JP2021095346A - Method for producing modified fibroin crosslinked body - Google Patents

Abstract

To provide a method for producing efficiently a crosslinked body with modified fibroin intermolecularly crosslinked while inhibiting a side reaction such as gelation.SOLUTION: The present disclosure provides a method for producing a modified fibroin crosslinked body with intermolecularly crosslinked modified fibroin having a cysteine residue. The method includes a crosslinking step for irradiating modified fibroin with light and forming a disulfide bond by an oxidation reaction, to obtain a modified fibroin crosslinked body.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a modified fibroin crosslinked product.

A method of molecular cross-linking a protein is widely known for the purpose of improving the durability and physical properties of the protein. Specifically, for example, there is a report on a method of cross-linking a protein using a predetermined cross-linking agent such as a saccharide (for example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2018-150364

By the way, modified fibroin has a plurality of structural factors that hinder the progress of the cross-linking reaction. For example, modified fibroin has a crystalline portion in which molecules are regularly arranged and an amorphous portion having an irregular structure, and forms a complex higher-order structure. In addition, the modified fibroin has a relatively large number of regions related to interactions such as ionic bonds, electrostatic bonds, and hydrogen bonds, so that the force of binding molecules is relatively strong. Due to these structural factors, it was difficult to control the reaction of cross-linking modified fibroin between molecules.

In the cross-linking reaction of modified fibroin, if the reaction becomes uncontrollable, uncontrolled cross-linking may be repeated to produce a solvent-insoluble gel-like substance, and as a result, the cross-linked product of the desired modified fibroin is made efficient. It was difficult to get it well.

An object of the present invention is to provide a method for efficiently producing a crosslinked product in which modified fibroin is crosslinked between molecules while suppressing side reactions such as gelation.

The present invention relates to, for example, the following inventions.
[1]
A method for producing a modified fibroin crosslinked product in which modified fibroin having a cysteine residue is crosslinked between molecules.
A method comprising a cross-linking step of obtaining the modified fibroin crosslinked product by irradiating the modified fibroin with light to form a disulfide bond by an oxidation reaction.
[2]
The method according to [1], wherein the cross-linking step is performed by irradiating the modified fibroin with light in the presence of a photosensitizer.
[3]
The method according to [2], wherein the photosensitizer is a ketone.
[4]
The method according to any one of [1] to [3], wherein the cross-linking step is performed by irradiating a modified fibroin solution containing the modified fibroin and a solvent for dissolving the modified fibroin with light.
[5]
The method according to [4], wherein the solvent is dimethyl sulfoxide or hexafluoro-2-propanol.
[6]
The method according to any one of [1] to [5], wherein the modified fibroin has a molecular weight of 90 kDa or more.
[7]
The method according to any one of [1] to [6], wherein the modified fibroin is modified spider silk fibroin.
[8]
The modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n Motif-REP] _m or Formula 2: [(A) _n Motif-REP] _m- (A) _{n Motif.}
The domain sequence has an amino acid sequence corresponding to the insertion of a cysteine residue in a REP located near the N-terminus and / or C-terminus of the domain sequence as compared to naturally occurring fibroin. The method according to any one of [1] to [7].
[In Formulas 1 and 2, (A) _n motif indicates an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% of the total number of amino acid residues in _{n motif.} That is all. REP shows an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences. ]
[9]
The modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n Motif-REP] _m or Formula 2: [(A) _n Motif-REP] _m- (A) _{n Motif.}
Described in any of [1] to [8], wherein the domain sequence has an amino acid sequence corresponding to the insertion of 1 or more and less than 16 cysteine residues in REP as compared with naturally occurring fibroin. the method of.
[In Formulas 1 and 2, (A) _n motif indicates an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% of the total number of amino acid residues in _{n motif.} That is all. REP shows an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences. ]
[10]
The method according to any one of [1] to [9], wherein the modified fibroin contains a histidine tag sequence at either or both of the N-terminal and the C-terminal.
[11]
The method according to any one of [1] to [10], wherein the wavelength of the light is 315 to 400 nm.
[12]
The method according to any one of [1] to [11], wherein the contact time is 15 minutes or more.

According to the present invention, it is possible to provide a method for efficiently producing a crosslinked product in which modified fibroin is crosslinked between molecules while suppressing side reactions such as gelation. According to the production method of the present invention, it is possible to selectively react cysteine residues with each other even in a modified fibroin containing other amino acid residues such as histidine residues. Therefore, the production method of the present invention can be suitably used for cross-linking modified fibroin containing a histidine tag.

It is a schematic diagram which shows an example of the domain sequence of fibroin. It is a graph which shows the measurement result of the molecular weight by GPC in Test Example 1. It is a graph which shows the measurement result of the molecular weight by SDS-PAGE in Test Example 1. It is a graph which shows the measurement result of the molecular weight by GPC in Test Example 2. It is a graph which shows the measurement result of the molecular weight by SDS-PAGE in Test Example 2. It is a graph which shows the measurement result of the molecular weight by GPC in Test Example 3. It is a graph which shows the measurement result of the molecular weight by GPC in Test Example 4.

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

[Method for producing modified fibroin crosslinked product]
The production method according to the present embodiment includes a cross-linking step of irradiating modified fibroin having a cysteine residue with light to form a disulfide bond by an oxidation reaction to obtain a modified fibroin crosslinked product. The modified fibroin cross-linked product is one in which modified fibroin having a cysteine residue is cross-linked between molecules. The thiol group (-SH group) of the cysteine residue is activated by the light irradiated in the cross-linking step, and the activated -SH group forms a disulfide bond, whereby the modified fibroins having the cysteine residue are separated from each other. Cross-linking produces a modified fibroin cross-linked product.

According to the production method according to the present embodiment, even in a modified fibroin containing a cysteine residue and an amino acid residue having a property capable of participating in a photoreaction (for example, a histidine residue or a tyrosine residue), the cysteine residue is also present. Can be selectively reacted. Therefore, a selective reaction using a cysteine residue as a reaction target becomes possible. As a result, a modified fibroin crosslinked product can be efficiently obtained.

In the method of cross-linking a protein using a cross-linking agent such as glutaraldehyde, isocyanate, carbodiimide, or N-hydroxysuccinimide, an operation of removing the unreacted cross-linking agent may be performed after the reaction. In the production method according to the above, since a photoreaction is used, for example, it is not necessary to use a highly toxic cross-linking agent, and there is an advantage that a cross-linked product can be obtained more easily. Further, the production method according to the present embodiment has an advantage that the time for the crosslinking reaction can be further shortened.

<Crosslinking process>
In the cross-linking step, a modified fibroin crosslinked product is obtained by irradiating the modified fibroin having a cysteine residue (modified fibroin containing a cysteine residue) with light to form a disulfide bond by an oxidation reaction.

The cross-linking step may be carried out by irradiating the modified fibroin containing a cysteine residue with light in the presence of a photosensitizer from the viewpoint that the cross-linking reaction is more easily promoted. By using a photosensitizer, the thiol group at the cysteine residue is specifically activated, and the reaction tends to proceed more selectively.

As the photosensitizer, general ones can be used, and there is no particular limitation. The photosensitizer is preferably a ketone from the viewpoint of further increasing the reaction efficiency. Ketones include benzophenone, anthraquinone, camphaquinone, 4- (dimethylamino) benzophenone, thioxanthone derivative, substituted benzoylbenzoic acid, 2,4-diethyloxanthen-9-one, anthraquinone-containing carboxylic acid, bromomethyl-anthraquinone, anthraquinone. Examples include sulfonic acid and dibenzosverenone.

The amount of the photosensitizer used in the crosslinking step may be 0.05 to 20 parts by mass and 0.5 to 5 parts by mass with respect to 100 parts by mass of the modified fibroin containing a cysteine residue.

In the cross-linking step, the time for irradiating light (photoreaction time) is preferably 15 minutes or more, more preferably 30 minutes or more, for example, 1 hour or more, 5 hours or more, 10 hours or more, 15 It may be more than an hour or more than 18 hours. The photoreaction time may be, for example, 30 hours or less, or 25 hours or less.

In the cross-linking step, the wavelength of the light to be irradiated is not particularly limited, but is preferably 200 to 400 nm, which is an ultraviolet region, and more preferably 315 to 400 nm.

The scale of the cross-linking reaction is not particularly limited, but from the viewpoint of industrial production, the amount of the modified fibroin containing a cysteine residue is preferably 10 mg or more, more preferably 100 mg or more, and more preferably 500 mg or more. Is even more preferable.

In the cross-linking step, the temperature at which light is irradiated is not particularly limited, but may be, for example, 25 ° C. or higher, 40 ° C. or higher, or 80 ° C. or higher, and is 25 ° C. or lower, 10 ° C. or lower, or 0 ° C. or lower. It may be there.

Examples of the method of irradiating the cysteine residue-containing modified fibroin with light include a method of irradiating the cysteine residue-containing modified fibroin itself with light in a solid state, and a solvent in which the cysteine residue-containing modified fibroin is dissolved. Examples thereof include a method of irradiating a modified fibroin solution with light and a method of irradiating a gel containing a modified fibroin containing a cysteine residue with light.

In the production method according to one embodiment, the cross-linking step is performed by, for example, irradiating a modified fibroin solution containing a cysteine residue-containing modified fibroin and a solvent in which the cysteine residue-containing modified fibroin is dissolved with light. May be done. The production method according to one embodiment may further include a dissolution step of dissolving the modified fibroin containing a cysteine residue in a solvent to obtain a modified fibroin solution before the crosslinking step.

