JP7573254B2 - Method for producing crosslinked modified fibroin - Google Patents

Description

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

Methods of molecular cross-linking proteins for the purpose of improving the durability and physical properties of proteins are widely known. Specifically, for example, there have been reports of methods of cross-linking proteins using a specific cross-linking agent such as a sugar (for example, Patent Document 1).

JP 2018-150364 A

However, modified fibroin has several structural factors that impede the progress of the cross-linking reaction. For example, modified fibroin contains crystalline parts in which molecules are regularly arranged, and amorphous parts with irregular structures, forming a complex higher-order structure. In addition, modified fibroin has a relatively large number of regions related to interactions such as ionic bonds, electrostatic bonds, and hydrogen bonds, so the molecular binding force is relatively strong. These structural factors have made it difficult to control the reaction that cross-links modified fibroin between molecules.

In the crosslinking reaction of modified fibroin, if the reaction becomes uncontrollable, uncontrolled crosslinking may occur repeatedly, resulting in the production of a solvent-insoluble gel-like substance. As a result, it is difficult to efficiently obtain the desired crosslinked modified fibroin.

The object of the present invention is to provide a method for efficiently producing a crosslinked body 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 crosslinked modified fibroin in which modified fibroin having cysteine residues is crosslinked between molecules, comprising the steps of:
The method comprises a crosslinking step of irradiating the modified fibroin with light to form a disulfide bond by an oxidation reaction, thereby obtaining a crosslinked product of the modified fibroin.
[2]
The method according to [1], wherein the crosslinking step is carried out 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 crosslinking step is carried out by irradiating a modified fibroin solution containing the modified fibroin and a solvent capable of 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 molecular weight of the modified fibroin is 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 method according to any one of [1] to [7], wherein the domain sequence has an amino acid sequence corresponding to an insertion of a cysteine residue in REP located near the N-terminus and/or C-terminus of the domain sequence, compared to naturally occurring fibroin.
[In formula 1 and formula 2, (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in (A) _n motif is 80% or more. REP represents an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other 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;
The method according to any one of [1] to [8], wherein the domain sequence has an amino acid sequence corresponding to an insertion of 1 to less than 16 cysteine residues in REP, as compared with naturally occurring fibroin.
[In formula 1 and formula 2, (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in (A) _n motif is 80% or more. REP represents an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other or different amino acid sequences.]
[10]
The method according to any one of [1] to [9], wherein the modified fibroin comprises a histidine tag sequence at either or both of the N-terminus and the C-terminus.
[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 longer.

The present invention provides a method for efficiently producing a crosslinked body in which modified fibroin molecules are crosslinked while suppressing side reactions such as gelation. According to the production method of the present invention, it is possible to selectively react cysteine residues even in modified fibroin that contains other amino acid residues such as histidine residues. Therefore, the production method of the present invention can be suitably used for crosslinking modified fibroin that contains a histidine tag.

FIG. 2 is a schematic diagram showing an example of a fibroin domain arrangement. 1 is a graph showing the results of molecular weight measurement by GPC in Test Example 1. 1 is a graph showing the results of molecular weight measurement by SDS-PAGE in Test Example 1. 1 is a graph showing the results of molecular weight measurement by GPC in Test Example 2. 1 is a graph showing the results of molecular weight measurement by SDS-PAGE in Test Example 2. 1 is a graph showing the results of molecular weight measurement by GPC in Test Example 3. 1 is a graph showing the results of molecular weight measurement by GPC in Test Example 4.

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

[Method for producing crosslinked modified fibroin]
The manufacturing method according to this embodiment includes a crosslinking step in which modified fibroin having cysteine residues is irradiated with light to form disulfide bonds by oxidation reaction, thereby obtaining a crosslinked modified fibroin. The crosslinked modified fibroin is formed by intermolecular crosslinking of modified fibroin having cysteine residues. The thiol group (-SH group) of the cysteine residue is activated by the light irradiated in the crosslinking step, and the activated -SH group forms a disulfide bond, thereby crosslinking modified fibroins having cysteine residues to each other, thereby producing a crosslinked modified fibroin.

According to the manufacturing method of this embodiment, even in modified fibroin that contains cysteine residues and amino acid residues that can participate in photoreactions (e.g., histidine residues, tyrosine residues), the cysteine residues can be selectively reacted. This makes it possible to selectively react with the cysteine residues as the reaction target. As a result, modified fibroin crosslinked bodies can be efficiently obtained.

In methods of crosslinking proteins using crosslinking agents such as glutaraldehyde, isocyanate, carbodiimide, and N-hydroxysuccinimide, an operation of removing unreacted crosslinking agent may be performed after the reaction, whereas the production method according to the present embodiment utilizes a photoreaction, and therefore has the advantage that it is not necessary to use a highly toxic crosslinking agent, for example, and a crosslinked product can be obtained more easily. Furthermore, the production method according to the present embodiment has the advantage that the time for the crosslinking reaction can be further shortened.

<Crosslinking step>
In the crosslinking step, modified fibroin having cysteine residues (cysteine residue-containing modified fibroin) is irradiated with light to form disulfide bonds by oxidation reaction, thereby obtaining a crosslinked modified fibroin.

The crosslinking step may be carried out by irradiating the modified fibroin containing cysteine residues with light in the presence of a photosensitizer, from the viewpoint of further promoting the crosslinking reaction. By using a photosensitizer, the thiol groups in the cysteine residues are specifically activated, which tends to make it easier to proceed with the reaction more selectively.

Any common photosensitizer can be used, and there is no particular limitation. From the viewpoint of further increasing the reaction efficiency, the photosensitizer is preferably a ketone. Ketones include benzophenone, anthraquinone, camphorquinone, 4-(dimethylamino)benzophenone, thioxanthone derivatives, substituted benzoylbenzoic acid, 2,4-diethyloxanthene-9-one, anthraquinone-containing carboxylic acid, bromomethyl-anthraquinone, anthraquinone sulfonic acid, and dibenzosuberenone.

The amount of photosensitizer used in the crosslinking step may be 0.05 to 20 parts by mass, or 0.5 to 5 parts by mass, per 100 parts by mass of the cysteine residue-containing modified fibroin.

In the crosslinking step, the time for irradiating light (photoreaction time) is preferably 15 minutes or more, more preferably 30 minutes or more, and may be, for example, 1 hour or more, 5 hours or more, 10 hours or more, 15 hours or more, or 18 hours or more. The photoreaction time may be, for example, 30 hours or less, or 25 hours or less.

In the crosslinking process, the wavelength of the light to be irradiated is not particularly limited, but it is preferably in the ultraviolet range of 200 to 400 nm, and more preferably 315 to 400 nm.

The scale of the crosslinking reaction is not particularly limited, but from the viewpoint of industrial production, the amount of the modified fibroin containing cysteine residues is preferably 10 mg or more, more preferably 100 mg or more, and even more preferably 500 mg or more.

In the crosslinking process, 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 25°C or lower, 10°C or lower, or 0°C or lower.

Methods for irradiating modified fibroin containing cysteine residues with light include, for example, a method of irradiating the modified fibroin containing cysteine residues itself in a solid state with light, a method of irradiating a modified fibroin solution containing a solvent in which the modified fibroin containing cysteine residues is dissolved with light, and a method of irradiating a gel containing modified fibroin containing cysteine residues with light.

In one embodiment of the manufacturing method, the crosslinking step may be performed, for example, by 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. The manufacturing method according to one embodiment may further include a dissolving step of dissolving the cysteine residue-containing modified fibroin in a solvent to obtain a modified fibroin solution prior to the crosslinking step.

