is not particularly limited as long as it is a general chromatographic device that is resistant to solvents (especially organic solvents) and capable of appropriately controlling the flow rate and the like.

The flow rate at which the recombinant structural protein solution is introduced into the column packed with the porous gel is not particularly limited as long as it can be introduced into the column packed with the porous gel without any problem. When the gel is silica gel, the column capacity is 2 cm in inner diameter, 40 cm in column length, and the target recombinant structural protein is PRT799 (molecular weight: 211.4 kDa), 0.1 mL/min to 100 mL/min, preferably 0.1 mL/min to 50 mL/min, more preferably 0.1 mL/min to 6 mL/min.

The recombinant structural protein solution preferably adjusts the concentration of the solute (preferably, the recombinant structural protein) according to the type of column packed with the porous gel. Methods for adjusting the concentration include methods such as concentration and dilution. Examples of methods for concentration include distillation, and examples for dilution include a method of diluting with the same solvent as the solvent for the recombinant structural protein solution. mentioned.

The production method of this embodiment may further include, before step (C), a step of removing an insoluble fraction from the recombinant structural protein solution prepared in step (B). Methods for removing the insoluble fraction include, for example, common methods such as centrifugation and filtration through filters such as drum filters and press filters. In the case of filter filtration, a method using a Teflon filter, a method using a filter aid such as celite or diatomaceous earth and a pre-coating agent, etc. can more efficiently collect the soluble fraction containing the recombinant structural protein. The collected soluble fraction containing the recombinant structural protein can be used in step (C) as a recombinant structural protein solution.

[Step (D)]
Step (D) is a step of collecting the fraction containing the recombinant structural protein having the desired molecular weight.

The fraction containing the recombinant structural protein having the desired molecular weight may be set arbitrarily, but it is preferably a fraction containing a protein with a molecular weight similar to that of the target recombinant structural protein. The molecular weight of the target recombinant structural protein can be estimated from the amino acid sequence of the structural protein. Minutes may be specified.

In addition, a fraction other than the fraction containing a protein having a molecular weight similar to that of the target recombinant structural protein may be set as the "fraction containing a recombinant structural protein having a desired molecular weight". As a result, for example, degradation products of the target recombinant structural protein can also be recovered.

The method for collecting the fraction containing the recombinant structural protein having the desired molecular weight is not particularly limited. For example, the fraction containing the protein having the desired molecular weight is discharged from a column filled with porous gel collection in another container at the time of collection. Moreover, when step (D) is performed using a chromatography device, for example, it can be collected using a fraction collector of the chromatography device or the like.

The time for the fraction containing the recombinant structural protein having the desired molecular weight to be discharged from the column packed with the porous gel is appropriately determined depending on the flow rate, column capacity, porous gel and type of the target recombinant structural protein. can be set.

Specifically, the flow rate is 0.3 mL/min, the column capacity is 4.6 mm in the column inner diameter, the column length is 250 mm, the two columns are directly connected, the porous gel is silica gel, and the target recombinant structural protein is In the case of PRT918 (molecular weight 52.7 kDa), fractions such as 7 minutes to 12 minutes and 8 minutes to 11 minutes after introduction of the recombinant structural protein solution into a column filled with porous gel, and more The fraction with high purity of the recombinant structural protein of interest is the 9-10 minute fraction.

When increasing the recovery amount, it is possible to increase the column capacity, increase the number of times, etc. When increasing the number of times, it is possible to obtain a fraction of about 60 mL by performing 50 times under the same conditions as above.

When the column volume is 20 mm in column inner diameter, 250 mm in column length, silica gel as the porous gel, and PRT918 (molecular weight 52.7 kDa) as the target recombinant structural protein, the treatment is performed at a flow rate of 6 mL/min.

According to this method, a recombinant structural protein having a predetermined molecular weight can be obtained in high purity by a simple method. Moreover, by using a solvent mainly composed of an organic solvent, it can be easily recovered by an operation such as distillation, which is economical.

A recombinant structural protein powder can be obtained by freeze-drying a recombinant structural protein having a predetermined molecular weight recovered by an operation such as distillation, and the recombinant structural protein powder is dissolved to be processed into protein molding. can also

[Recombinant Structural Protein]
According to the method for producing a recombinant structural protein having a desired molecular weight according to the present invention, a recombinant structural protein with a narrow molecular weight distribution (hereinafter, particularly referred to as a "single molecular weight recombinant structural protein") can be obtained. be done.

