JP2020121958A - Protein molding manufacturing method - Google Patents

Abstract

To provide a method allowing simple manufacture of a protein molding.SOLUTION: A protein molding manufacturing method comprises obtaining a protein molding by contacting a molding precursor with water. The molding precursor is a reaction mixture of a dope solution, which contains a modified fibroin and a solvent, and a reactant having a reactive group which can react with a protein to form a bond. The modified fibroin comprises a domain sequence represented by formula 1, [(A)motif-REP], or formula 2, [(A)motif-REP]-(A)motif. The domain sequence has an amino acid sequence such that a lysine residue is inserted in REP compared to a naturally occurring fibroin. [In formulas 1 and 2, (A)motif represents an amino acid sequence comprising 4-27 amino acid residues, where the number of alanine residues is 80% or more of the total number of amino acid residues in (A)motif; REP represents an amino acid sequence comprising 10-200 amino acid residues; m represents an integer from 10 to 300; the multiple occurrences of (A)motif may be amino acid sequences identical to or different from each other; and the multiple occurrences of REP may be amino acid sequences identical to or different from each other.]SELECTED DRAWING: None

Description

The present invention relates to a method for producing a protein molded body.

In recent years, the materialization of biodegradable proteins has been actively researched in response to increasing awareness of environmental conservation. For example, Non-Patent Document 1 reports a method of obtaining a bioplastic by heating and compressing silk powder or wool powder to make it a resin. In Patent Document 1, a powdered artificial spider silk protein is heated and compressed. By doing so, it is described that a resin molded body made of an artificial spider silk protein is obtained. These so-called artificial protein molded bodies are expected to be more and more demanded because, unlike molded bodies made of petroleum-based materials such as synthetic fibers and synthetic resins, the energy required for production and processing is small. Under such circumstances, realization of a method for more easily obtaining an artificial protein molded body is expected.

International Publication No. 2017/047504

Shinji Hirai, Monthly Journal of Functional Materials, June 2014, "Environmentally Friendly Silk and Wool Resins Using Waste-Derived Animal Proteins"

An object of the present invention is to provide a method capable of easily producing a protein molded body.

The present invention relates to the following inventions, for example.
[1]
A step of contacting a molded body precursor with water to obtain a protein molded body,
The molded body precursor is a reaction mixture of a dope solution containing modified fibroin and a solvent, and a reactive agent having a reactive group capable of reacting with a protein to form a bond,
The modified fibroin comprises a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , or the formula 2: [(A) _n motif-REP] _m -(A) _n motif,
A method for producing a molded protein, wherein the domain sequence has an amino acid sequence corresponding to insertion of a lysine residue in REP as compared with naturally-occurring fibroin.
[In Formula 1 and Formula 2, the (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues is 80% of the total number of amino acid residues in the (A) _n motif. That is all. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m shows the integer of 10-300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences. ]
[2]
The domain sequence contains a GPGXX (where X represents an amino acid residue other than a glycine residue) motif in REP, and the content rate of the GPGXX motif is 10% or more. Production method.
[3]
The domain sequence contains a glycine residue, a serine residue, or an alanine residue in REP, and a lysine residue is inserted at a position adjacent to the glycine residue, the serine residue, or the alanine residue in REP. The method for producing a protein molded product according to [1] or [2], which has an amino acid sequence corresponding to the above.
[4]
Any of [1] to [3], wherein the domain sequence has an amino acid sequence corresponding to insertion of a lysine residue at or near the center of the amino acid sequence in REP, as compared with naturally-occurring fibroin. A method for producing a protein molded body according to item 1.
[5]
The domain sequence has a hydrophobic amino acid residue in REP, and has an amino acid sequence corresponding to insertion of a lysine residue at a position adjacent to the hydrophobic amino acid residue in REP, [1] ~ The method for producing a protein molded product according to any one of [4].
[6]
The method for producing a protein molded product according to any of [1] to [5], wherein the naturally-derived fibroin is fibroin derived from insects or arachnids.
[7]
The method for producing a protein molded product according to claim 1, wherein the naturally-derived fibroin is a large bottle-shaped spider protein (MaSp) or a small bottle-shaped spider protein (MiSp) of arachnids.

According to the present invention, it becomes possible to provide a method capable of easily producing a protein molded body.