The modified fibroin solution contains a modified fibroin containing cysteine residues and a solvent in which it is dissolved (solvent for dissolving). The modified fibroin solution may contain unavoidable components, such as impurities contained in proteins. The modified fibroin solution may further contain the above-mentioned photosensitizer. The modified fibroin solution may contain a modified fibroin containing cysteine residues, a photosensitizer, and a solvent in which they are dissolved.

Examples of the solvent include water, hexafluoroisopropanol, dimethyl sulfoxide (DMSO), formic acid, etc. From the viewpoint of facilitating purification after the reaction, being able to dissolve the cysteine residue-containing modified fibroin without adding a denaturant, and being able to more easily suppress undesired side reactions, it is preferable that the solvent is dimethyl sulfoxide, hexafluoroisopropanol, or a combination of these. From the industrial viewpoint, the viewpoint of easily dissolving the modified fibroin, and the viewpoint of having a low boiling point and making it easier to perform reduced pressure drying in the recovery process described below, it is preferable that the solvent is hexafluoroisopropanol.

As the cysteine residue-containing modified fibroin to be dissolved in the solvent, a purified cysteine residue-containing modified fibroin may be used, or a cysteine residue-containing modified fibroin in a host cell expressing the desired cysteine residue-containing modified fibroin may be used. The purified cysteine residue-containing modified fibroin may be a cysteine residue-containing modified fibroin purified from a host cell expressing a cysteine residue-containing modified fibroin. When dissolving the cysteine residue-containing modified fibroin in the host cell as the desired cysteine residue-containing modified fibroin, the host cell is contacted with a solvent to dissolve the cysteine residue-containing modified fibroin in the host cell in the solvent. The host cell may be one that expresses the desired cysteine residue-containing modified fibroin, and may be, for example, an intact cell or a cell that has been subjected to a treatment such as a destruction treatment. It may also be a cell that has already been subjected to a simple purification treatment.

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

The modified fibroin containing cysteine residues may be dissolved, for example, at room temperature, or may be held at various heating temperatures to dissolve the modified fibroin containing cysteine residues in the solvent. The holding time at the heating temperature is not particularly limited, but may be 1 minute or more, and in consideration of industrial production, 1 to 120 minutes is preferable, 5 to 60 minutes is more preferable, and 10 to 30 minutes is even more preferable. The holding time at the heating temperature may be appropriately set, for example, under conditions in which the modified fibroin containing cysteine residues is sufficiently dissolved and little dissolution of impurities (things other than the desired modified fibroin containing cysteine residues) is observed.

When dissolving the cysteine residue-containing modified fibroin in host cells expressing the cysteine residue-containing modified fibroin, the amount of solvent added may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times the ratio of the solvent (mL) to the weight (g) of the host cells (volume (mL)/weight (g)).

The content of the cysteine residue-containing modified fibroin in the modified fibroin solution may be, 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.

The formation of crosslinked modified fibroin (crosslinking of modified fibroin containing cysteine residues) can be confirmed by GPC. When a solution containing modified fibroin containing cysteine residues is dissolved in the column, larger molecules are eluted earlier because they travel a shorter distance in the column in a given time, while smaller molecules are eluted later because they travel a longer distance in the column. As described above, the progress of the crosslinking reaction can be confirmed by separating the molecules according to their size.

The cross-linking of the 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 the polymerized modified fibroin containing cysteine residues before the reaction can be confirmed.

In the crosslinking step, the modified fibroin crosslinked body 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 bodies containing the modified fibroin crosslinked body, or, if necessary, other components may be separated (removed) from the reaction mixture before use as a raw material for the molded body.

In the manufacturing method according to this embodiment, gelation can be easily suppressed by controlling the position where the cysteine residue is introduced, the number of cysteine residues to be introduced, etc., and the method can be suitably used for industrial production of crosslinked modified fibroin.

<Recovery process>
The production method according to this embodiment may further include a recovery step of recovering the crosslinked modified fibroin. For example, when the crosslinked modified fibroin is obtained as a reaction mixture containing other components in the crosslinking step, the recovery step may include removing the other components (e.g., solvent, photosensitizer) from the reaction mixture.

Depending on the conditions, concentration or other procedures may cause amino acid residues (e.g., amino acid residues other than cysteine residues) to cause side reactions (e.g., further cross-linking reactions), so it is desirable to separate some or all of the other components (e.g., photosensitizer, solvent) from the reaction mixture.

When the crosslinking step is carried out by irradiating the modified fibroin solution with light, after the crosslinking step, the solvent may be removed from the reaction mixture containing the modified fibroin crosslinked body and the solvent as a recovery step.

Methods for removing the solvent from the reaction mixture include, for example, volatilizing the solvent by drying under reduced pressure.

One method for removing the solvent is, for example, to add a poor solvent for the crosslinked modified fibroin to a reaction mixture containing the crosslinked modified fibroin and other components such as a solvent, thereby precipitating the crosslinked modified fibroin. The type of poor solvent may be one that has the property of being difficult to dissolve the crosslinked modified fibroin, and may be appropriately selected depending on the type of crosslinked modified fibroin. Examples of poor solvents include alcohols other than hexafluoroisopropanol, ketones, esters, ethers, benzene, and toluenes.

Before removing the solvent, insoluble matter may be removed from the reaction mixture, if necessary. Methods for removing insoluble matter from the reaction mixture include common methods such as centrifugation and filtration using a filter such as a drum filter or press filter. When using filter filtration, insoluble matter can be removed more efficiently from the reaction mixture by using a filter aid such as celite or diatomaceous earth in combination with a precoat agent.

After molding, the crosslinked modified fibroin may be washed with water or the like if necessary to remove other components (e.g., photosensitizer, solvent).

(Modified fibroin)
The modified fibroin according to this embodiment is a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. The modified fibroin may further have amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically, but are not limited to, regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of about 100 amino acid residues.

As used herein, "modified fibroin" refers to a fibroin whose amino acid sequence is different from that of naturally occurring fibroin. As used herein, "naturally occurring fibroin" refers to a fibroin whose amino acid sequence is identical to that of fibroin produced by naturally occurring insects, arachnids, etc. Naturally occurring fibroin is also a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif.

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

Fibroin produced by insects includes, for example, Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia cynthia ricini, Samia cynthia, Caligra japonica, Antheraea mylitta, and Antheraea japonica. Examples include silk proteins produced by silkworms such as Bombyx mori (Bombyx mori), and hornet silk proteins excreted by hornet larvae (Vespa simillima xanthoptera).

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

Fibroin produced by spiders includes, for example, spiders belonging to the Araneus genus, such as the Japanese raven spider, the Japanese garden raven spider, the red raven spider, the green raven spider, and the Japanese bean raven spider; spiders belonging to the Neoscona genus, such as the Japanese mountain raven spider, the Japanese house raven spider, and the Japanese Satsuma spider; spiders belonging to the Pronus genus, such as the Japanese dwarf raven spider; spiders belonging to the Cyrtarachne genus, such as the Japanese spine spider and the Japanese giant spine spider; spiders belonging to the Gaster genus, such as the Japanese spine spider and the Japanese spine spider; spiders belonging to the genus Acantha, spiders belonging to the genus Ordgarius such as Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Argiope such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Arachnura such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Acusilas such as Orbweaver Spider, spiders belonging to the genus Cytophora such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Poltys such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the bush spider, the four-headed bush spider, the round bush spider, and the black bush spider, and spiders belonging to the genus Chorizopes, such as the Japanese bush spider, as well as spiders belonging to the genus Tetragnatha, such as the long-legged spider, the long-legged spider, the broad-legged spider, and the scale-like long-legged spider, spiders belonging to the genus Leucauge, such as the large white-legged spider, the medium-legged spider, and the small white-legged spider, spiders belonging to the genus Orb Weaver, such as the golden orb spider and the giant orb spider, Examples of spider silk proteins include those produced by spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as the golden spider, spiders belonging to the genus Dyschiriognatha such as the small long-legged spider, spiders belonging to the genus Latrodectus such as the black widow spider, the redback spider, the gray widow spider and the three-spotted latrodectus, and spiders belonging to the family Tetragnathiidae such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

More specific examples of fibroins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession numbers AAC04504 (amino acid sequence), U37520 (base sequence)), major angu11ate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [derived from Eurosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [derived from Eurosthenops australis] (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephila cruentata] (GenBank accession number ABR37278.1 (amino acid sequence) and the like.