The single-molecular-weight recombinant structural protein according to the present embodiment has a narrow molecular weight distribution, and thus can be said to have high purity. Here, purity means the ratio of recombinant structural proteins having a given molecular weight (ie, the molecular weight with the highest frequency) to the total amount of recombinant structural proteins. The single-molecular-weight recombinant structural protein according to this embodiment preferably has a purity of 90% or higher, more preferably 95% or higher, even more preferably 97% or higher, and 99% or higher. It is even more preferred to have

The molecular weight of the single molecular weight recombinant structural protein is not particularly limited as long as it is a molecular weight that can be separated in a column filled with porous gel. When the recombinant structural proteins are fibroin, collagen, resilin, elastin, keratin, proteins derived from these, etc., the molecular weights thereof include, for example, 50 kDa, 100 kDa, 150 kDa, 200 KDa, 250 KDa, 300 KDa, etc. .

[Protein molded product and method for producing the same]
(Dope Solution Containing Single Molecular Weight Recombinant Structural Protein)
The dope solution according to this embodiment comprises a single molecular weight recombinant structural protein according to the invention. The dope solution according to the present embodiment can be obtained, for example, by dissolving the single-molecular-weight recombinant structural protein according to the present invention in a solvent, or by carrying out the method for producing the recombinant structural protein according to the present invention. , can also be obtained as a result of step (D). The result of step (D) can be subjected to further purification steps to further refine the purity of the single molecular weight recombinant structural protein. Further purification steps to be performed separately are not particularly limited, and include ethanol precipitation, affinity chromatography, ion exchange chromatography and the like.

The concentration of the recombinant structural protein with a single molecular weight in the dope solution according to the present embodiment may be 1% by mass or more, 2% by mass or more, or 3% by mass or more based on the total amount of the dope solution. may be 4% by mass or more, may be 5% by mass or more, may be 10% by mass or more, may be 20% by mass or more, may be 30% by mass or more, It may be 40% by mass or more, 50% by mass or more, or 60% by mass or more. The upper concentration limit for single molecular weight recombinant structural proteins may be, for example, 70% by weight or less.

As the solvent for the dope solution, those commonly used as solvents for dissolving structural proteins can be used. Examples include N-dimethylformamide (DMF), formic acid, and aqueous solutions containing urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide, calcium chloride, lithium thiocyanate, and the like. These solvents may be used singly or in combination of two or more.

The dope liquid may contain a dissolution accelerator. Examples of the dissolution accelerator include inorganic salts composed of a Lewis acid and a Lewis base shown below. Lewis bases include, for example, oxoacid ions (nitrate ion, perchlorate ion, etc.), metal oxoacid ions (permanganate ion, etc.), halide ions, thiocyanate ions, cyanate ions, and the like. Examples of Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions. Specific examples of inorganic salts composed of a Lewis acid and a 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 salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron thiocyanate; Aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate and aluminum thiocyanate, potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate and potassium thiocyanate Potassium salts such as sodium chloride, sodium bromide, sodium iodide, sodium nitrate, sodium perchlorate and sodium thiocyanate, zinc chloride, zinc bromide, zinc iodide, zinc nitrate, perchloric acid Zinc and zinc salts such as zinc thiocyanate, magnesium salts such as magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate and magnesium thiocyanate, barium chloride, barium bromide, barium iodide, 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 enhancer is 1.0 parts by mass or more, 5.0 parts by mass or more, 9.0 parts by mass or more, 15 parts by mass or more, or 20.0 parts by mass with respect to the total amount of 100 parts by mass of the recombinant structural protein. It may be at least parts by mass. The content of the dissolution enhancer 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 recombinant structural protein.

It may be heated to 30 to 90° C. during the production of the dope solution according to this embodiment. The dissolving temperature may be appropriately set according to the solvent used, the type of recombinant structural protein, and the like. Shaking and stirring may be used to facilitate dissolution.