The modified fibroin solution contains a modified fibroin containing a cysteine residue and a solvent for dissolving the modified fibroin (solvent for dissolution). The modified fibroin solution may contain unavoidable components such as impurities contained in the protein. The modified fibroin solution may further contain the photosensitizer described above. The modified fibroin solution may contain a modified fibroin containing a cysteine residue, a photosensitizer, and a solvent in which these are dissolved.

Examples of the solvent include water, hexafluoroisopropanol, dimethyl sulfoxide (DMSO), formic acid and the like. From the viewpoint that purification after the reaction is easy, cysteine residue-containing modified fibroin can be dissolved without adding a denaturing agent, and unwanted side reactions can be more easily suppressed. It is preferably dimethyl sulfoxide, hexafluoroisopropanol or a combination thereof. The solvent is preferably hexafluoroisopropanol from an industrial point of view, from the viewpoint of easily dissolving the modified fibroin, and from the viewpoint of having a low boiling point and facilitating vacuum drying in the recovery step described later.

As the cysteine residue-containing modified fibroin to be dissolved in a solvent, purified cysteine residue-containing modified fibroin may be used, and the cysteine residue-containing modified fibroin in the host cell expressing the desired cysteine residue-containing modified fibroin may be used. May be used. The purified cysteine residue-containing modified fibroin may be a cysteine residue-containing modified fibroin purified from a host cell expressing the cysteine residue-containing modified fibroin. When lysing a cysteine residue-containing modified fibroin in a host cell as a target cysteine residue-containing modified fibroin, the host cell is brought into contact with a solvent to dissolve the cysteine residue-containing modified fibroin in the host cell in the solvent. Let me. The host cell may be any cell that expresses the desired modified fibroin containing a cysteine residue, and may be, for example, an intact cell or a cell that has undergone a treatment such as a destruction treatment. Further, the cells may have already undergone a simple purification treatment.

The method for purifying the modified fibroin containing a cysteine residue from the host cell expressing the modified fibroin containing a cysteine residue is not particularly limited, and is described in, for example, known methods (for example, Japanese Patent No. 6077570 and Japanese Patent No. 6077569). The described method) can be used.

The cysteine residue-containing modified fibroin may be dissolved, for example, at room temperature, or may be maintained at various heating temperatures to dissolve the cysteine residue-containing modified fibroin in a solvent. The holding time of the heating temperature is not particularly limited, but may be 1 minute or more, and considering industrial production, 1 to 120 minutes is preferable, 5 to 60 minutes is more preferable, and 10 to 30 minutes is further preferable. The holding time of the heating temperature may be appropriately set, for example, under the condition that the cysteine residue-containing modified fibroin is sufficiently dissolved and the contaminants (other than the target cysteine residue-containing modified fibroin) are less dissolved. ..

When lysing the cysteine residue-containing modified fibroin in the host cell expressing the cysteine residue-containing modified fibroin, the amount of the solvent added is the ratio of the solvent (mL) to the weight (g) of the host cell (volume (mL)). / Weight (g)) may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, 1 to 5 times. It's okay.

The content of the modified fibroin containing a cysteine residue in the modified fibroin solution is, for example, 5% by mass or more and 35% by mass or less, or 0.1% by mass or more and 50% by mass or less based on the total mass of the modified fibroin solution. Good.

The formation of the modified fibroin cross-linked product (cross-linking of the modified fibroin containing a cysteine residue) can be confirmed by GPC. When a solution of modified fibroin containing cysteine residues flows through the column, large molecules elute in a short time due to the short movement distance in the column in a certain period of time, and small molecules move in the column. The longer the distance, the slower the elution time. As described above, the progress of the cross-linking reaction can be confirmed by separating the molecules according to their size.

Cross-linking of modified fibroin containing cysteine residues can also be confirmed by SDS-PAGE. When the reaction mixture obtained after the reaction is analyzed, a band corresponding to a multimer of the modified fibroin containing a cysteine residue before the reaction can be confirmed.

In the cross-linking step, the modified fibroin cross-linked product may be obtained as a reaction mixture containing other components. The reaction mixture may be used as it is as a raw material for various molded products containing a modified fibroin crosslinked product, or if necessary, it may be used as a raw material for a molded product after separating (removing) other components from the reaction mixture. Good.

In the production method according to the present embodiment, gelation can be easily suppressed by controlling the position where the cysteine residue is introduced, the number of cysteine residues to be introduced, and the like, and it is suitable for producing an industrially modified fibroin crosslinked product. It can be preferably used.

<Recovery process>
The production method according to the present embodiment may further include a recovery step of recovering the modified fibroin crosslinked product. For example, if the modified fibroin crosslinked product is obtained as a reaction mixture containing other components in the cross-linking step, the recovery step may remove the other components (eg, solvent, photosensitizer) from the reaction mixture. May include.

Depending on the conditions, amino acid residues (for example, amino acid residues other than cysteine residues) may cause side reactions (for example, further cross-linking reaction) by concentrating or the like. Therefore, other components from the reaction mixture It is desirable to separate some or all of (eg, photosensitizers, solvents).

When the cross-linking step is performed by irradiating the modified fibroin solution with light, the solvent may be removed from the reaction mixture containing the modified fibroin cross-linked product and the solvent as a recovery step after the cross-linking step.

Examples of the method for removing the solvent from the reaction mixture include a method of volatilizing the solvent by a method such as drying under reduced pressure.

Examples of the method for removing the solvent include a method of precipitating the modified fibroin crosslinked product by adding a poor solvent for the modified fibroin crosslinked product to the reaction mixture containing the modified fibroin crosslinked product and other components such as a solvent. Be done. The type of the poor solvent may have the property of being difficult to dissolve the modified fibroin crosslinked product, and may be appropriately selected depending on the type of the modified fibroin crosslinked product and the like. Examples of the poor solvent include alcohols other than hexafluoroisopropanol, ketones, esters, ethers, benzene, and toluene.

If necessary, the insoluble material may be removed from the reaction mixture before removing the solvent. Examples of the method for removing the insoluble matter from the reaction mixture include general methods such as centrifugation, filter filtration such as a drum filter and a press filter. In the case of filter filtration, insoluble matter can be removed more efficiently from the reaction mixture by using a filtration aid such as Celite and diatomaceous earth and a precoating agent in combination.

The modified fibroin crosslinked product may be molded if necessary, and then washed with water or the like to remove other components (for example, a photosensitizer, a solvent).

(Modified fibroin)
The modified fibroin according to the present embodiment has a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein contained. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.

As used herein, the term "modified fibroin" means fibroin whose amino acid sequence is different from that of naturally occurring fibroin. As used herein, "naturally-derived fibroin" means fibroin whose amino acid sequence is the same as that of fibroin produced by naturally occurring insects, spiders, and the like. Naturally-derived fibroin is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} Is.

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

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

More specific examples of fibroin produced by insects include, for example, silk moth fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).

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

More specific examples of fibroins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleic acid sequence)), fibroin-. 4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (nucleic acid sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession number A) U37520 (nucleic acid sequence)), major angu11ate protein 1 [derived from Laterectus hesperus] (GenBank accession number ABR68856 (nucleic acid sequence), EF595246 (nucleic acid sequence)), dragline silk protein2 (Amino acid sequence), AF441245 (Nucleotide sequence)), major antagonist protein 1 [derived from Euprosthenops protein] (GenBank accession number CAJ00428 (nucleic acid sequence), AJ973155 (nucleic acid sequence)), and major amplifier Protein 2 Accession number CAM32249.1 (nucleic acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila protein] (GenBank accession number AAC14589.1 (nucleic acid sequence)), minor complete silk protein (GenBank Accession No. AAC14591.1 (Amino Acid Sequence)), minor amplify spidroin-like protein [Nefilengys cruisentata] (GenBank Accession No. ABR) 3778.1 (amino acid sequence) and the like can be mentioned.

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

If the "modified fibroin" has the amino acid sequence specified in the present invention, the amino acid sequence of the "modified fibroin" is modified by relying on the naturally occurring fibroin (for example, the gene sequence of the cloned naturally occurring fibroin is modified). The amino acid sequence may be modified by the above (for example, by chemically synthesizing a nucleic acid encoding the designed amino acid sequence) by artificially designing the amino acid sequence regardless of naturally occurring fibroin. It may have a desired amino acid sequence). A modified fibroin amino acid sequence is also included in the modified fibroin if its amino acid sequence is different from that of naturally occurring fibroin.

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically _{corresponding to (A) n} motif of amino acid sequence) and an amorphous region (typically, REP of amino acid sequence). An amino acid sequence that produces (corresponding.)), _{Which is represented by the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, (A) _n motif shows an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% or more of the total number of amino acid residues in _{n motif.} REP shows an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. m is preferably an integer of 20 to 300, and more preferably an integer of 30 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

For the (A) _n motif, the _{number of alanine residues with respect to the total number of amino acid residues in the (A) n} motif may be 80% or more, preferably 85% or more, and 90% or more. It is more preferably 95% or more, and even more preferably 100% (meaning that it is composed of only alanine residues). It is preferable that at least seven _{(A) n} motifs present in the domain sequence are composed of only alanine residues. When it is composed only of alanine residues, (A) _n motif is (Ala) _k (Ala indicates an alanine residue, k is an integer of 4 to 27, preferably an integer of 4 to 20, more preferably. It means that it has an amino acid sequence represented by (indicating an integer of 4 to 16).