More specific examples of naturally derived fibroin include fibroins whose sequence information is registered in NCBI GenBank. For example, from among the sequences registered in NCBI GenBank that contain INV as a division, it can be confirmed by extracting sequences in which spidroin, amplify, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION, a specific product character string from CDS, and a specific character string in TISSUE TYPE from SOURCE.

"Modified fibroin" may be one whose amino acid sequence has been modified based on naturally occurring fibroin (e.g., one whose amino acid sequence has been modified by modifying the genetic sequence of a cloned naturally occurring fibroin) so long as it has the amino acid sequence specified in the present invention, or one whose amino acid sequence has been artificially designed without relying on naturally occurring fibroin (e.g., one having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid that codes for a designed amino acid sequence). Note that modified fibroin whose amino acid sequence has been modified is also included in the category of modified fibroin, so long as the amino acid sequence differs from that of naturally occurring fibroin.

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

The (A) _n motif may have a number of alanine residues of 80% or more relative to the total number of amino acid residues in the (A) _n motif, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning composed only of alanine residues). Of the (A) _n motifs present in a plurality in the domain sequence, at least seven are preferably composed only of alanine residues. "Composed only of alanine residues" means that the (A) _n motif has an amino acid sequence represented by (Ala) _k (Ala represents an alanine residue, and k represents an integer of 4 to 27, preferably an integer of 4 to 20, more preferably 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 selected from the group consisting of glycine residues, serine residues, and alanine residues. That is, REP may contain amino acid residues selected from the group consisting of glycine residues, serine residues, and alanine residues.

One or more of the amino acid residues constituting REP may be hydrophobic amino acid residues. In other words, REP preferably contains hydrophobic amino acid residues. A hydrophobic amino acid residue means an amino acid residue with a positive hydrophobic index. For the hydrophobic index (Hydrophilic Index, hereinafter also referred to as "HI") of an amino acid residue, a publicly known index (Hydrophilic Index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132) is used. Examples of hydrophobic amino acid residues include isoleucine (HI: 4.5), valine (HI: 4.2), leucine (HI: 3.8), phenylalanine (HI: 2.8), methionine (HI: 1.9), and alanine (HI: 1.8).

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

The domain sequence preferably has an amino acid sequence corresponding to a cysteine residue inserted at a position adjacent to a glycine residue, serine residue, or alanine residue in REP, and more preferably has an amino acid sequence corresponding to a cysteine residue inserted at a position adjacent to a glycine residue in REP. In this case, since the side chain of the cysteine residue has a wide range of mobility, the mercapto group (-SH) present in the side chain of the cysteine residue is more likely to form intramolecular or intermolecular disulfide bonds, and the physical properties can be further improved. The cysteine residue in REP may be located between a glycine residue, serine residue, or alanine residue and a glycine residue, serine residue, or alanine residue, or may be located between a serine residue and a glycine residue.

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 REP. In this case, the hydrophobic amino acid residues are fixed to each other by hydrophobic interaction between molecules, so that the mercapto group (-SH) in the cysteine residue is more likely to form a disulfide bond, and the physical properties can be further improved. The cysteine residue in REP may be located next to the hydrophobic amino acid residue, may be located between the hydrophobic amino acid residue and an amino acid residue other than the hydrophobic amino acid residue, may be located between the hydrophobic amino acid residue and a glycine residue, a serine residue, or an alanine residue, or may be located between the hydrophobic amino acid residue and a glycine residue. The hydrophobic amino acid residue may be one selected from the group consisting of an isoleucine residue, a valine residue, a leucine residue, a phenylalanine residue, a methionine residue, and an alanine residue.

The domain sequence may have an amino acid sequence corresponding to the insertion of a cysteine residue in the REP located near the N-terminus and/or C-terminus of the domain sequence, as compared with naturally occurring fibroin. In this case, the molecular chain becomes longer, and therefore improved physical properties can be expected. In this specification, the REP located near the N-terminus of the domain sequence means the REP located 1 to 3 from the N-terminus of the domain sequence. For example, the cysteine residue may be located in the REP located 1 to 2 from the N-terminus of the domain sequence. In this specification, the REP located near the C-terminus of the domain sequence means the REP located 1 to 3 from the C-terminus of the domain sequence. For example, the cysteine residue may be located in the REP located 1 to 2 from the C-terminus of the domain sequence. It is preferable that the cysteine residue is located in the REP located at the most N-terminus and/or the most C-terminus of the domain sequence.

The domain sequence may have an amino acid sequence in which a cysteine residue has been inserted in the center or near the center of the REP compared to naturally occurring fibroin. In this specification, near the center of the amino acid sequence in the REP refers to the first to fifth positions from the amino acid residue located in the center of the REP (the amino acid residue on the N-terminal side if there are two amino acid residues located in the center) toward the N-terminal side, or the first to fifth positions from the amino acid residue located in the center of the REP (the amino acid residue on the C-terminal side if there are two amino acid residues located in the center). For example, the cysteine residue may be located in the center of the REP, or may be located in the first to third or first to second positions from the amino acid residue located in the center of the REP toward the N-terminal side or C-terminal side.

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

When the modified fibroin contains a GPGXX motif in the REP, the GPGXX motif content is not particularly limited, but may be 1% or more, 3% or more, 5% or more, 10% or more, or 20% or more. In this case, the stress of the modified fibroin fiber becomes even higher. There is no particular upper limit to the GPGXX motif content, and it 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 a fibroin containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , or Formula 2: [(A) _n motif-REP] _m- (A) _n motif, the GPGXX motif content is calculated as c/d, where c is three times the total number of GPGXX motifs contained in the region of all REPs contained in the sequence obtained by removing from the domain sequence the sequence from the (A) _{n motif} located at the most C-terminal side to the C-terminus of the domain sequence (i.e., equivalent to the total number of G and P in the GPGXX motif), and d is the total number of amino acid residues of all REPs removed from the domain sequence from the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence and further removing the (A)n motif.

In calculating the GPGXX motif content, the target is "the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because the "sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" (sequence corresponding to REP) may contain a sequence that has low correlation with the sequence characteristic of fibroin, and when m is small (i.e., when the domain sequence is short), this affects the calculation result of the GPGXX motif content, and this influence is to be eliminated. Note that when the "GPGXX motif" is located at the C-terminus of 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. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. 1. First, in the domain sequence of fibroin shown in FIG. 1 (which is of the "[(A) _n motif-REP] _m -(A) _n motif" type), all REPs are included in the "sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence shown in "Region A" in FIG. 1), so the number of GPGXX motifs for calculating c is 7, and c is 7×3=21. Similarly, all REPs are included in the "sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" (the sequence shown in "Region A" in FIG. 1), so the total number d of amino acid residues of all REPs obtained by further removing 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, which is 21/150=14.0% in the case of the fibroin in FIG.