It is a schematic diagram which shows an example of the domain sequence of fibroin. 3 is a graph showing the results of viscosity measurement when producing a molded body precursor in Example 1. 1 is a photograph showing a molded body precursor and a protein molded body in Example 1. 5 is a graph showing the results of measuring the viscosity of the molded body precursor in Example 2. 5 is a photograph showing a protein molded body in Example 2.

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

[Method for producing molded protein]
The method for producing a protein molded body according to the present embodiment includes a step (contact step) of bringing a molded body precursor into contact with water to obtain a protein molded body. The molded body precursor is a reaction mixture of a dope solution containing modified fibroin (details will be described later) and a solvent, and a reactive agent having a reactive group capable of reacting with a protein to form a bond.

(Modified fibroin)
The modified fibroin according to the present embodiment has a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. It is a protein containing. The modified fibroin may have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically, but not limited to, regions having no repeat of the amino acid motif characteristic of fibroin, and consist of about 100 amino acids.

As used herein, the term "modified fibroin" means fibroin whose amino acid sequence differs from that of naturally-occurring fibroin. As used herein, the term "naturally-derived fibroin" means fibroin whose amino acid sequence is the same as fibroin produced by naturally occurring insects, arachnids and the like. Naturally-derived 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. Is.

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

Examples of fibroin produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamai cya cya pyramid, Anteraea pyramyi, Ori a pu rye, Pomegranate (Anteraea periyi), Pomegranate (Anteraea periyi), Anteraea peryany (Etera peri ny) ), silkworm (Samia cynthia), chrysanthemum (Caligura japonica), chusser silkworm (Antheraea mylitta), silk proteins produced by silkworms such as Anthera ea limata (Antheraea assama). Silk protein is mentioned.

Specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence), AAA278840.1 (amino acid sequence)).

Examples of fibroin produced by spiders include spiders belonging to the genus Araneus (genus Araneus) such as Onigumo, Niwaonigamo, Akanonigumo, Aonigumo and Maeoniguimo, spiders such as Yamashiroonigomo, Jononigu spp. Spiders belonging to the genus Proton, spiders belonging to the genus Pronus, spiders belonging to the genus Cyrtarachne (genus Cyrtarachne), such as spider spider, spider spider Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius such as Mamei Taiseki spider and Mutsugai spider, and belonging to the genus Argiopsis such as Argiogiope, Argiope spp. , Spiders belonging to the genus Cytophora, such as Spiders belonging to the genus Acusilas, such as Spiders belonging to the genus Arachnura, Spiders belonging to the genus Cytophora such as Spiders, Spiders, and Black Spiders. Spider silk protein produced by spiders belonging to the genus Cyclosa (genus Cyclosa), such as spiders belonging to the category ), spider silk proteins, and spider silk spiders belonging to the genus Chorizopes, such as the spider silk spider. Spiders belonging to the genus Tetragnatha, such as the herring-tailed spider, sword-tailed duck spider, and urocore, and the spider belonging to the genus Tetragnatha, the genus Leucaug, which belongs to the genus Leucaug, such as the genus Leucagu Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira, such as black spiders, spiders belonging to the genus Dyschiriognatha, such as dwarf spiders, such as the black-breasted spider, black widow spider, and the spider, Latrodectus mactans. Spiders produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus (genus Latrorectus) and spiders belonging to the genus Euprosthenops (genus Euprosthenops). Examples include pider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of fibroin produced by spiders include, for example, fibroin-3 (adf-3) [from Araneus diadematus] (GenBank Accession No. AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-. 4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenACank4 accession number Amino acid sequence ANC50). U37520 (base sequence)), major angu11ate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spirabel2a2A2c [Negative]. (Amino acid sequence), AF441245 (base sequence), major ampulate spidroin 1 [derived from Euprothenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampulthrates epiroin 2 [Europrosperus entropus]. Accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampulate silk NP 2[N] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys cruenta] (GenBank Accession No. ABR 37278.1 (amino acid sequence) and the like.

More specific examples of naturally-derived fibroin include fibroin whose sequence information is registered in NCBI GenBank. For example, among sequences containing INV as DIVISION among the sequence information registered in NCBI GenBank, spidroin, complete, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific product from CDS, and a sequence in which a specific character string is described from SOURCE to TISSUE TYPE.