REP is composed of 10 to 200 amino acid residues. One or more of the amino acid residues constituting REP may be an amino acid residue selected from the group consisting of glycine residue, serine residue, and alanine residue. That is, the REP may contain an amino acid residue selected from the group consisting of a glycine residue, a serine residue, and an alanine residue.

One or more of the amino acid residues constituting REP may be hydrophobic amino acid residues. That is, the REP preferably contains a hydrophobic amino acid residue. The hydrophobic amino acid residue means an amino acid residue having a positive hydrophobicity index. Regarding the hydrophobicity index of amino acid residues (hydropathy index, hereinafter also referred to as “HI”), a known index (Hydrotherapy index: Kyte J, & Doolittle R (1982) “A single method for protein)” "Character of a protein", J. Mol. Biol., 157, pp. 105-132) is used. Examples of the hydrophobic amino acid residue include isoleucine (HI: 4.5), valine (HI: 4.2), leucine (HI: 3.8), phenylalanine (HI: 2.8), and methionine (HI: HI:). 1.9), alanine (HI: 1.8) can be mentioned.

In the modified fibroin according to the present embodiment, the domain sequence has an amino acid sequence corresponding to the insertion of a cysteine residue in REP as compared with naturally occurring fibroin.

The domain sequence preferably has an amino acid sequence corresponding to the insertion of a cysteine residue at a position adjacent to a glycine residue, a serine residue, or an alanine residue in the REP. It is more preferable to have an amino acid sequence corresponding to the insertion of the cysteine residue at a position adjacent to the glycine residue. In this case, since the movable range of the side chain of the cysteine residue is wide, the mercapto group (-SH) existing in the side chain of the cysteine residue easily forms a disulfide bond within or between the molecules, which further improves the physical properties. It can be further improved. The cysteine residue in REP may be located between the glycine residue, serine residue, or alanine residue and the glycine residue, serine residue, or alanine residue, and the glycine residue and glycine residue. It may be located between the residues.

The domain sequence preferably has an amino acid sequence corresponding to the insertion of a cysteine residue at a position adjacent to the hydrophobic amino acid residue in the REP. In this case, the hydrophobic amino acid residues are fixed between the molecules by hydrophobic interaction, so that the mercapto group (-SH) at the cysteine residue easily forms a disulfide bond, further improving the physical properties. Can be done. The cysteine residue in REP may be located next to the hydrophobic amino acid residue and may be located between the hydrophobic amino acid residue and an amino acid residue other than the hydrophobic amino acid residue. , It may be located between a hydrophobic amino acid residue and a glycine residue, a serine residue, or an alanine residue, and may be located between a hydrophobic amino acid residue and a glycine residue. The hydrophobic amino acid residue may be one selected from the group consisting of isoleucine residue, valine residue, leucine residue, phenylalanine residue, methionine residue, and alanine residue.

The domain sequence has an amino acid sequence corresponding to the insertion of a cysteine residue in the REP located near the N-terminal and / or C-terminal of the domain sequence as compared with naturally occurring fibroin. Good. In this case, since the molecular chain becomes long, improvement in physical properties can be expected. In the present specification, the REP located near the N-terminal of the domain sequence means the REP located at the 1st to 3rd positions from the N-terminal of the domain sequence. For example, the cysteine residue may be located in the REP located 1-2 from the N-terminus of the domain sequence. In the present specification, the REP located near the C-terminal of the domain sequence means the REP located at the 1st to 3rd positions from the C-terminal of the domain sequence. For example, the cysteine residue may be located in the REP located 1-2 from the C-terminus of the domain sequence. The cysteine residue is preferably located in the REP located on the most N-terminal side and / or the most C-terminal side of the domain sequence.

The domain sequence may have an amino acid sequence corresponding to the insertion of a cysteine residue in or near the center of the REP as compared to naturally occurring fibroin. In the present specification, the vicinity of the center of the amino acid sequence in REP means N from the amino acid residue located in the center of REP (if there are two amino acid residues located in the center, the amino acid residue on the N-terminal side). The 1st to 5th positions from the 1st to 5th positions toward the terminal side, or the 1st to 5th amino acid residues located in the center of the REP (if there are two amino acid residues located in the center, the amino acid residue on the C-terminal side). Indicates the position of. For example, the cysteine residue may be located in the center of the REP, and is located at the 1st to 3rd or 1st to 2nd positions toward the N-terminal side or the C-terminal side from the amino acid residue located in the center of the REP. You may be doing it.

The modified fibroin preferably contains a GPGXX motif (G indicates a glycine residue, P indicates a proline residue, and X indicates an amino acid residue other than a glycine residue) in the amino acid sequence of REP. By including this motif in the REP, the elongation of the modified fibroin can be improved.

When the modified fibroin contains the GPGXXX motif in the REP, the content of the GPGXXX motif is not particularly limited, but may be 1% or more, 3% or more, 5% or more, 10% or more, or 20. It may be% or more. In this case, the stress of the modified fibroin fiber becomes even higher. The upper limit of the GPGXX motif content is not particularly limited and may be 50% or less, or 30% or less.

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

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

FIG. 1 is a schematic diagram showing a domain sequence of fibroin. The calculation method of the GPGXX motif content will be specifically described with reference to FIG. First, in the fibroin domain sequence shown in FIG. 1 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are “located closest to the C-terminal side (). A) _{GPGXX for calculating c because it is included in the "sequence obtained by removing the sequence from the n} motif to the C-terminal of the domain sequence from the domain sequence" (the sequence shown by "region A" in FIG. 1). The number of motifs is 7, and c is 7 × 3 = 21. _{Similarly, all REPs are "sequences obtained by removing the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (the sequence shown in "Region A" in FIG. 1). Since it is contained in.), The total number d of amino acid residues in all REPs excluding the _{(A) n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150.} Next, c / d (%) can be calculated by dividing c by d, and in the case of fibroin in FIG. 1, 21/150 = 14.0%.

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

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

The domain sequence may have an amino acid sequence corresponding to the insertion of 1 or more and less than 16 cysteine residues in the REP as compared to naturally occurring fibroin. This makes it easier to control the reaction via cysteine residues, and makes it easier to suppress the progress of side reactions including gelation. Therefore, in order to suppress side reactions including gelation, the total number of cysteine residues corresponding to the insertion into the REP is preferably 1 or more and less than 16. The total number of cysteine residues corresponding to the insertion in REP is 1 or more and 12 or less, 1 or more and 10 or less, 1 or more and 8 or less, 1 or more and less than 8, 1 or more and 6 or less, 1 or more and less than 4, or 2 or more and 4 It may be as follows. The number of cysteine residues per REP in the domain sequence may be, for example, 1-3, 1-2, or 1.

The total number of cysteine residues of the modified fibroin according to the present embodiment may be 1 or more and less than 16, 1 or more and 12 or less, 1 or more and 10 or less, 1 or more and 8 or less, 1 or more and 6 or less, or 2 or more and 4 or less. ..

In the modified fibroin according to the present embodiment, in addition to the above-mentioned modification relating to the cysteine residue in REP, one or more amino acid residues were substituted, deleted, inserted and / or added as compared with the naturally occurring fibroin. There may be further modifications of the amino acid sequence corresponding to this.

The molecular weight of the modified fibroin according to the present embodiment is not particularly limited, but may be, for example, 10 kDa or more and 700 kDa or less. The molecular weight of the modified fibroin according to the present embodiment may be, for example, 10 kDa or more, 30 kDa or more, 60 kDa or more, 90 kDa or more, 150 kDa or more, 200 kDa or more, 300 kDa or more, or 500 kDa or more, and 600 kDa or less, 500 kDa or less, 400 kDa or less. , 300 kDa or less, or 200 kDa or less. It is expected to create a situation in which only the activated amino acids among a plurality of types of amino acids react, in which the energy required for the reaction increases and the activation energy increases as the molecular weight increases. In addition, fibroin having a high molecular weight is preferably used in terms of obtaining mechanical properties such as strength, and 90 kDa or more, 150 kDa or more, 200 kDa or more, 300 kDa or more, or 500 kDa or more is preferable.

The molecular weight of the modified fibroin is a measured value obtained by SDS-PAGE.

As a more specific example of the modified fibroin according to the present embodiment, (i) includes the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. , Modified fibroin, comprising the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, modified fibroin, or comprising the amino acid sequence represented by (iii) SEQ ID NO: 28. A modified fibroin can be mentioned.

The amino acid sequence (PRT1) shown in SEQ ID NO: 1 is obtained by inserting one cysteine residue into the REP located on the N-terminal side of the domain sequence with respect to the amino acid sequence (PRT11) shown in SEQ ID NO: 11. is there.

The amino acid sequence (PRT2) shown in SEQ ID NO: 2 is obtained by inserting one cysteine residue into the REP located on the N-terminal side of the domain sequence with respect to the amino acid sequence (PRT12) shown in SEQ ID NO: 12. is there.

The amino acid sequence (PRT3) shown in SEQ ID NO: 3 has cysteine in the REP located on the most N-terminal side and the REP located on the most C-terminal side of the domain sequence with respect to the amino acid sequence (PRT12) shown in SEQ ID NO: 12. Each residue is inserted in one place.