The modified fibroin preferably has a hydrophobicity of REP of -0.8 or more, more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of REP, and it 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: Formula 1: [(A) _n motif-REP] _m , or Formula 2: [(A) _n motif-REP] _m- (A) _n motif. In a fibroin containing a domain sequence represented by the (A)n motif, the hydrophobicity of REP is calculated as e/f when the sum of the hydrophobicity indices of each amino acid residue in all REPs contained in a sequence (sequence corresponding to "region A" in Figure 1) obtained by removing from the domain sequence the sequence from the ₍ A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence, and the total number of amino acid residues in all REPs obtained by removing from the domain sequence the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence and further removing the (A) _n motif. The reason for the calculation of the hydrophobicity of REP to be targeted is the same as that described above, in that "the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is the same as that described above.

The domain sequence may have an amino acid sequence corresponding to 1 to less than 16 cysteine residues inserted into REP compared to naturally-occurring fibroin. This makes it easier to control reactions mediated by cysteine residues, and makes it easier to suppress the progression of side reactions including gelation. Therefore, in order to suppress side reactions including gelation, it is preferable that the total number of cysteine residues corresponding to those inserted into REP is 1 to less than 16. The total number of cysteine residues corresponding to those inserted into REP may be 1 to 12, 1 to 10, 1 to 8, 1 to less than 8, 1 to 6, 1 to less than 4, or 2 to 4. The number of cysteine residues per REP in the domain sequence may be, for example, 1 to 3, 1 to 2, or 1.

The total number of cysteine residues in the modified fibroin of this 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 addition to the above-mentioned modifications of the cysteine residues in the REP, the modified fibroin according to this embodiment may further have modifications in the amino acid sequence equivalent to the substitution, deletion, insertion and/or addition of one or more amino acid residues compared to naturally occurring fibroin.

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

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

More specific examples of modified fibroin according to this embodiment include (i) modified fibroin containing 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, (ii) modified fibroin containing the amino acid sequence represented by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or (iii) modified fibroin containing the amino acid sequence represented by SEQ ID NO:28.

The amino acid sequence shown in SEQ ID NO:1 (PRT1) is the amino acid sequence shown in SEQ ID NO:11 (PRT11) in which one cysteine residue has been inserted into the REP located at the most N-terminal end of the domain sequence.

The amino acid sequence shown in SEQ ID NO:2 (PRT2) is the amino acid sequence shown in SEQ ID NO:12 (PRT12) in which one cysteine residue has been inserted into the REP located at the most N-terminal end of the domain sequence.

The amino acid sequence shown in SEQ ID NO:3 (PRT3) is the amino acid sequence shown in SEQ ID NO:12 (PRT12) in which one cysteine residue has been inserted in each of the REP located at the most N-terminus and the REP located at the most C-terminus of the domain sequence.

The amino acid sequence shown in SEQ ID NO:4 (PRT4) is the amino acid sequence shown in SEQ ID NO:12 (PRT12) in which one cysteine residue has been inserted in each of the REPs located at the first and second positions from the N-terminus and C-terminus of the domain sequence.

The amino acid sequence shown in SEQ ID NO:5 (PRT5) is the amino acid sequence shown in SEQ ID NO:12 (PRT12) in which one cysteine residue has been inserted in each of the REPs located at positions 1 to 4 from the N-terminus and C-terminus of the domain sequence.

The amino acid sequence shown in SEQ ID NO:6 (PRT6) is the amino acid sequence shown in SEQ ID NO:12 (PRT12) in which one cysteine residue has been inserted into each of the REPs located at positions 1 to 8 from the N-terminus and C-terminus of the domain sequence.

The amino acid sequence shown in SEQ ID NO:7 (PRT7) is the amino acid sequence shown in SEQ ID NO:13 (PRT13) in which one cysteine residue has been inserted into each of the REPs located at the most N-terminus and the most C-terminus of the domain sequence.

The amino acid sequence shown in SEQ ID NO:8 (PRT8) is the amino acid sequence shown in SEQ ID NO:14 (PRT14) in which one cysteine residue has been inserted into each of the REPs located at the most N-terminus and the most C-terminus of the domain sequence.

The amino acid sequence shown in SEQ ID NO:9 (PRT9) is the amino acid sequence shown in SEQ ID NO:14 (PRT14) in which one cysteine residue has been inserted in each of the REPs located at positions 1 to 4 from the N-terminus and C-terminus of the domain sequence.

The amino acid sequence shown in SEQ ID NO:10 (PRT10) is the amino acid sequence shown in SEQ ID NO:14 (PRT14) in which one cysteine residue has been inserted in each of the REPs located at positions 1 to 8 from the N-terminus and C-terminus of the domain sequence.

The amino acid sequence shown in SEQ ID NO:28 (PRT28) is the amino acid sequence shown in SEQ ID NO:30 (PRT30) in which one cysteine residue has been inserted into the REP located at the most C-terminal end of the domain sequence.

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

The modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This allows the modified fibroin to be isolated, immobilized, detected, visualized, etc.

Examples of tag sequences include affinity tags that utilize specific affinity (binding ability, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). A His tag is a short peptide with about 4 to 10 histidine residues lined up, and has the property of specifically binding to metal ions such as nickel, and can be used to isolate modified fibroin by chelating metal chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 26 (amino acid sequence including a His tag).

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

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

Furthermore, a tag sequence that can be cleaved with 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 cleaved can be recovered.

More specific examples of modified fibroins containing a tag sequence include (iii) modified fibroins containing the amino acid sequence shown in 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); (iv) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 21 (PRT21), SEQ ID NO: 22 (PRT22), SEQ ID NO: 23 (PRT23), or SEQ ID NO: 24 (PRT24); and (v) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 29 (PRT29).

The modified fibroin (iii) may have only the amino acid sequence shown 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.

The GPGXX motif content of modified fibroins containing the amino acid sequences shown in 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) is 40.2%, 39.9%, 39.9%, 39.7%, 39.3%, and 38.6%, respectively, all of which are 10% or more.

The modified fibroin (iv) may have only the amino acid sequence shown 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 fibroins containing the amino acid sequences shown in SEQ ID NO:21 (PRT21), SEQ ID NO:22 (PRT22), SEQ ID NO:23 (PRT23), or SEQ ID NO:24 (PRT24) is 39.9%, 39.9%, 39.3%, and 38.6%, respectively, all of which are 10% or more.

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

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

[Nucleic Acid]
In one embodiment, the nucleic acid encodes the modified fibroin. Specific examples of the nucleic acid include a nucleic acid encoding a modified fibroin containing the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:28, or a modified fibroin having the amino acid sequence shown in SEQ ID NO:25 or SEQ ID NO:26 (tag sequence) bound to either or both of the N-terminus and C-terminus of the amino acid sequence.

A nucleic acid according to one embodiment is a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid encoding the above-mentioned modified fibroin, and encodes a modified fibroin comprising 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 of the modified fibroin encoded by the nucleic acid has an amino acid sequence corresponding to the insertion of a cysteine residue into REP, as compared with naturally-occurring fibroin.

"Stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. "Stringent conditions" may be low stringent conditions, medium stringent conditions, or high stringent conditions. Low stringent conditions mean that hybridization occurs only when there is at least 85% identity between the sequences, for example, 5xSSC containing 0.5% SDS is used and hybridization is performed at 42°C. Medium stringent conditions mean that hybridization occurs only when there is at least 90% identity between the sequences, for example, 5xSSC containing 0.5% SDS is used and hybridization is performed at 50°C. High stringent conditions mean that hybridization occurs only when there is at least 95% identity between the sequences, for example, 5xSSC containing 0.5% SDS is used and hybridization is performed at 60°C.