The viscosity of the dope liquid according to this embodiment may be appropriately set according to the use of the dope liquid. For example, when the dope solution according to the present embodiment is used as a spinning stock solution, the viscosity thereof may be appropriately set according to the spinning method. (centipoise) or the like. The viscosity of the spinning stock solution can be measured, for example, using a product name "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

(Method for producing protein compact)
In the method for producing a protein compact according to the present embodiment, in addition to steps (A) to (D) of the method for producing a recombinant structural protein having a desired molecular weight, step (E): A step of molding the recombinant structural protein to obtain a molded product is further provided. The molding may be performed by a method known to those skilled in the art, for example, using the dope solution described above.

The resulting protein compact is a single-molecular-weight recombinant structural protein. The shape of the molded article is not particularly limited, and may be, for example, fibers, films, porous bodies, particles, molded articles, and the like.

A film-like molded body (protein film) can be obtained, for example, by forming a film of the dope solution described above and removing the solvent from the formed film.

A fibrous molded body (protein fiber) can be obtained, for example, by spinning the dope solution described above and removing the solvent from the spun dope solution.

A porous molded body (porous protein body) is basically obtained by a method for producing a porous body from a fibroin-derived protein, which is described in International Publication No. 2014/175178.

Particle-shaped compacts (protein particles) can be obtained, for example, by using the dope solution described above and replacing the solvent in the dope solution with a water-soluble solvent to obtain an aqueous protein solution, and drying the aqueous protein solution. obtained by a method comprising A water-soluble solvent refers to a solvent containing water, and includes, for example, water, a water-soluble buffer solution, physiological saline, and the like. The step of replacing with the water-soluble solvent is preferably carried out by a method of putting the dope solution into the dialysis membrane, immersing it in the water-soluble solvent, and replacing the water-soluble solvent one or more times. Specifically, it is more preferable to put the dope solution into the dialysis membrane, leave the solution in an amount of the water-soluble solvent that is 100 times or more the amount of the dope solution for 3 hours, and repeat this replacement of the water-soluble solvent. Any dialysis membrane may be used as long as it does not permeate proteins, and may be, for example, a cellulose dialysis membrane. By repeating replacement of the water-soluble solvent, the amount of solvent present in the dope can be brought close to zero. A dialysis membrane may not be used in the second half of the step of replacing with a water-soluble solvent. The step of drying the aqueous protein solution preferably uses vacuum freeze-drying. The degree of vacuum during vacuum freeze-drying is preferably 200 pascals (Pa) or less, more preferably 150 pascals or less, and even more preferably 100 pascals or less. The moisture content of the freeze-dried particles is preferably 5.0% or less, more preferably 3.0% or less.

For example, the specification of WO 2017/047504 describes a method for producing a molded article from a fibroin-derived protein, and the molded article is basically obtained by this method. When producing a molded product from a fibroin-derived protein, for example, the following operations are carried out. That is, first, a composition containing protein (only protein or containing other components) is introduced into the mold of a pressure molding machine, and then the mold is heated and pressed against the composition. Heating and pressing are continued until the protein reaches a predetermined temperature under a predetermined pressure to obtain a heat-pressed composition. Next, the temperature of the mold is lowered using a cooler (for example, a spot cooler), and when the composition reaches a predetermined temperature, the contents are taken out to obtain a molded product. Heating is preferably carried out at 80 to 300°C, more preferably 100 to 180°C, even more preferably 100 to 130°C. The pressure is preferably 5 kN or higher, more preferably 10 kN or higher, and even more preferably 20 kN or higher. In addition, after the predetermined heating and pressurizing conditions are reached, the time for which the treatment is continued under those conditions (warming conditions) is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, and even more preferably 5 to 30 minutes.

[Diluent for poorly soluble protein solution and poorly soluble protein diluent]
The diluent for the poorly soluble protein solution according to the present embodiment is a diluent for the poorly soluble protein solution and includes a mixed solvent containing water. A poorly soluble protein solution is a solution in which a poorly soluble protein is dissolved in a dissolution solvent. The dissolution solvent and mixed solvent are as described above. Also, the diluent for the sparingly soluble protein solution may be the same as the mixed solvent for dilution described above.