The amino acid sequence (PRT4) shown in SEQ ID NO: 4 is located in the REP located at the first and second positions from the N-terminal side and the C-terminal side of the domain sequence with respect to the amino acid sequence (PRT12) shown in SEQ ID NO: 12, respectively. , One cysteine residue is inserted in each.

The amino acid sequence (PRT5) shown in SEQ ID NO: 5 is contained in the REP located at the 1st to 4th positions from the N-terminal side and the C-terminal side of the domain sequence with respect to the amino acid sequence (PRT12) shown in SEQ ID NO: 12. One cysteine residue is inserted in each.

The amino acid sequence (PRT6) shown in SEQ ID NO: 6 is contained in the REP located at the 1st to 8th positions from the N-terminal side and the C-terminal side of the domain sequence with respect to the amino acid sequence (PRT12) shown in SEQ ID NO: 12. One cysteine residue is inserted in each.

The amino acid sequence (PRT7) shown in SEQ ID NO: 7 has a cysteine residue in the REP located on the N-terminal side and the C-terminal side of the domain sequence with respect to the amino acid sequence (PRT13) shown in SEQ ID NO: 13. It is inserted one by one.

The amino acid sequence (PRT8) shown in SEQ ID NO: 8 has a cysteine residue in the REP located on the N-terminal side and the C-terminal side of the domain sequence with respect to the amino acid sequence (PRT14) shown in SEQ ID NO: 14. It is inserted one by one.

The amino acid sequence (PRT9) shown in SEQ ID NO: 9 is contained in the REP located at the 1st to 4th positions from the N-terminal side and the C-terminal side of the domain sequence with respect to the amino acid sequence (PRT14) shown in SEQ ID NO: 14. One cysteine residue is inserted in each.

The amino acid sequence (PRT10) shown in SEQ ID NO: 10 is contained in the REP located at the 1st to 8th positions from the N-terminal side and the C-terminal side of the domain sequence with respect to the amino acid sequence (PRT14) shown in SEQ ID NO: 14. One cysteine residue is inserted in each.

The amino acid sequence shown by SEQ ID NO: 28 (PRT28) is obtained by inserting one cysteine residue into the REP located at the most C-terminal side of the domain sequence with respect to the amino acid sequence (PRT30) shown by SEQ ID NO: 30. is there.

The modified fibroin of (i) may have only the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. The modified fibroin of (ii) may have only the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. The modified fibroin of (iii) may have only the amino acid sequence shown in SEQ ID NO: 28.

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

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography). Can be used for. Specific examples of the tag sequence include the amino acid sequence shown by SEQ ID NO: 25 or SEQ ID NO: 26 (amino acid sequence including His tag).

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

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

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

As a more specific example of the modified fibroin containing the tag sequence, (iii) SEQ ID NO: 15 (PRT15), SEQ ID NO: 16 (PRT16), SEQ ID NO: 17 (PRT17), SEQ ID NO: 18 (PRT18), SEQ ID NO: 19 (PRT19). ), Or a modified fibroin containing the amino acid sequence set forth in SEQ ID NO: 20 (PRT20), (iv) SEQ ID NO: 21 (PRT21), SEQ ID NO: 22 (PRT22), SEQ ID NO: 23 (PRT23), or SEQ ID NO: 24 (PRT24). ), Or modified fibroin (v) containing the amino acid sequence represented by SEQ ID NO: 29 (PRT29).

The modified fibroin of (iii) may have only the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

Includes the amino acid sequence set forth by SEQ ID NO: 15 (PRT15), SEQ ID NO: 16 (PRT16), SEQ ID NO: 17 (PRT17), SEQ ID NO: 18 (PRT18), SEQ ID NO: 19 (PRT19), or SEQ ID NO: 20 (PRT20). The GPGXX motif content of the modified fibroin was 40.2%, 39.9%, 39.9%, 39.7%, 39.3%, and 38.6%, respectively, all of which were 10% or more. ..

The modified fibroin of (iv) may have only the amino acid sequence set forth in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

The GPGXX motif content of the modified fibroin containing the amino acid sequence set forth in SEQ ID NO: 21 (PRT21), SEQ ID NO: 22 (PRT22), SEQ ID NO: 23 (PRT23), or SEQ ID NO: 24 (PRT24) was 39.9%, respectively. , 39.9%, 39.3%, and 38.6%, all of which are 10% or more.

The modified fibroin of (iii) may have only the amino acid sequence set forth in SEQ ID NO: 29 (PRT29). The GPGXX motif content of the modified fibroin, including the amino acid sequence set forth in SEQ ID NO: 29 (PRT29), is 32.

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

[Nucleic acid]
The nucleic acid according to one embodiment encodes the modified fibroin described above. Specific examples of nucleic acids include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 28. Modified fibroin containing the amino acid sequence represented by, or modified fibroin in which the amino acid sequence (tag sequence) represented by SEQ ID NO: 25 or SEQ ID NO: 26 is bound to either or both of the N-terminal and C-terminal of these amino acid sequences. Nucleic acids encoding such as.

The nucleic acid according to one embodiment hybridizes with the complementary strand of the nucleic acid encoding the modified fibroin under stringent conditions, and formula 1: [(A) _n motif-REP] _m , or formula 2: [(. A) _n motif-REP] _m- (A) A nucleic acid encoding modified fibroin containing a domain sequence represented by the _{n motif.} The domain sequence of the modified fibroin encoded by the nucleic acid has an amino acid sequence corresponding to the insertion of a cysteine residue in REP as compared to naturally occurring fibroin.

The "stringent condition" refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. The "stringent condition" may be any of a low stringent condition, a medium stringent condition and a high stringent condition. Low stringent conditions mean that hybridization occurs only when at least 85% or more identity is present between the sequences, eg, using a 5 × SSC containing 0.5% SDS at 42 ° C. Conditions for hybridization can be mentioned. Medium stringent conditions mean that hybridization occurs only when at least 90% or more identity is present between the sequences, eg, using a 5 × SSC containing 0.5% SDS at 50 ° C. Conditions for hybridization can be mentioned. High stringent conditions mean that hybridization occurs only when at least 95% or more identity is present between the sequences, eg, using a 5 × SSC containing 0.5% SDS at 60 ° C. Conditions for hybridization can be mentioned.

[Host and expression vector]
The expression vector according to one embodiment has the above nucleic acid sequence and one or more regulatory sequences operably linked to the nucleic acid sequence. The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector.

The host according to one embodiment is transformed with the above expression vector. As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

As the expression vector, a vector that can be autonomously replicated in a host cell or can be integrated into the chromosome of the host and contains a promoter at a position where the nucleic acid according to one embodiment can be transcribed is preferably used.

When a prokaryote such as a bacterium is used as a host, the expression vector according to the embodiment is capable of self-sustaining replication in the prokaryote, and at the same time, a promoter, a ribosome binding sequence, a nucleic acid and a transcription termination sequence according to the embodiment. It is preferable that the vector contains. It may contain genes that control promoters.

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

Microorganisms belonging to the genus Escherichia include, for example, Escherichia coli BL21 (Novagen), Escherichia coli BL21 (DE3) (Life Technologies), Escherichia coli BLR (DE3) (Merck Millipore), Escherichia coli DH1, Escherichia.・ Kori GI698, Escherichia Kori HB101, Escherichia Kori JM109, Escherichia Kori K5 (ATCC 23506), Escherichia Kori KY3276, Escherichia Kori MC1000, Escherichia Kori MG1655 (ATCC 47076), Escherichia Kori MG1655 (ATCC 47076) 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W1485, Escherichia coli W1485 ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue and the like can be mentioned.

Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus bolsterensis, Brevibacillus centroporus Brevibacillus formosas, Brevibacillus invocatus, Brevibacillus lachilosporus, Brevibacillus limnofilus, Brevibacillus parabis. , Brevibacillus Reus Zeri, Brevibacillus Thermolber, Brevibacillus Brevis 47 (FERM BP-1223), Brevibacillus Brevis 47K (FERM BP-2308), Brevibacillus Brevis 47-5 (FERM BP-1664), Brevi Brevibacillus chocinensis 47-5Q (JCM8975), Brevibacillus chocinensis HPD31 (FERM BP-1087), Brevibacillus chocinensis HPD31-S (FERM BP-6623), Brevibacillus chocinensis HPD31-OK (FERM BP-) 4573), Brevibacillus chocinensis SP3 strain (manufactured by Takara), and the like.

Examples of microorganisms belonging to the genus Serratia include, for example, Serratia liquiefacience ATCC14460, Serratia entomophila, Serratia ficiaria, Serratia ficiaria, Serratia ficaria, Serratia ficaria, Serratia ficaria. (Serratia grimesii), Serratia proteamaculans, Serratia odorifera, Serratia primushika (Serratia primusica), Serratia primusica, etc.

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

Examples of microorganisms belonging to the genus Microbacterium include Microbacterium / Ammonia Philum ATCC15354 and the like.

Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corinebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corinebacterium glutamicum ATCC14067) ATCC13826, ATCC14067, and Brevibacterium immariophyllum. (Brevibacterium immunophilium) ATCC14068, Brevibacterium lactofermentum (Corinebacterium glutamicum ATCC13869) ATCC13665, ATCC13869, Brevibacterium roseum ATCC13825, Brevibacterium saccharolitamic, Brevibacterium Thiogenitalis ATCC19240, Brevibacterium album ATCC15111, Brevibacterium serinum ATCC15112 and the like can be mentioned.

Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC6781, ATCC6782, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC140. Lamb (Corynebacterium acetoacidophilum) ATCC13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanolitamicum ATCC21511, Corynebacterium carnae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC130 Examples thereof include Corynebacterium melanocera ATCC17965, Corynebacterium thermoaminogenes AJ12340 (FERMBP-1539), Corynebacterium herculis ATCC13868 and the like.

Pseudomonas microorganisms belonging to the genus Pseudomonas include, for example, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas esudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas (Pseudomonas sp.) D-0110 and the like can be mentioned.

As a method for introducing an expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open No. 63-248394), or the methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979). Can be mentioned.

Transformation of microorganisms belonging to the genus Brevibacillus is carried out, for example, by the method of Takahashi et al. (J. Bacteriol., 1983, 156: 1130-1134) or the method of Takagi et al. (Agric. Biol. Chem., 1989, 53:30). -3100), or by the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61: 202-203).

Examples of the vector into which the nucleic acid according to one embodiment is introduced (hereinafter, simply referred to as “vector”) include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), and pSE280. (Manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53,277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [Escherichia ), PGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), JP-A 60-22901 Publication], pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. coli. Bacteriol. , 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), and the like.

When Escherichia coli is used as a host, pUC18, pBluescriptII, pSupex, pET22b, pCold and the like can be mentioned as suitable vectors.

Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (Japanese Patent Laid-Open No. 2-31682), pNY700 (Japanese Patent Laid-Open No. 4-278901), pHY4831 (J). .Vector., 1987, 1239-1245), pNU200 (Shigezo Utaka, Journal of the Japanese Society of Agricultural Chemistry, 1987, 61: 669-676), pNU100 (Appl. Microbial. Biotechnol., 1989, 30: 75-80), pNU211 (J. Biochem., 1992, 112: 488-491), pNU211R2L5 (Japanese Unexamined Patent Publication No. 7-170984), pNH301 (Appl.Environ.Microbiol., 1992, 58: 525-531), pNH326, pNH400 (J. Biochem. Vector., 1995, 177: 745-749), pHT210 (Japanese Patent Laid-Open No. 6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42: 358-363), or microorganisms belonging to Escherichia coli and the genus Brevibacillus. Examples thereof include pNCO2 (Japanese Patent Laid-Open No. 2002-238569), which is a shuttle vector with and.

The promoter is not limited as long as it functions in the host cell. For example, promoters derived from Escherichia coli or phages such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter can be mentioned. Further, an artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series (Ptrp × 2), a tac promoter, a lacT7 promoter, and a let I promoter can also be used.

It is preferable to use a plasmid in which the Shine-Dalgarno sequence, which is a ribosome-binding sequence, and the start codon are adjusted to an appropriate distance (for example, 6 to 18 bases). In the expression vector, the transcription termination sequence is not always necessary for the expression of the nucleic acid, but it is preferable to arrange the transcription termination sequence directly under the structural gene.

Eukaryotic hosts include, for example, yeast, filamentous fungi (molds, etc.) and insect cells.

Examples of yeast include the genus Saccharomyces, the genus Schizoccharomyces, the genus Kluyveromyces, the genus Trichosporon, the genus Trichosporon, the genus Sichaniomyces, the genus Schanda , Yeasts belonging to the genus Yarowia, the genus Hansenula, etc. can be mentioned. More specifically, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces marxianus (Kluyveromyces marxianus), Trichosporon pullulans (Trichosporon pullulans), Shiwaniomaisesu - Albius (Schwanniomyces alluvius), Cyaniomyces occidentalis, Candida utilis, Pichia pastris (Pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia (Pichia polymorpha), Pichia stypitis, Yarrowia lipolytica, Hansenula polymorpha and the like.

Expression vectors when yeast is used as a host cell are usually the origin of replication (when amplification in the host is required) and a selection marker for vector growth in E. coli, a promoter for recombinant protein expression in yeast and It preferably contains a terminator, as well as a selection marker for yeast.

When the expression vector is a non-integrated vector, it preferably further contains a self-replicating sequence (ARS). This can improve the stability of the expression vector in cells (Myers, AM, et al. (1986) Gene 45: 299-310).

Vectors for using yeast as a host include, for example, YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, pGAPZα. And so on.

The promoter is not limited as long as it can be expressed in yeast. For example, promoters of glycolytic genes such as hexogen kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα 1 promoter, CUP 1 promoter, pGAP promoter, Examples include the pGCW14 promoter, AOX1 promoter, MOX promoter and the like.

As a method for introducing an expression vector into yeast, any method for introducing DNA into yeast can be used. For example, an electroporation method (Methods Enzymol., 194,182 (1990)), spheroplast. Method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), Lithium Acetate Method (J. Bacteriol., 153, 163 (1983)), Proc. Natl. Acad. Sci. The method described in USA, 75, 1929 (1978) and the like can be mentioned.

Filamentous fungi include, for example, the genus Acremonium, the genus Aspergillus, the genus Ustylago, the genus Trichoderma, the genus Neurospora, the genus Fusalium, the genus Fusaria, and the genus Fusaria. Penicillium, Myceliophtra, Botryts, Magnaporthe, Mucor, Metalithium, Monascus, Monascus , And fungi belonging to the genus Risomcoa.

Specific examples of filamentous fungi include Aspergillus aspergillus, Aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus Aspergillus oryzae, Aspergillus sake, Aspergillus sojae, Aspergillus tubignis, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus Parashichikusu (Aspergillus parasiticus), Aspergillus Fikumu (Fikyuumu) (Aspergillus ficuum), Aspergillus Fenikusu (Aspergillus phoeicus), Aspergillus Foechizusu (Fechidasu) (Aspergillus foetidus), Aspergillus Furabusu (Aspergillus flavus), Aspergillus fumigatus (Aspergillus fumigatus ), Aspergillus japonics, Aspergillus japonicus, Trichoderma viride, Trichoderma harzianum, Trichoderma harzianum, Trichoderma risseim Aspergillus, Aspergillus, Aspergillus cellulophyllum, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus. ora crassa), fusalium oxysporus, fusalium graminearum, fusalium venenatum, fumicola insolence penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium (Penicillium camemberti), Penicillium Kanesensu (Penicillium canescens), Penicillium Emerusoni (Penicillium emersonii), Penicillium funiculosum (Penicillium funiculosum), Penicillium Gurizeorozeumu (Penicillium griseoroseum), Penicillium Papurogenamu (Penicillium purpurogenum), Penicillium Rokeforuchi (Penicillium roqueforti), Myceriophtaora thermofilm, Mucor ambigus, Mucor cyllineroides, Mucor cyrcinelloides, Mucor cyrcinelloides, Mucoa filalis・ Inaequisporus, Mucor inaequisporus, Mucor oblongillilipticus, Mucor racemosus, Mucor recurbus, Mucor recurvus, Mucor recurvus, Mucor recurvus, Mucor recurvus Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium arrhizus) and the like.

When the host is a filamentous fungus, the promoter may be any of a gene related to glycolysis, a gene related to constitutive expression, an enzyme gene related to hydrolysis, and the like, and specifically, amyB, glaA, agdA, glaB, and TEF1. , XynF1tanasegene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, catB and the like can be mentioned.

The introduction of the expression vector into the filamentous fungus can be carried out by using a conventionally known method. For example, the method of Cohen et al. (Calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)], Protoplast method [Mol. Gen. Genet. , 168: 111 (1979)], Competent Method [J. et al. Mol. Biol. , 56: 209 (1971)], electroporation method and the like.

Examples of the insect cells include, for example, scaly insect cells, more specifically, insect cells derived from Prodenia frugiperda such as Sf9 and Sf21, and cabbage looper (Trichoplus) such as High 5. ) Derived insect cells and the like.

Examples of vectors when using insect cells as a host include baculoviridae virus (Baculoviridae virus) such as Augurapha californica nuclear polyhedrosis virus, which is a virus that infects night-stealing insects. , A Laboratory Manual, WH Freeman and Company, New York (1992).

When using insect cells as a host, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W. et al. H. Polypeptides can be expressed by the methods described in Freeman and Company, New York (1992), Bio / Technology, 6, 47 (1988) and the like. That is, after co-introducing a recombinant gene transfer vector and baculovirus into insect cells to obtain a recombinant virus (expression vector) in the insect cell culture supernatant, the recombinant virus is further infected with the insect cells to form a polypeptide. Can be expressed. Examples of the gene transfer vector used in this method include pVL1392, pVL1393, and pBlueBacIII (all manufactured by Invitrogen).

Examples of the method for co-introducing the recombinant gene transfer vector into insect cells and baculovirus for preparing the recombinant virus include the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075) and the lipofection method (Proc. Natl. Acad). Sci. USA, 84, 7413 (1987)) and the like.