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

In one embodiment, the host 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 suitably used.

As an expression vector, one that is capable of autonomous replication in a host cell or that can be integrated into a host chromosome and contains a promoter at a position where the nucleic acid of 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 one embodiment is preferably a vector capable of autonomous replication in the prokaryote and including a promoter, a ribosome binding sequence, a nucleic acid according to one embodiment, and a transcription termination sequence. A gene that controls the promoter may also be included.

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

Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli BL21 (Novagen), Escherichia coli BL21(DE3) (Life Technologies), Escherichia coli BLR(DE3) (Merck Millipore), Escherichia coli DH1, Escherichia coli GI698, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli K5 (ATCC 23506), Escherichia coli KY3276, Escherichia coli MC1000, Escherichia coli MG1655 (ATCC 47076), and Escherichia coli No. 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W3110 (ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, etc.

Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus borstelensis, Brevibacillus centroporas, Brevibacillus formosus, Brevibacillus invocatus, Brevibacillus lathyrosporus, Brevibacillus limnophilus, Brevibacillus parabrevis, Brevibacillus reuszeri, Brevibacillus thermolvar, Brevibacillus brevis 47 (FERM BP-1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevibacillus brevis 47-5Q (JCM8975), Brevibacillus choshinensis HPD31 ... BP-1087), Brevibacillus choshinensis HPD31-S (FERM BP-6623), Brevibacillus choshinensis HPD31-OK (FERM BP-4573), Brevibacillus choshinensis SP3 strain (manufactured by Takara Biosciences), etc.

Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens ATCC14460, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesi, Serratia proteamaculans, Serratia odorifera, Serratia plymuthica, and Serratia rubidae. rubidaea) etc.

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

An example of a microorganism belonging to the genus Microbacterium is Microbacterium ammoniaphyllum ATCC15354.

Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC14067) ATCC13826, ATCC14067, Brevibacterium immariophilum (Brevibacterium immariophilum) ATCC14068, Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC13869) ATCC13665, ATCC13869, Brevibacterium roseum ATCC13825, Brevibacterium saccharolyticum (Brevibacterium saccharolyticum ATCC 14066, Brevibacterium thiogenitalis ATCC 19240, Brevibacterium album ATCC 15111, Brevibacterium serinum ATCC 15112, etc.

Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC6871, ATCC6872, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium acetoacidophilum ATCC14067, Corynebacterium acetos ... acetacidophilum) ATCC13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanolyticum ATCC21511, Corynebacterium callunae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060, Corynebacterium lilium ATCC15990, Corynebacterium melassecora ATCC17965, Corynebacterium thermoaminogenes AJ12340 (FERMBP-1539), Corynebacterium herculis ATCC13868, etc.

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

The method for introducing an expression vector into the host cell can be any method for introducing DNA into the host cell. For example, the method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP Patent Publication 63-248394), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979) can be mentioned.

Transformation of microorganisms belonging to the genus Brevibacillus can be carried out, for example, by the method of Takahashi et al. (J. Bacteriol., 1983, 156:1130-1134), the method of Takagi et al. (Agric. Biol. Chem., 1989, 53:3099-3100), or the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61:202-203).

Examples of vectors (hereinafter simply referred to as "vectors") into which the nucleic acid of one embodiment is introduced include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP10 (JP Patent Publication 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(-) (Stratagene), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), JP 60-221091 A, pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), etc.

When Escherichia coli is used as a host, suitable vectors include pUC18, pBluescriptII, pSupex, pET22b, and pCold.

Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (JP Patent Publication No. 2-31682), pNY700 (JP Patent Publication No. 4-278091), pHY4831 (J. Bacteriol., 1987, 1239-1245), pNU200 (Udaka Shigezo, Journal of the Japanese Society of Agricultural Chemistry, 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211 (J. Biochem., 1992, 112: 488-491), pNU2 Examples of such vectors include 11R2L5 (JP Patent Publication No. 7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58: 525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177: 745-749), pHT210 (JP Patent Publication No. 6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42: 358-363), and pNCO2 (JP Patent Publication No. 2002-238569), which is a shuttle vector between Escherichia coli and microorganisms belonging to the genus Brevibacillus.

There are no limitations on the promoter, so long as it functions in the host cell. Examples include promoters derived from Escherichia coli or phages, such as the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. Artificially designed and modified promoters, such as a promoter with two Ptrp promoters in series (Ptrp x 2), tac promoter, lacT7 promoter, and let I promoter, can also be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases). In an expression vector, a transcription termination sequence is not necessarily required for the expression of the above-mentioned nucleic acid, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.

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

Examples of yeasts include those belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia, and Hansenula. More specifically, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Trichosporon pullulans, Schwanniomyces alluvius, Schwanniomyces occidentalis, Candida utilis, utilis, Pichia pastoris, Pichia angusta, Pichia methanolica, Pichia polymorpha, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, etc.

When yeast is used as a host cell, the expression vector typically preferably contains an origin of replication (if amplification in the host is required) and a selectable marker for propagation of the vector in E. coli, a promoter and terminator for recombinant protein expression in yeast, and a selectable marker for yeast.

When the expression vector is a non-integrative vector, it is preferable that it further contains an autonomously replicating sequence (ARS). This can improve the stability of the expression vector in the cell (Myers, A.M., et al. (1986) Gene 45:299-310).

When yeast is used as a host, examples of vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, and pPICZ B.

There are no limitations on the promoter, so long as it can be expressed in yeast. Examples include promoters of glycolytic genes such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP 1 promoter, pGAP promoter, pGCW14 promoter, AOX1 promoter, MOX promoter, etc.

As a method for introducing an expression vector into yeast, any method for introducing DNA into yeast can be used, such as electroporation (Methods Enzymol., 194, 182 (1990)), spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), lithium acetate method (J. Bacteriol., 153, 163 (1983)), and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

Examples of filamentous fungi include the genus Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, Penicillium, and the like. Examples of fungi that may be included include those belonging to the genera Myceliophtora, Botrytis, Magnaporthe, Mucor, Metarhizium, Monascus, Rhizopus, and Rhizomucor.

Specific examples of filamentous fungi include Acremonium alabamense, Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus oryzae, Aspergillus sake, Aspergillus sojae, Aspergillus tubigensis ... Aspergillus tubigensis, Aspergillus niger, Aspergillus nidulans, Aspergillus parasiticus, Aspergillus ficum, Aspergillus phoeicus, Aspergillus foetidus, Aspergillus flavus, Aspergillus fumigatus fumigatus, Aspergillus japonicus, Trichoderma viride, Trichoderma harzianum, Trichoderma reesei, Chrysosporium lucknowense, Thermoascus, Sporotrichum, Sporotrichum cellulophilum cellulophilum, Talaromyces, Thielavia terrestris, Thielavia, Neurospora crassa, Fusarium oxysporus, Fusarium graminerum, Fusarium venenatum, Humicola insolens, Penicillium chrysogenum, Penicillium camemberti camemberti, Penicillium canescens, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium purpurogenum, Penicillium roqueforti, Myceliophtaora thermophilum, Mucor ambigus Mucor circinelloides, Mucor fragilis, Mucor hiemalis, Mucor inaequisporus, Mucor oblongiellipticus, Mucor racemosus, Mucor recurvus, Mucor saturninus, Mucor subtilismius Examples of such species include Rhizopus arrhizus, ...