The sparingly soluble protein is not particularly limited as long as it is sparingly soluble in water, and examples thereof include fibroin, collagen, resilin, elastin and keratin, and proteins derived from these, particularly spider silk proteins. is preferably By diluting the sparingly soluble protein solution with the diluent, precipitation of the sparingly soluble protein can be suppressed while suppressing gelation of the sparingly soluble protein solution.

The poorly soluble protein diluent according to this embodiment includes a poorly soluble protein, a dissolving solvent for dissolving the poorly soluble protein, and a diluent for the poorly soluble protein solution. The poorly soluble protein, the solvent for dissolving the poorly soluble protein, and the diluent are as described above. The poorly soluble protein diluted solution is obtained by dissolving the poorly soluble protein in a dissolution solvent to form a poorly soluble protein solution, and then diluting the poorly soluble protein solution with a diluent while the poorly soluble protein remains dissolved. . The poorly soluble protein may be dissolved by adding a solvent for dissolution to the poorly soluble protein, or adding the poorly soluble protein to the solvent for dissolution. and dilution may be performed at the same time, or the solvent for dissolution may be added first to dissolve the sparingly soluble protein, and then the mixed solvent may be added for dilution. The poorly soluble protein diluent according to the present embodiment can be used as it is for purifying the poorly soluble protein. For example, the diluted solution is subjected to molecular weight fractionation using a porous gel and fractionated, whereby a target protein having a very high degree of purity and a desired molecular weight can be purified.

[Method for Analyzing Recombinant Structural Protein with Desired Molecular Weight]
The method for analyzing a recombinant structural protein having a desired molecular weight according to the present embodiment includes steps (A) to (D) of the method for producing a recombinant structural protein having a desired molecular weight, in addition to steps (F) and (D). ) analyzing the collected fractions with a detector.

Any detector can be used as long as it can analyze the molecular weight of the collected fraction, and examples thereof include quantitative detectors such as UV (ultraviolet) spectrophotometers. It is also preferred that the detector further comprises a mass spectrometer. Examples of mass spectrometers include MS (mass spectrometer).

According to the above analysis method, whether or not a recombinant structural protein having a desired molecular weight can be obtained, and its quantification and purity can be analyzed, thereby providing an optimal method for producing a recombinant structural protein having a desired molecular weight. can be provided.

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

[Reference Example 1: Production of recombinant structural protein]
(1) Preparation of target recombinant structural protein-expressing strain (recombinant cell) Having a spider silk-derived sequence having the amino acid sequence shown in SEQ ID NO: 29 (Met-PRT918) and SEQ ID NO: 9 (Met-PRT799) synthesized fibroin. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. The hydropathic index and molecular weight (kDa) of each protein are shown in Table 5.

Each nucleic acid encoding the protein of interest was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was digested with restriction enzymes NdeI and EcoRI, excised, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector. Escherichia coli BLR (DE3) was transformed with each of the pET-22b(+) expression vectors in which the four kinds of nucleic acids were respectively recombined to obtain transformed Escherichia coli (recombinant cells) expressing the desired protein. .

(2) Expression of Recombinant Structural Protein of Interest 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 6) containing ampicillin to an OD ₆₀₀ of 0.005. The temperature of the culture solution was kept at 30° C., and flask culture was performed until OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 7) to an OD ₆₀₀ of 0.05. The temperature of the culture medium was maintained at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture medium 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 medium was maintained at 37° C. and the pH was constantly controlled to 6.9 for culturing. In addition, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and culture was carried out for 20 hours. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce the expression of the target protein. After 20 hours from the addition of IPTG, the culture solution was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture medium before and after the addition of IPTG, and the appearance of a band of the desired protein size depending on the addition of IPTG indicated that the desired protein was expressed as an insoluble form. I confirmed that

(3) Removal of contaminants and purification of the _target protein DMSO (first protic polar solvent)) 500 μL. After adding the dry cells, after confirming that the cells were not clumped together, they were dissolved by heating under the conditions of 60° C., 2000 rpm, and 30 minutes while stirring. After completion of heating and dissolving, the solution was allowed to cool to 45° C. or lower, and after cooling to 45° C., acetone (second aprotic polar solvent) was added as a second solvent while stirring. The ratio of the amount of DMSO added to the amount of acetone added (amount of DMSO added: amount of acetone added) was 5 (vol/vol):1 (vol/vol). About 5 to 10 minutes after the addition, the stirring was stopped and the mixture was allowed to stand at room temperature (including the stirring time of 60 minutes). After the desired standing time had passed, centrifugation was performed at 500 xg for 10 minutes at 20°C.