The recombinant vector according to one embodiment preferably further contains a selectable marker gene for transformant selection. For example, in Escherichia coli, as the selectable marker gene, a resistance gene to various drugs such as tetracycline, ampicillin, and kanamycin can be used. Recessive selectable markers that can complement gene mutations involved in auxotrophy can also be used. In yeast, a resistance gene to geneticin can be used as a selectable marker gene, and a gene that complements a gene mutation involved in auxotrophy, a selectable marker such as LEU2, URA3, TRP1, or HIS3 can also be used. In filamentous fungi, as selectable marker genes, niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6,386-389, (1984)), sC (Gene, Gene, 84,329-334, (1989)), ptrA (BiosciBiotechnol Biochem, 64,1416-1421, (2000)), pyrG (BiochemBiophyss Res Commun, 112,284-289, (1983)), amdS (Gene, Gene, 26) 205-221, (1983)), aureobasidin resistance gene (Mol Gen Genet, 261,290-296, (1999)), benomil resistance gene (Proc Natl Acad Sci USA, 83, 4869-4873 (1986)) A marker gene selected from the group consisting of the hyglomycin resistance gene (Gene, 57, 21-26, (1987)), a leucine-requiring complementary gene, and the like can be mentioned. When the host is an auxotrophic mutant, a wild-type gene that complements the auxotrophy can be used as a selectable marker gene.

The host transformed with the expression vector according to one embodiment can be selected by plaque hybridization, colony hybridization or the like using a probe that selectively binds to the nucleic acid. As the probe, a partial DNA fragment amplified by the PCR method modified with a radioisotope or digoxigenin based on the sequence information of the nucleic acid can be used.

[Manufacturing method of modified fibroin]
The modified fibroin according to the present embodiment can be produced by a method including a step of expressing the nucleic acid by a host transformed with the expression vector. As an expression method, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. When expressed by yeast, animal cells, or insect cells, modified fibroin can be obtained as a polypeptide to which sugar or sugar chain is added.

For the modified fibroin according to the present embodiment, for example, a host transformed with the above expression vector is cultured in a culture medium, the modified fibroin according to the present embodiment is generated and accumulated in the culture medium, and the modified fibroin according to the present embodiment is collected from the culture medium. It can be manufactured by. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic medium such as yeast, the culture medium of the host contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and the host is cultured. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently carry out the above.

The carbon source may be any assimilated by the host, for example, glucose, fructose, sucrose, carbohydrates containing them such as molasses, starch and starch hydrolysates, and organic acids such as acetic acid and propionic acid. , And alcohols such as ethanol and propanol can be used.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof can be used.

As the inorganic salt, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

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

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

As insect cell culture media, commonly used TNM-FH medium (manufactured by Harmingen), Sf-900 II SFM medium (manufactured by Life Technologies), ExCell400, ExCell405 (all manufactured by JRH Biosciences), Race' s Insect Medium (Nature, 195, 788 (1962)) and the like can be used.

Insect cells can be cultured under conditions such as a culture medium having a pH of 6 to 7 and a culture temperature of 25 to 30 ° C. for a culture time of 1 to 5 days. In addition, an antibiotic such as gentamicin may be added to the culture medium as needed during the culture.

When the host is a plant cell, the transformed plant cell may be cultured as it is, or it can be differentiated into a plant organ and cultured. As the medium for culturing the plant cells, a commonly used Murashige and Scoog (MS) medium, White medium, or a medium obtained by adding plant hormones such as auxin and cytokinin to these media is used. be able to.

The culture of animal cells can be carried out under conditions such as a culture medium pH of 5 to 9 and a culture temperature of 20 to 40 ° C. for a culture time of 3 to 60 days. In addition, antibiotics such as kanamycin and hygromycin may be added to the medium as needed during culturing.

As a method for producing modified fibroin using a host transformed with the above expression vector, a method for producing the modified fibroin inside the host cell, a method for secreting the modified fibroin outside the host cell, and a method for producing the modified fibroin on the outer membrane of the host cell. There is a way. Each of these methods can be selected by varying the structure of the host cell used and the modified fibroin produced.

For example, when modified fibroin is produced in the host cell or on the extracellular membrane of the host cell, the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Rowe et al. (Proc. Natl. Acad). By applying the method described in Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990), JP-A-5-336963, International Publication No. 94/23021, etc. mutatis mutandis. , The modified fibroin can be modified to be actively secreted outside the host cell. That is, the modified fibroin can be positively secreted outside the host cell by expressing the modified fibroin in the form of adding a signal peptide to the polypeptide containing the active site of the modified fibroin by using a gene recombination method.

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

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

When the modified fibroin is expressed by forming an insoluble matter in the cells, the insoluble matter of the modified fibroin is recovered as a precipitation fraction by similarly collecting the host cell, crushing the host cell, and centrifuging the cell. The insoluble form of the recovered modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.

When the modified fibroin or a derivative having a sugar chain added to the modified fibroin is secreted extracellularly, the modified fibroin or a derivative thereof can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

<Use of modified fibroin crosslinked product>
The modified fibroin crosslinked product can be used as a raw material for various molded products, for example. The molded product containing the modified fibroin crosslinked product can be produced, for example, by a method including a step of molding a protein solution containing the modified fibroin crosslinked product (molding step). The shape of the molded product is not particularly limited, and examples thereof include fibers, films, molded products, gels, porous materials, and particles.

The protein solution can be obtained, for example, by dissolving the modified fibroin crosslinked product in a solvent. Examples of the solvent include formic acid, dimethyl sulfoxide, hexafluoro-2-propanol, N, N-dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and water. The protein solution may contain, for example, a modified fibroin crosslinked product and other components other than the solvent. Examples of other components include proteins other than modified fibroin crosslinked products.

The content of the modified fibroin crosslinked product in the protein solution and the total content of the protein in the protein solution may be appropriately adjusted according to the shape of the molded product and the like. The method for adjusting these is not particularly limited, but for example, a method for increasing the modified fibroin crosslinked product concentration or the protein concentration by volatilizing the solvent by distilling a protein solution, or a method having a high protein concentration in the dissolution step is used. Examples thereof include a method or a method in which the amount of the solvent added is smaller than the amount of the protein.

When the molded product containing the modified fibroin crosslinked product is a fiber, the molding step may be performed, for example, by spinning a protein solution. The protein solution in this case may be adjusted to a viscosity suitable for spinning. Viscosities suitable for spinning may generally be 1000-50,000 cP (centipoise) at 40 ° C. The viscosity can be measured using, for example, the trade name "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

When the molded product containing the modified fibroin crosslinked product is a fiber, the total content (concentration) of the protein in the protein solution may be adjusted to a concentration and viscosity capable of spinning, if necessary. The method for adjusting the total protein content and the viscosity of the protein solution is not particularly limited. Moreover, as a spinning method, wet spinning and the like can be mentioned. When a protein solution adjusted to a concentration and viscosity suitable for spinning is applied to the coagulating solution as a doping solution, the protein coagulates. At this time, by applying the protein solution to the coagulating liquid as a thread-like liquid, the protein coagulates into a thread-like form and a thread (undrawn thread) can be formed. The undrawn yarn can be formed, for example, according to the method described in Japanese Patent No. 5584932.

Hereinafter, examples of wet spinning will be shown, but the spinning method is not particularly limited, and may be wet spinning, dry spinning, dry wet spinning, or the like.

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

The method of applying the protein solution as a filamentous liquid is not particularly limited, and examples thereof include a method of extruding (discharging) the protein solution from a spinning mouthpiece into a coagulating liquid in a solvent removal tank. Undrawn yarn is obtained by coagulation of the protein. The extrusion (discharge) speed when extruding (discharging) the protein solution into the coagulating liquid can be appropriately set according to the diameter of the mouthpiece, the viscosity of the protein solution, etc., and for example, a nozzle having a diameter of 0.1 to 0.6 mm is used. In the case of a syringe pump having a syringe pump, the extrusion (discharge) speed may be 0.2 to 6.0 mL / h per hole from the viewpoint of spinning stability, and 1.4 to 4.0 mL / h per hole. may be h. The length of the desolvation tank (coagulant tank) in which the coagulant is placed is not particularly limited, but may be, for example, 200 to 500 mm. The take-up speed of the undrawn yarn formed by the coagulation of the protein may be, for example, 1 to 14 m / min, and the residence time may be, for example, 0.01 to 0.15 minutes. The take-up speed of the undrawn yarn may be 1 to 3 m / min from the viewpoint of the efficiency of desolvation. The undrawn yarn formed by coagulation of the protein may be further stretched (pre-stretched) in the coagulation liquid, but from the viewpoint of suppressing evaporation of the lower alcohol used in the coagulation liquid, the coagulation liquid is kept at a low temperature and is not yet drawn. It may be taken from the coagulating liquid in the state of drawn yarn.

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

When the molded product produced by using the modified fibroin crosslinked product is a film (raw material film), the protein solution may be adjusted to a concentration and viscosity capable of forming a film, if necessary. The method for forming a protein into a film is not particularly limited, but a predetermined method is obtained by applying a protein solution to a flat plate resistant to a solvent to a predetermined thickness to form a coating film, and removing the solvent from the coating film. A method of obtaining a film having a thickness of the above can be mentioned.

Examples of the method for forming a film having a predetermined thickness include a casting method. When forming a film by the casting method, a protein solution is cast on a flat plate to a thickness of several microns or more using a jig such as a doctor coat or a knife coater to form a cast film, and then dried under reduced pressure or desolvated. A raw material film (polypeptide film) can be obtained by removing the solvent by immersing it in a tank. The raw material film can be formed according to the method described in Japanese Patent No. 5678283.

When the molded product produced by using the modified fibroin crosslinked structure is a molded product (raw material molded product), the method for forming the raw material molded product is not particularly limited. For example, by introducing a dry protein powder containing a modified fibroin crosslinked product into a pressure molding machine and then pressurizing and heating using a hand press machine or the like, the dried protein powder reaches the temperature required for molding. A raw material molded product can be obtained. Further, the raw material molded product can be formed according to the method described in Patent Document (WO2017 / 047503).