When the host is a filamentous fungus, the promoter may be any of genes related to glycolysis, genes related to constitutive expression, and enzyme genes related to hydrolysis, and specific examples include amyB, glaA, agdA, glaB, TEF1, xynF1 tannasegene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, and catB.

Introduction of an expression vector into a filamentous fungus can be carried out using a conventional method. Examples of such methods include the method of Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69:2110 (1972)], the protoplast method [Mol. Gen. Genet., 168:111 (1979)], the competent method [J. Mol. Biol., 56:209 (1971)], and the electroporation method.

Examples of insect cells include lepidopteran insect cells, more specifically insect cells derived from Spodoptera frugiperda such as Sf9 and Sf21, and insect cells derived from Trichoplusia ni such as High 5.

When insect cells are used as hosts, examples of vectors include baculoviruses such as Autographa californica nuclear polyhedrosis virus, which is a virus that infects insects of the family Noctuidae (Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)).

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

Examples of methods for co-introducing a recombinant gene transfer vector and a baculovirus into insect cells to prepare a recombinant virus include the calcium phosphate method (JP Patent Publication 2-227075) and the lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).

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

A host transformed with an 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. The probe can be a partial DNA fragment amplified by PCR based on the sequence information of the nucleic acid and modified with a radioisotope or digoxigenin.

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

The modified fibroin according to this embodiment can be produced, for example, by culturing a host transformed with the expression vector in a culture medium, producing and accumulating the modified fibroin according to this embodiment in the culture medium, and harvesting it from the culture medium. The method for culturing the host in the culture medium can be performed according to a method typically used for culturing a host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for the host may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host and allows efficient cultivation of the host.

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

As nitrogen sources, for example, ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria and their digestions can be used.

Examples of inorganic salts that can be used include potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

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

If necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium 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 necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a trp promoter.

As culture media for insect cells, commonly used TNM-FH medium (manufactured by Pharmingen), Sf-900 II SFM medium (manufactured by Life Technologies), ExCell 400, ExCell 405 (both manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788 (1962)), etc. can be used.

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

When the host is a plant cell, the transformed plant cell may be cultured as is, or may be differentiated into a plant organ and then cultured. As a medium for culturing the plant cell, the commonly used Murashige and Skoog (MS) medium, White medium, or a medium to which a plant hormone such as auxin or cytokinin has been added can be used.

Animal cells can be cultured for 3 to 60 days under conditions such as a culture medium pH of 5 to 9 and a culture temperature of 20 to 40°C. In addition, antibiotics such as kanamycin and hygromycin may be added to the medium during culture as necessary.

Methods for producing modified fibroin using a host transformed with the above expression vector include a method for producing the modified fibroin within 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. Each of these methods can be selected by changing the host cell used and the structure of the modified fibroin to be produced.

For example, when the modified fibroin is produced inside a host cell or on the outer membrane of the host cell, the modified fibroin can be modified to be actively secreted outside the host cell by applying, mutatis mutandis, the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Rowe et al. (Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)), or the methods described in JP-A-5-336963 and WO 94/23021. That is, the modified fibroin can be actively secreted outside the host cell by expressing a polypeptide containing the active site of the modified fibroin in a form in which a signal peptide has been added using a genetic recombination technique.

The modified fibroin produced by the host transformed with the expression vector can be isolated and purified by methods commonly used for isolating and purifying proteins. For example, when the modified fibroin is expressed in a dissolved state within the cells, after the culture is completed, the host cells are collected by centrifugation and suspended in an aqueous buffer solution, and then disrupted using an ultrasonic homogenizer, French press, Manton Gaulin homogenizer, Dyno Mill, or the like to obtain a cell-free extract. The supernatant obtained by centrifuging the cell-free extract can be used to obtain a purified sample by using methods commonly used for isolating and purifying proteins, such as solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing, either alone or in combination.

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

In addition, when the modified fibroin is expressed by forming an insoluble body within the cells, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble body of the modified fibroin as a precipitate fraction. The recovered insoluble body of the modified fibroin can be solubilized with a protein denaturant. After this operation, a purified preparation of the modified fibroin can be obtained by the same isolation and purification method as above.

When the modified fibroin or a derivative in which a sugar chain has been added to the modified fibroin is secreted outside the cells, the modified fibroin or its derivative can be recovered from the culture supernatant. That is, the culture is treated by a method such as centrifugation to obtain a culture supernatant, and a purified specimen can be obtained from the culture supernatant by the same isolation and purification method as described above.

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

The protein solution can be obtained, for example, by dissolving the crosslinked modified fibroin 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 other components in addition to the crosslinked modified fibroin and the solvent. Examples of the other components include proteins other than the crosslinked modified fibroin.

The content of the crosslinked modified fibroin in the protein solution and the total content of the protein in the protein solution may be adjusted as appropriate depending on the shape of the molded body, etc. Methods for adjusting these include, but are not limited to, increasing the concentration of the crosslinked modified fibroin or the protein concentration by volatilizing the solvent by distilling the protein solution, using a solution with a high protein concentration in the dissolving process, or reducing the amount of solvent added relative to the amount of protein.

When the molded article containing the crosslinked modified fibroin is a fiber, the molding process may be carried out, for example, by spinning a protein solution. In this case, the protein solution may be adjusted to a viscosity suitable for spinning. The viscosity suitable for spinning may generally be 1000 to 50,000 cP (centipoise) at 40°C. The viscosity can be measured, for example, using an "EMS Viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd.

When the molded article containing the crosslinked modified fibroin is a fiber, the total protein content (concentration) in the protein solution may be adjusted to a concentration and viscosity that allows spinning, if necessary. The method for adjusting the total protein content and the viscosity of the protein solution is not particularly limited. In addition, examples of spinning methods include wet spinning. When a protein solution adjusted to a concentration and viscosity suitable for spinning is applied as a dope liquid to a coagulation liquid, the protein coagulates. In this case, by applying the protein solution as a thread-like liquid to the coagulation liquid, the protein coagulates into a thread-like shape, and a thread (undrawn thread) can be formed. The formation of the undrawn thread can be performed, for example, in accordance with the method described in Patent Publication No. 5,584,932.

Below, an example of wet spinning is shown, but the spinning method is not particularly limited and may be wet spinning, dry spinning, dry-wet spinning, etc.

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

The method of applying the protein solution as a thread-like liquid is not particularly limited, but for example, it can be extruded (discharged) from a spinning nozzle into the coagulation liquid in a desolvation tank. The protein coagulates to obtain an undrawn thread. The extrusion (discharge) speed when the protein solution is extruded (discharged) into the coagulation liquid can be set appropriately depending on the diameter of the nozzle and the viscosity of the protein solution, but for example, in the case of a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm, the extrusion (discharge) speed may be 0.2 to 6.0 mL/h per hole, or 1.4 to 4.0 mL/h per hole, from the viewpoint of spinning stability. The length of the desolvation tank (coagulation liquid tank) in which the coagulation liquid is placed is not particularly limited, but for example, the length may be 200 to 500 mm. The take-up speed of the undrawn thread formed by protein coagulation may be, for example, 1 to 14 m/min, and the residence time may be, for example, 0.01 to 0.15 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 protein coagulation may be further drawn (pre-drawn) in the coagulation liquid, but from the viewpoint of suppressing evaporation of the lower alcohol used in the coagulation liquid, the coagulation liquid may be kept at a low temperature and the yarn may be taken up from the coagulation liquid in the undrawn state.