After centrifugation, the extract (soluble fraction) was added to an equal volume of ethanol (a poor solvent for the target recombinant protein) to precipitate hydrophobic proteins (Met-PRT799 and Met-PRT918). After standing for 2 hours, the precipitated fibroin was collected by centrifugation (conditions: 20°C, 500 xg, 30 minutes). The precipitated fibroin was washed three times with the same amount of RO water as the extract. After washing was completed, the samples were lyophilized. After freeze-drying, the purified powder was recovered. The powder obtained was analyzed by SDS-PAGE. The results are shown in FIG.

In FIG. 4, Met-PRT799 was applied to lanes 1 and 3, Met-PRT918 was applied to lanes 2 and 4, and molecular weight marker protein was applied to lane M, respectively. Lanes 1 and 2 are stained with Oriole (trademark) fluorescent gel stain (manufactured by Bio-Rad) capable of staining all proteins after electrophoresis, lanes 3 and 4 are Met-PRT799 and Met- It was stained with an InVision™ His-tagged in-gel staining reagent (manufactured by Thermo Fisher Scientific) that reacts with the His-tagged region of PRT918. PRT799 with a theoretical molecular weight of 211.4 kDa was detected as a band between the 250 kDa and 150 kDa molecular weight markers, and PRT918 with a theoretical molecular weight of 52.7 kDa was detected as a band near the 50 kDa molecular weight marker.

Example 1 Purification of Recombinant Structural Proteins of Interest of Defined Molecular Weight - Purified Powder
10 mg of the purified powder was dissolved in 82 μL of formic acid, the solution was diluted to 0.1 w/v% (with a mixed solvent of 1 mL of acetonitrile and water (H ₂ O) per 1 mg of protein), and passed through a Teflon filter with a 0.5 μm pore size. Perform suction filtration.

Using two silica gel columns (column length: 250 mm, column inner diameter: 4.6 mm (manufactured by Imtakt)) in which the column carrier is silica gel, molecular weight fractionation is performed using size exclusion chromatography by preparative HPLC (flow rate: 0 .3 mL/min). Protein components running down were collected with a retention time of 9-10 minutes.

[Example 2 Examination of the ratio of H ₂ O in the mixed solvent]
10 mg of the purified powder obtained in Example 1 was dissolved in 82 μL of formic acid, and the solution was diluted with a mixed solvent containing H ₂ O to a 0.1 mg protein/mL mixed solvent (0.1 wt %). H ₂ O is mixed with solvent X (methanol, 2-propanol, acetonitrile, DMSO, or NMP) in the proportions shown in Table 8 below for each mixed solvent, and the dispersibility of the solution is measured by the precipitate after centrifugation. Evaluated by presence or absence. Specifically, the solution was centrifuged at 11000 g for 5 minutes at 20° C., and the presence of precipitate was visually confirmed. The results are shown in Table 8 below.

Claims (14)