When the molded product produced by using the modified fibroin crosslinked product is a gel, the method for forming the gel is not particularly limited. For example, a method including a step of dissolving the modified fibroin crosslinked product in a dissolving solvent to obtain a protein solution (dissolution step) and a step of substituting the dissolving solvent in the obtained protein solution with a water-soluble solvent (substitution step). Can produce a gel containing a modified fibroin crosslinked product. The method for producing a gel containing the modified fibroin crosslinked product may include a step of holding the protein solution in a predetermined shape between the dissolution step and the replacement step, or after the replacement step, the obtained gel may be used. It may include a step of cutting into a predetermined shape. The step of holding the protein solution in a predetermined shape can be performed, for example, by pouring the protein solution into a mold having a predetermined shape. The formation of the gel can be carried out, for example, according to the method described in Japanese Patent No. 5782580.

When the molded product produced by using the modified fibroin crosslinked product is a porous body, the protein solution may be adjusted to a concentration and viscosity capable of making it porous, if necessary. The method for forming the porous body is not particularly limited. For example, a method of obtaining a raw material porous body by adding an appropriate amount of a foaming agent to a protein solution adjusted to a concentration and viscosity suitable for porosity and removing the solvent, or a method described in Japanese Patent No. 5796147. It can be done according to the above.

When the molded product produced by using the modified fibroin crosslinked product is particles, the method for forming the particles is not particularly limited. The particles are obtained, for example, by a method including a step of obtaining an aqueous solution of a protein by substituting a solvent in the dope solution with a water-soluble solvent using the above-mentioned dope solution, and a step of drying the aqueous solution of the protein. The water-soluble solvent refers to a solvent containing water, and examples thereof include water, a water-soluble buffer solution, and physiological saline. The step of substituting with a water-soluble solvent is preferably carried out by a method in which the dope solution is placed in a dialysis membrane, immersed in the water-soluble solvent, and the water-soluble solvent is replaced at least once. Specifically, the dope solution is placed in a dialysis membrane, allowed to stand in a water-soluble solvent (one dose) 100 times or more the amount of the dope solution for 3 hours, and this water-soluble solvent replacement is repeated 3 times or more in total. Is more preferable. The dialysis membrane may be a cellulose dialysis membrane or the like as long as it does not permeate the protein. By repeating the substitution of the water-soluble solvent, the amount of the solvent existing in the doping solution can be brought close to zero. The dialysis membrane may not be used in the latter half of the step of substituting with a water-soluble solvent. Vacuum freeze-drying is preferably used in the step of drying the aqueous protein solution. 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 particles after freeze-drying is preferably 5.0% or less, more preferably 3.0% or less.

In the production method according to the present embodiment, by controlling the cross-linking reaction, for example, the degree of protein physical properties (for example, protein durability) can be controlled.

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

[Manufacturing of modified fibroin]
(1) Preparation of expression vector As modified fibroin having a cysteine residue, modified fibroin having the amino acid sequence shown by SEQ ID NO: 16 (PRT16), modified fibroin having the amino acid sequence shown by SEQ ID NO: 29 (PRT29), and sequence. A modified fibroin (PRT32) having the amino acid sequence represented by number 32 was designed.

As modified fibroin having no cysteine residue, modified fibroin (PRT27) having the amino acid sequence shown by SEQ ID NO: 27 and modified fibroin (PRT30) having the amino acid sequence shown by SEQ ID NO: 30 were designed.

The amino acid sequence (PRT16) shown in SEQ ID NO: 16 is obtained by inserting one cysteine residue into the REP of the domain sequence with respect to the amino acid sequence (PRT27) shown in SEQ ID NO: 27. The total number of cysteine residues of the modified fibroin having the amino acid sequence shown in SEQ ID NO: 16 is 1. The molecular weight of the modified fibroin having the amino acid sequence shown in SEQ ID NO: 16 is 100 kDa.

The modified fibroin (PRT29) having the amino acid sequence shown by SEQ ID NO: 29 has one cysteine residue in the REP located at the most C-terminal side of the domain sequence with respect to the amino acid sequence (PRT30) shown in SEQ ID NO: 30. It is the one that was inserted. The total number of cysteine residues of the modified fibroin having the amino acid sequence shown in SEQ ID NO: 29 is 1. The modified fibroin having the amino acid sequence shown in SEQ ID NO: 29 has a molecular weight of 300 kDa.

The amino acid sequence (PRT32) shown in SEQ ID NO: 32 is obtained by inserting two cysteine residues into the REP of the domain sequence with respect to the amino acid sequence (PRT27) shown in SEQ ID NO: 27. The total number of cysteine residues of the modified fibroin having the amino acid sequence shown in SEQ ID NO: 32 is 2. The molecular weight of the modified fibroin having the amino acid sequence shown in SEQ ID NO: 32 is 50 kDa.

Nucleic acids encoding modified fibroin having each of the above amino acid sequences were synthesized. An NdeI site was added to the nucleic acid at the 5'end, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into a protein expression vector pET-22b (+) to obtain an expression vector.

(2) Production of protein Escherichia coli BLR (DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 1) containing ampicillin so that OD _{600 was 0.005.} The culture solution temperature was maintained at 30 ° C., _{and flask culture was carried out until the OD 600} reached 5, (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 2) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

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

(3) Purification of protein The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL at 60 ° C. Was stirred with a stirrer for 30 minutes to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis is recovered by centrifugation, water is removed by a lyophilizer, and the lyophilized powder is recovered to obtain powdered modified fibroin (PRT16, PRT29, PRT27, and PRT32). It was.

[Test Example 1: Production of modified fibroin crosslinked product 1]
(Reaction using modified fibroin with cysteine residue)
Example 1
Modified fibroin having a cysteine residue (modified fibroin having an amino acid sequence containing one cysteine residue in REP) 20 mg of PRT16 and hexafluoroisopropanol (manufactured by Central Glass Co., Ltd.) were added in a 1 mL glass container, and the modified fibroin was added. PRT16 was dissolved in hexafluoroisopropanol to prepare a modified fibroin solution. The reaction was carried out by irradiating the modified fibroin solution with ultraviolet rays at 365 nm for 20 hours (MODELEN-160 l / J, 100 V, manufactured by SPECTROLINE) while stirring at room temperature (25 ° C.). After distilling off the solvent, it was dried under reduced pressure to obtain a product. This was used as a sample of Example 1 for later analysis.

Comparative Example 1-1
Modified fibroin having a cysteine residue (modified fibroin having an amino acid sequence containing one cysteine residue in REP) PRT16 was used as a sample of Comparative Example 1-1 for subsequent analysis.

(Reaction using modified fibroin without cysteine residue)
Comparative Example 1-2
The reaction was carried out in the same manner as in Example 1 except that 20 mg of modified fibroin PRT27 having no cysteine residue was used. After distilling off the solvent, it was dried under reduced pressure to obtain a product. This was used as a sample of Comparative Example 1-2 for later analysis.

Comparative Example 1-3
Modified fibroin PRT27 having no cysteine residue was used as a sample of Comparative Example 1-3 for the subsequent analysis.

(Confirmation of molecular weight of product by GPC)
The molecular weight was measured by GPC (Gel Permeation Chromatograph) under the following conditions. The dry powder of modified fibroin obtained in the above Examples and Comparative Examples was dissolved in a solvent (hexafluoro-2-propanol (HFIP) containing 2 mM trifluoroacetate) so as to have a concentration of 0.2 w / v%. The obtained modified fibroin solution was suction-filtered with a Teflon (registered trademark) filter having a pore size of 0.45 μm to prepare a GPC sample.

The molecular weight fractionation of the sample was performed using size exclusion chromatography (Forte, manufactured by YMC) by GPC. The conditions are as shown below. FIG. 2 shows the measurement results.
Column: YMC-GPC T10M-40 (polystyrene gel, manufactured by YMC)
Flow rate: 10 mL / min Carrier: HFIP containing 2 mM sodium trifluoroacetate

By GPC measurement, the sample obtained by irradiating the modified fibroin having a cysteine residue with light (Example 1) had an increased molecular weight as compared with the sample not irradiated with light (Comparative Example 1-1). It was confirmed that there was. On the other hand, in the sample obtained by irradiating the modified fibroin having no cysteine residue with light (Comparative Example 1-2), no increase in molecular weight was confirmed, and decomposition of the modified fibroin molecule was confirmed (Comparative Example 1). -2 and Comparative Example 1-3).

(Confirmation of molecular weight of product by SDS-PAGE)
The molecular weight of the product was analyzed by SDS-PAGE on the samples of Examples and Comparative Examples. The results are shown in FIG. In FIG. 3, lane 1 is a standard product (STD), lane 2 is the result of Example 1, lane 3 is the result of Comparative Example 1-1, lane 4 is the result of Comparative Example 1-2, and lane 5 is the result of Comparative Example 1-3. Shown.

The product obtained by irradiating the modified fibroin PRT16 having a cysteine residue with light (Example 1) was analyzed by GPC and SDS-PAGE to form a disulfide bond between sulfur atoms at the cysteine residue. It was confirmed that the molecular weight increased due to.