(b) Drawing The undrawn yarn obtained by the above method may further include a step of drawing. The drawing may be a one-stage drawing or a multi-stage drawing of two or more stages. Multi-stage drawing is suitable for producing a fiber with high toughness because the molecules are oriented in multiple stages and the total draw ratio can be increased.

When the molded product produced using the crosslinked modified fibroin is a film (raw material film), the protein solution may be adjusted to a concentration and viscosity that allows film formation, if necessary. Methods for forming a protein film include, but are not limited to, a method in which the protein solution is applied to a solvent-resistant flat plate to a predetermined thickness to form a coating film, and the solvent is removed from the coating film to obtain a film of the predetermined thickness.

One example of a method for forming a film of a predetermined thickness is the casting method. When forming a film by the casting method, a protein solution is cast onto a flat plate using a tool such as a doctor coater or knife coater to a thickness of several microns or more to form a cast film, and then the solvent is removed by drying under reduced pressure or immersion in a desolvation tank to obtain a raw film (polypeptide film). The raw film can be formed in accordance with the method described in Japanese Patent No. 5678283.

When the molded product produced using the crosslinked modified fibroin is a molded product (raw material molded product), the method for forming the raw material molded product is not particularly limited. For example, after introducing a dry protein powder containing a crosslinked modified fibroin into a pressure molding machine, the dry protein powder can reach the temperature required for molding by applying pressure and heat using a hand press or the like to obtain a raw material molded product. In addition, the raw material molded product can be formed in accordance with the method described in the patent document (WO2017/047503).

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

When the molded body produced using the crosslinked modified fibroin is a porous body, the protein solution may be adjusted to a concentration and viscosity that allows for porosity, if necessary. The method for forming the porous body is not particularly limited. For example, a method of 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 to obtain a raw porous body, or a method similar to that described in Patent No. 5796147, etc. may be used.

When the molded body produced using the modified fibroin crosslinked body is a particle, the method of forming the particle is not particularly limited. The particle can be obtained, for example, by a method including a step of obtaining an aqueous solution of a protein by replacing the 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 of such solvent include water, a water-soluble buffer solution, and physiological saline. The step of replacing with a water-soluble solvent is preferably carried out by putting the dope solution into a dialysis membrane, immersing it in the water-soluble solvent, and replacing the water-soluble solvent one or more times. Specifically, it is more preferable to put the dope solution into the dialysis membrane, leave it in a water-soluble solvent (for one time) that is 100 times or more the amount of the dope solution for three hours, and repeat this water-soluble solvent replacement three or more times in total. The dialysis membrane may be any membrane that does not allow proteins to pass through, and may be, for example, a cellulose dialysis membrane. By repeating the replacement of the water-soluble solvent, the amount of the solvent present in the dope solution can be brought close to zero. In the latter half of the step of replacing with a water-soluble solvent, the dialysis membrane does not need to be used. The step of drying the aqueous protein solution preferably uses vacuum freeze-drying. The degree of vacuum during vacuum freeze-drying is preferably 200 Pascals (Pa) or less, more preferably 150 Pascals or less, and even more preferably 100 Pascals or less. The moisture content of the particles after freeze-drying is preferably 5.0% or less, more preferably 3.0% or less.

In the manufacturing method according to this embodiment, the degree of the physical properties of the protein (e.g., the durability of the protein) can be controlled by controlling the crosslinking reaction.

The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples.

[Production of modified fibroin]
(1) Preparation of expression vectors As modified fibroins having cysteine residues, modified fibroin having the amino acid sequence shown in SEQ ID NO: 16 (PRT16), modified fibroin having the amino acid sequence shown in SEQ ID NO: 29 (PRT29), and modified fibroin having the amino acid sequence shown in SEQ ID NO: 32 (PRT32) were designed.

As modified fibroins without cysteine residues, modified fibroin (PRT27) having the amino acid sequence shown in SEQ ID NO:27 and modified fibroin (PRT30) having the amino acid sequence shown in SEQ ID NO:30 were designed.

The amino acid sequence shown in SEQ ID NO:16 (PRT16) is obtained by inserting one cysteine residue into the REP of the domain sequence of the amino acid sequence shown in SEQ ID NO:27 (PRT27). The total number of cysteine residues in 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 having the amino acid sequence shown in SEQ ID NO:29 (PRT29) is obtained by inserting one cysteine residue into the REP located at the most C-terminal end of the domain sequence of the amino acid sequence shown in SEQ ID NO:30 (PRT30). The total number of cysteine residues in the modified fibroin having the amino acid sequence shown in SEQ ID NO:29 is 1. The molecular weight of the modified fibroin having the amino acid sequence shown in SEQ ID NO:29 is 300 kDa.

The amino acid sequence shown in SEQ ID NO:32 (PRT32) is obtained by inserting two cysteine residues into the REP of the domain sequence of the amino acid sequence shown in SEQ ID NO:27 (PRT27). The total number of cysteine residues in 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 fibroins having the above-mentioned amino acid sequences were synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. This nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Protein Production Escherichia coli BLR (DE3) was transformed with the obtained expression vector. The transformed Escherichia 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 the OD ₆₀₀ became 0.005. The culture solution temperature was kept at 30° C., and flask culture was continued until the OD ₆₀₀ became 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 2) so that the OD ₆₀₀ was 0.05. The culture temperature was kept at 37° C., and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture 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 culture temperature was kept at 37°C, and the culture was controlled to a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 1 mM to induce the expression of the modified fibroin. 20 hours after the addition of IPTG, the culture was centrifuged and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of a band of the desired modified fibroin size depending on the addition of IPTG.

(3) Protein purification The cells harvested 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg/mL, and stirred with a stirrer at 60 ° C. for 30 minutes to dissolve. After dissolution, the solution was dialyzed against water using a dialysis tube (Cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.) The white aggregated protein obtained after dialysis was collected by centrifugation, and the water was removed using a freeze-dryer to collect the freeze-dried powder, thereby obtaining powdered modified fibroins (PRT16, PRT29, PRT27, and PRT32).

[Test Example 1: Production of crosslinked modified fibroin 1]
(Reaction using modified fibroin having cysteine residues)
Example 1
A modified fibroin solution was prepared by adding 20 mg of modified fibroin having a cysteine residue (modified fibroin having an amino acid sequence in which one cysteine residue is included in REP) PRT16 and 1 mL of hexafluoroisopropanol (manufactured by Central Glass Co., Ltd.) to a glass container, and dissolving the modified fibroin PRT16 in hexafluoroisopropanol. The modified fibroin solution was irradiated with 365 nm ultraviolet light (manufactured by Spectroline, MODELEN-1601/J, 100V) for 20 hours while stirring at room temperature (25°C) to cause a reaction. After distilling off the solvent, the product was obtained by drying under reduced pressure. This was used as the sample of Example 1 for subsequent analysis.

Comparative Example 1-1
A modified fibroin having a cysteine residue (a modified fibroin having an amino acid sequence in which one cysteine residue is included in REP) PRT16 was used as a sample of Comparative Example 1-1 in the subsequent analysis.

(Reaction using modified fibroin having no 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, the product was dried under reduced pressure to obtain a product. This product was used in the subsequent analysis as a sample of Comparative Example 1-2.

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

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

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

GPC measurements confirmed that the sample obtained by irradiating light to modified fibroin having cysteine residues (Example 1) had an increased molecular weight compared to the sample not irradiated with light (Comparative Example 1-1). On the other hand, in the sample obtained by irradiating light to modified fibroin not having cysteine residues (Comparative Example 1-2), no increase in molecular weight was confirmed, and decomposition of the modified fibroin molecules was confirmed (Comparative Example 1-2 vs. Comparative Example 1-3).