(A) preparing a recombinant structural protein solution in which the recombinant structural protein is dissolved in a solvent for dissolution;
(B) diluting the recombinant structural protein solution prepared in step (A) with a mixed solvent containing water;
(C) a step of passing the recombinant structural protein solution diluted in step (B) through a column filled with a porous gel to fractionate the molecular weight;
(D) recovering a fraction containing the recombinant structural protein having the desired molecular weight, comprising:
The dissolution solvent is formic acid,
The mixed solvent is a mixed solvent of water and acetonitrile with an acetonitrile:water weight ratio of 2:8 to 6:4, and a mixed solvent of water and isopropanol with an isopropanol:water weight ratio of 4:6 to 7:3. , N-methyl-2-pyrrolidone (NMP):water in a mixed solvent of water and NMP in a weight ratio of 3:7 to 9:1, or dimethylsulfoxide (DMSO):water in a weight ratio of 5:5 to selected from the group consisting of a 10:0 mixed solvent of water and DMSO;
manufacturing method . 2. The production method according to claim 1 , wherein step (A) includes collecting the recombinant structural protein solution and removing or reducing host cells and/or host cell-derived contaminants. 3. The production method according to claim 1 , wherein the dissolution solvent and/or the mixed solvent further contain a salt. The production method according to any one of claims 1 to 3 , wherein the porous gel is silica gel. The production method according to any one of claims 1 to 4 , wherein the recombinant structural protein is a spider silk protein. The production method according to any one of claims 1 to 5 , wherein the recombinant structural protein recovered in step (D) has a purity of 90% or higher. (A) preparing a recombinant structural protein solution in which the recombinant structural protein is dissolved in a solvent for dissolution;
(B) diluting the recombinant structural protein solution prepared in step (A) with a mixed solvent containing water;
(C) a step of passing the recombinant structural protein solution diluted in step (B) through a column filled with a porous gel to fractionate the molecular weight;
(D) collecting the fraction containing the recombinant structural protein having the desired molecular weight;
(E) A method for producing a protein molded product, comprising the step of molding the recombinant structural protein recovered in step (D) to obtain a molded product,
The dissolution solvent is formic acid,
The mixed solvent is a mixed solvent of water and acetonitrile with an acetonitrile:water weight ratio of 2:8 to 6:4, and a mixed solvent of water and isopropanol with an isopropanol:water weight ratio of 4:6 to 7:3. , N-methyl-2-pyrrolidone (NMP):water in a mixed solvent of water and NMP in a weight ratio of 3:7 to 9:1, or dimethylsulfoxide (DMSO):water in a weight ratio of 5:5 to selected from the group consisting of a 10:0 mixed solvent of water and DMSO;
manufacturing method . 8. The method for producing a protein molded product according to claim 7 , wherein in step (E), the recombinant structural protein is molded into protein fibers. A diluent for a poorly soluble protein solution containing a mixed solvent containing water, wherein the poorly soluble protein solution is a solution in which a poorly soluble protein is dissolved in a dissolution solvent, and the diluent is a poorly soluble protein A diluent that can dilute the solution while the poorly soluble protein is dissolved ,
The dissolution solvent is formic acid,
The mixed solvent is a mixed solvent of water and acetonitrile with an acetonitrile:water weight ratio of 2:8 to 6:4, and a mixed solvent of water and isopropanol with an isopropanol:water weight ratio of 4:6 to 7:3. , N-methyl-2-pyrrolidone (NMP):water in a mixed solvent of water and NMP in a weight ratio of 3:7 to 9:1, or dimethylsulfoxide (DMSO):water in a weight ratio of 5:5 to selected from the group consisting of a 10:0 mixed solvent of water and DMSO;
Diluent . 10. The diluent of claim 9 , wherein said sparingly soluble protein is spider silk protein. A poorly soluble protein diluent, comprising a poorly soluble protein, a dissolving solvent for dissolving the poorly soluble protein, and the diluent according to claim 9 or 10 . (A) preparing a recombinant structural protein solution in which the recombinant structural protein is dissolved in a solvent for dissolution;
(B) diluting the recombinant structural protein solution prepared in step (A) with a mixed solvent containing water;
(C) a step of passing the recombinant structural protein solution diluted in step (B) through a column filled with a porous gel to fractionate the molecular weight;
(D) collecting the fraction containing the recombinant structural protein having the desired molecular weight;
(F) analyzing the fraction collected in step (D) with a detector, the method for analyzing a recombinant structural protein having a desired molecular weight ,
The dissolution solvent is formic acid,
The mixed solvent is a mixed solvent of water and acetonitrile with an acetonitrile:water weight ratio of 2:8 to 6:4, and a mixed solvent of water and isopropanol with an isopropanol:water weight ratio of 4:6 to 7:3. , N-methyl-2-pyrrolidone (NMP):water in a mixed solvent of water and NMP in a weight ratio of 3:7 to 9:1, or dimethylsulfoxide (DMSO):water in a weight ratio of 5:5 to An analytical method selected from the group consisting of a 10:0 mixed solvent of water and DMSO . 13. The method of analysis according to claim 12 , wherein said detector comprises a mass spectrometer. 14. The analytical method according to claim 12 or 13 , wherein said recombinant structural protein is a spider silk protein.

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