On the other hand, even if the modified fibroin PRT27 having no cysteine residue was irradiated with light, the molecular weight did not increase (comparison between Comparative Example 1-2 and Comparative Example 1-3). From this, it was clarified that the formation of a disulfide bond between cysteines is necessary in the molecular weight increase reaction using the light of the modified fibroin. Furthermore, since cysteine can be selectively reacted in the presence of cysteine and histidine contained in the tag sequence, the reaction in the present invention is suitable for cross-linking an industrially available tag sequence-containing protein. It can be said that.

[Test Example 2: Crosslinking reaction in the presence of a photosensitizer]
20 mg of modified fibroin PRT16 (modified fibroin having a cysteine residue) having the amino acid sequence represented by SEQ ID NO: 16, 0.2 mg of 2,2-dimethoxy-2-phenylacetophenone, hexafluoroisopropanol (manufactured by Central Glass Co., Ltd.) ) Was placed in a 1 mL glass container, and modified fibroin and 2,2-dimethoxy-2-phenylacetophenone were dissolved in hexafluoroisopropanol to obtain a modified fibroin solution. The reaction was carried out by irradiating the modified fibroin solution with ultraviolet rays of 365 nm for a predetermined time (manufactured by SPECTROLINE, MODEL EN-160 l / J, 100 V) while stirring at room temperature (25 ° C.). After distilling off the solvent, washing with methanol was performed three times (50 mL × 3, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) to remove 2,2-dimethoxy-2-phenylacetophenone. Further, the product was obtained by drying under reduced pressure. Samples obtained by irradiating with ultraviolet rays for 15 minutes, 30 minutes, 45 minutes, 60 minutes and 90 minutes were subjected to Examples 2-1 and 2-2, 2-3, 2-3 and 2-4, respectively. As Example 2-5, it was used for later analysis.

(Confirmation of molecular weight of product by GPC)
The molecular weight of the product was confirmed by GPC under the same conditions as in Test Example 1 for the products obtained in Example 2-2, Example 2-3, Example 2-4, and Example 2-5. It was carried out in. The results are shown in FIG. Further, PRT16 was used as Comparative Example 2.

It was confirmed that when light was irradiated in the presence of the photosensitizer, the desired reaction was efficiently produced as compared with the condition in which the photosensitizer was not present. Up to the ultraviolet irradiation time of 45 minutes, the number of crosslinked products obtained by increasing the irradiation time increased, and then decreased.

[Confirmation of molecular weight of product by SDS-PAGE]
Measurement of molecular weight by SDS-PAGE was performed on the products obtained in Examples 2-1 and 2-2, 2-3, 2-4 and 2-5 in Test Examples. It was carried out under the same conditions as in 1. The results are shown in FIG. Moreover, PRT16 was used for analysis as Comparative Example 2. In FIG. 5, lane 1 is a standard product (STD), lane 2 is Example 2-1 (light irradiation time: 15 minutes), lane 3 is Example 2-2 (light irradiation time: 30 minutes), and lane 4 is. Example 2-3 (light irradiation time: 45 minutes), lane 5 is Example 2-4 (light irradiation time: 60 minutes), lane 6 is Example 2-5 (light irradiation time: 90 minutes), lane 7. Shows the result of Comparative Example 2 (without light irradiation).

Analysis by SDS-PAGE also confirmed an increase in molecular weight. From the results of Example 2-1 it can be seen that a crosslinked product can be obtained even from 15 minutes.

[Test Example 3: Production of modified fibroin crosslinked product 2]
500 mg of modified fibroin PRT29 having a cysteine residue, 5 mg of 2,2-dimethoxy-2-phenylacetophenone, and 25 mL of hexafluoroisopropanol (manufactured by Central Glass Co., Ltd.) were added to the modified fibroin and 2,2-dimethoxy. -2-Phenylacetophenone was dissolved in hexafluoroisopropanol to obtain a modified fibroin solution. The reaction was carried out by irradiating the modified fibroin solution with ultraviolet rays of 365 nm for 2 hours (manufactured by AS ONE, 1-5479-03 Handy UV Lamp Long Wave Wave 336 × 82.3 × 65 mm LUV16) while stirring at room temperature (25 ° C.). .. After distilling off the solvent, washing with methanol was performed three times (50 mL × 3, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) to remove 2,2-dimethoxy-2-phenylacetophenone. Further, the product was obtained by drying under reduced pressure. The obtained sample was used as a sample of Example 3 for later analysis. In addition, PRT29 was used as a sample of Comparative Example 3 for later analysis.

The above result shows that by improving the reactivity with a photosensitizer, an amino acid cross-linking reaction other than the intended one such as histidine occurs, and it is expected that the selectivity may decrease. On the contrary, the selection of the cysteine cross-linking reaction is expected. It shows that the sex has improved.

(Confirmation of molecular weight of product by GPC)
A modified fibroin PRT29 having a different sequence from the modified fibroin PRT16 used in Example 1 and having high hydrophilicity was used, and the desired crosslinked product was obtained even under the condition of increasing the scale. The results are shown in FIG.

[Test Example 4: Production of modified fibroin crosslinked product 3]
Modified by adding 20 mg of modified fibroin PRT32 having two cysteine residues in the molecule, 0.2 mg of 2,2-dimethoxy-2-phenylacetophenone, and 1 mL of hexafluoroisopropanol (manufactured by Central Glass Co., Ltd.) in a glass container. Fibroin and 2,2-dimethoxy-2-phenylacetophenone were dissolved in hexafluoroisopropanol to obtain a modified fibroin solution. The reaction was carried out by irradiating the modified fibroin solution with ultraviolet rays of 365 nm for 2 hours (manufactured by SPECTROLINE, MODEL EN-160 l / J, 100 V) while stirring at room temperature (25 ° C.). After distilling off the solvent, it was dried under reduced pressure to obtain a product. The obtained sample was used as Example 4 for later analysis.
In addition, PRT32 before the reaction was used for analysis as a sample of Comparative Example 4.

(Confirmation of molecular weight of product by GPC)
The molecular weight of the product was confirmed by GPC under the same conditions as in Test Example 1. The results are shown in FIG.

It was confirmed that even in the reaction using a molecule having two cysteines in one molecule, the reaction between the sulfur atoms contained in the cysteines in the protein proceeded and the protein having an increased molecular weight was produced.

Even in the reaction using a molecule having two cysteines in the same molecule, the reaction between sulfur atoms contained in the cysteine residue in the modified fibroin proceeds, and the modified fibroin (modified fibroin crosslinked product) having an increased molecular weight is produced. It was confirmed that it was generated.

[Test Example 5: Reaction using isocyanate]
Modified fibroin having a cysteine residue (modified fibroin having an amino acid sequence containing one cysteine residue in REP) PRT16 and hexamethylene diisocyanate are used, and these are used as a solvent (hexafluoro-2-propanol (HFIP)). Reacted in the presence. This produced a gel-like substance.

(result)
Under the condition that the modified fibroin PRT16 having a cysteine residue was irradiated with light, a desired crosslinked product was formed and could be isolated. On the other hand, under the condition using isocyanate, it was confirmed that a gel-like substance was formed, the yield was greatly reduced, a mixture of various kinds of products was formed, and isolation was difficult. In addition, the peak of the target crosslinked product could not be confirmed by GPC analysis.

Claims (12)

A method for producing a modified fibroin crosslinked product in which modified fibroin having a cysteine residue is crosslinked between molecules.
A method comprising a cross-linking step of obtaining the modified fibroin crosslinked product by irradiating the modified fibroin with light to form a disulfide bond by an oxidation reaction. The method according to claim 1, wherein the cross-linking step is performed by irradiating the modified fibroin with light in the presence of a photosensitizer. The method according to claim 2, wherein the photosensitizer is a ketone. The method according to any one of claims 1 to 3, wherein the cross-linking step is performed by irradiating a modified fibroin solution containing the modified fibroin and a solvent for dissolving the modified fibroin with light. The method according to claim 4, wherein the solvent is dimethyl sulfoxide or hexafluoro-2-propanol. The method according to any one of claims 1 to 5, wherein the modified fibroin has a molecular weight of 90 kDa or more. The method according to any one of claims 1 to 6, wherein the modified fibroin is modified spider silk fibroin. The modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n Motif-REP] _m or Formula 2: [(A) _n Motif-REP] _m- (A) _{n Motif.}
The domain sequence has an amino acid sequence corresponding to the insertion of a cysteine residue in a REP located near the N-terminus and / or C-terminus of the domain sequence as compared to naturally occurring fibroin. The method according to any one of claims 1 to 7.
[In Formulas 1 and 2, (A) _n motif indicates an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% of the total number of amino acid residues in _{n motif.} That is all. REP shows an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences. ] The modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n Motif-REP] _m or Formula 2: [(A) _n Motif-REP] _m- (A) _{n Motif.}
The invention according to any one of claims 1 to 8, wherein the domain sequence has an amino acid sequence corresponding to the insertion of 1 or more and less than 16 cysteine residues in REP as compared with naturally occurring fibroin. the method of.
[In Formulas 1 and 2, (A) _n motif indicates an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% of the total number of amino acid residues in _{n motif.} That is all. REP shows an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences. ] The method of any one of claims 1-9, wherein the modified fibroin comprises a histidine tag sequence at either or both of the N-terminus and the C-terminus. The method according to any one of claims 1 to 10, wherein the wavelength of the light is 315 to 400 nm. The method according to any one of claims 1 to 11, wherein in the cross-linking step, the light is irradiated for 15 minutes or more.

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