(Confirmation of molecular weight of product by SDS-PAGE)
The molecular weight of the product was analyzed by SDS-PAGE for the samples of the Examples and Comparative Examples. The results are shown in Figure 3. In Figure 3, lane 1 shows the results of the standard sample (STD), lane 2 shows the results of Example 1, lane 3 shows the results of Comparative Example 1-1, lane 4 shows the results of Comparative Example 1-2, and lane 5 shows the results of Comparative Example 1-3.

The product (Example 1) obtained by irradiating modified fibroin PRT16, which has cysteine residues, with light showed an increase in molecular weight due to disulfide bond formation between sulfur atoms in the cysteine residues, as confirmed by both GPC and SDS-PAGE analysis.

On the other hand, when modified fibroin PRT27, which does not have cysteine residues, was irradiated with light, no increase in molecular weight occurred (compare Comparative Example 1-2 and Comparative Example 1-3). This demonstrated that the formation of disulfide bonds between cysteines is necessary for the light-induced molecular weight increase reaction of modified fibroin. Furthermore, since cysteine can be selectively reacted in a situation where it coexists with histidine contained in the tag sequence, it can be said that the reaction in the present invention is suitable for crosslinking of tag sequence-containing proteins that have industrial applicability.

[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, and 1 mL of hexafluoroisopropanol (manufactured by Central Glass Co., Ltd.) were placed in a glass container, and the modified fibroin and 2,2-dimethoxy-2-phenylacetophenone were dissolved in hexafluoroisopropanol to obtain a modified fibroin solution. The modified fibroin solution was irradiated with 365 nm ultraviolet light for a predetermined time while stirring at room temperature (25 ° C.) (SPECTROLINE, MODEL EN-1601 / J, 100 V) to perform a reaction. After distilling off the solvent, the solution was washed three times with methanol (50 mL x 3, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) to remove 2,2-dimethoxy-2-phenylacetophenone. The product was obtained by further drying under reduced pressure. The samples obtained by irradiating with ultraviolet light for 15 minutes, 30 minutes, 45 minutes, 60 minutes and 90 minutes were designated as Examples 2-1, 2-2, 2-3, 2-4 and 2-5, respectively, and were used for the subsequent analysis.

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

It was confirmed that when light was irradiated in the presence of a photosensitizer, the target reaction occurred more efficiently than when the photosensitizer was not present. The amount of crosslinked bodies obtained increased with increasing UV irradiation time up to 45 minutes, and there was a tendency for the amount to decrease thereafter.

[Confirmation of molecular weight of product by SDS-PAGE]
Measurement of molecular weight by SDS-PAGE was carried out under the same conditions as in Test Example 1 for the products obtained in Examples 2-1, 2-2, 2-3, 2-4, and 2-5. The results are shown in FIG. 5. PRT16 was used in the analysis as Comparative Example 2. In FIG. 5, lane 1 shows the results of the standard (STD), lane 2 shows the results of Example 2-1 (light irradiation time: 15 minutes), lane 3 shows the results of Example 2-2 (light irradiation time: 30 minutes), lane 4 shows the results of Example 2-3 (light irradiation time: 45 minutes), lane 5 shows the results of Example 2-4 (light irradiation time: 60 minutes), lane 6 shows the results of Example 2-5 (light irradiation time: 90 minutes), and lane 7 shows the results of Comparative Example 2 (no light irradiation).

An increase in molecular weight was also confirmed by SDS-PAGE analysis. The results of Example 2-1 show that a crosslinked product can be obtained in as little as 15 minutes.

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

The above results show that while it is expected that improving reactivity with a photosensitizer could result in cross-linking reactions of amino acids other than the intended one, such as histidine, and thus a decrease in selectivity, the selectivity of the cross-linking reaction of cysteine was actually improved.

(Confirmation of molecular weight of product by GPC)
The modified fibroin PRT29, which has a different sequence and is highly hydrophilic from the modified fibroin PRT16 used in Example 1, was used, and the desired crosslinked product was obtained even under larger-scale conditions. The results are shown in FIG.

[Test Example 4: Production of crosslinked modified fibroin 3]
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.) were added to a glass container, and the modified fibroin and 2,2-dimethoxy-2-phenylacetophenone were dissolved in hexafluoroisopropanol to obtain a modified fibroin solution. The modified fibroin solution was irradiated with 365 nm ultraviolet light (SPECTROLINE, MODEL EN-1601/J, 100V) for 2 hours while stirring at room temperature (25°C) to cause a reaction. After distilling off the solvent, the product was obtained by drying under reduced pressure. The obtained sample was used for the subsequent analysis as Example 4.
In addition, PRT32 before the reaction was used as a sample of Comparative Example 4 for 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. The results are shown in FIG.

Even in this reaction using a molecule with two cysteines in one molecule, it was confirmed that the reaction between sulfur atoms contained in the cysteines in the protein proceeded, producing a protein with an increased molecular weight.

Even in the reaction using a molecule having two cysteines in the same molecule, it was confirmed that the reaction between the sulfur atoms contained in the cysteine residues in the modified fibroin proceeded, resulting in the production of modified fibroin with an increased molecular weight (modified fibroin crosslinked product).

Test Example 5: Reaction using isocyanate
A modified fibroin having a cysteine residue (modified fibroin having an amino acid sequence in which one cysteine residue is included in the REP) PRT16 was reacted with hexamethylene diisocyanate in the presence of a solvent (hexafluoro-2-propanol (HFIP)). This produced a gel-like substance.

(result)
When the modified fibroin PRT16 having cysteine residues was irradiated with light, the desired crosslinked product was generated and isolated. On the other hand, when isocyanate was used, a gel-like substance was formed, the yield was significantly reduced, and a mixture of various types of products was produced, making isolation difficult. Furthermore, the peak of the desired crosslinked product could not be confirmed by GPC analysis.

Claims (12)

A method for producing a modified fibroin crosslinked body in which modified fibroin molecules are crosslinked, comprising the steps of:
a crosslinking step of irradiating the modified fibroin with light to form a disulfide bond by an oxidation reaction to obtain the modified fibroin crosslinked product;
The modified fibroin is a fibroin whose amino acid sequence is different from the amino acid sequence of naturally occurring fibroin,
The modified fibroin has a cysteine residue,
The total number of cysteine residues in the modified fibroin is 1 or more and 6 or less ,
The method, wherein 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.
[In formula 1 and formula 2, (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in (A) _n motif is 80% or more. REP represents an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other or different amino acid sequences.] The method according to claim 1, wherein the crosslinking step is carried out by irradiating the modified fibroin with light in the presence of a photosensitizer. The method of claim 2, wherein the photosensitizer is a ketone. The method according to any one of claims 1 to 3, wherein the crosslinking step is carried out by irradiating a modified fibroin solution containing the modified fibroin and a solvent that dissolves 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 molecular weight of the modified fibroin is 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 method according to any one of claims 1 to 7, wherein the domain sequence has an amino acid sequence corresponding to the insertion of a cysteine residue in the REP located near the N-terminus and/or C-terminus of the domain sequence, compared to naturally occurring fibroin . The method according to any one of claims 1 to 8, wherein the domain sequence has an amino acid sequence corresponding to an insertion of 1 to 6 cysteine residues in REP compared to naturally occurring fibroin . The method according to any one of claims 1 to 9, wherein the modified fibroin contains a histidine tag sequence at either or both of the N-terminus and 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 the light is irradiated for 15 minutes or more in the crosslinking step.

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