“Modified fibroin” refers to a naturally-occurring fibroin that has an amino acid sequence specified in the present invention and has its amino acid sequence modified (for example, a gene sequence of a cloned naturally-derived fibroin is modified). By modifying the amino acid sequence), or by artificially designing the amino acid sequence independently of naturally-occurring fibroin (for example, by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). (Having a desired amino acid sequence). A modified fibroin having a modified amino acid sequence is also included in the modified fibroin as long as the amino acid sequence is different from the amino acid sequence of naturally-derived fibroin.

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

In the (A) _n motif, the number of alanine residues to the total number of amino acid residues in the (A) _n motif may be 80% or more, preferably 85% or more, and preferably 90% or more. It is more preferably 95% or more, still more preferably 100% (meaning that it is composed of only alanine residues). It is preferable that at least seven of the (A) _n motifs present in the domain sequence are composed of only alanine residues. It is composed of only alanine residues, and the (A) _n motif is (Ala) _k (Ala represents an alanine residue, k is an integer of 4 to 27, preferably an integer of 4 to 20, and more preferably It shows that it has an amino acid sequence represented by 4).

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

One or more of the amino acid residues that make up REP may be hydrophobic amino acid residues. That is, the REP preferably contains a hydrophobic amino acid residue. The hydrophobic amino acid residue means an amino acid residue having a positive hydrophobicity index. Regarding the hydrophobic index of amino acid residues (hydropathic index, hereinafter also referred to as “HI”), a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for dispensing thehydrophering”). character of a protein", J. Mol. Biol., 157, pp. 105-132). Examples of the hydrophobic amino acid residue 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 the present embodiment, the domain sequence has an amino acid sequence corresponding to insertion of a lysine residue in REP, as compared with naturally occurring fibroin.

The domain sequence preferably has an amino acid sequence corresponding to the insertion of a lysine residue at a position adjacent to a glycine residue, a serine residue, or an alanine residue in REP. It is more preferable to have an amino acid sequence corresponding to the insertion of a lysine residue at a position adjacent to the glycine residue. When a lysine residue exists at a position adjacent to a glycine residue, a serine residue, or an alanine residue, the side chain of the lysine residue has a wide range of motion, and therefore the amino group present in the side chain of the lysine residue Thus, the efficiency of the reaction via the is further improved. Thereby, a protein molded product having desired physical properties can be produced more advantageously. The lysine residue in REP may be located between a glycine residue, a serine residue, or an alanine residue, and a glycine residue, a serine residue, or an alanine residue. It may be located between the residues.

The domain sequence preferably has an amino acid sequence corresponding to insertion of a lysine residue at a position adjacent to a hydrophobic amino acid residue in REP. In this case, the hydrophobic amino acid residues are immobilized between the molecules by hydrophobic interaction, so that the efficiency of the reaction via the amino group present in the side chain of the lysine residue is further improved. The lysine residue in REP may be located next to the hydrophobic amino acid residue, or 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 a hydrophobic amino acid residue and a glycine residue, a serine residue, or an alanine residue, and may be located between a hydrophobic amino acid residue and a glycine residue. The hydrophobic amino acid residue may be one selected from the group consisting of isoleucine residue, valine residue, leucine residue, phenylalanine residue, methionine residue, and alanine residue.

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

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

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

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

In the present specification, the “GPGXX motif content rate” is a value calculated by the following method. Formula A: [(A) _n Motif-REP] _m , or Formula B: [(A) _n Motif-REP] _m- (A) In the fibroin containing the domain sequence represented by the _n motif, the most C-terminal side In all REPs contained in the sequence excluding the sequence from the located (A) _n motif to the C-terminal of the domain sequence from the domain sequence, the total number of GPGXX motifs contained in the region is tripled (ie, (Corresponding to the total number of G and P in the GPGXX motif) is c, and the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and further (A) _n motif is added. When the total number of amino acid residues of all REPs removed is d, the GPGXX motif content rate is calculated as c/d.

In the calculation of the GPGXX motif content, "the sequence in which the sequence from the (A) _n motif located at the most C-terminal to the C-terminal of the domain sequence is removed from the domain sequence" is "the most C-terminal side". The sequence (A) from the _n- motif to the C-terminal of the domain sequence" (sequence corresponding to REP) may include a sequence having low correlation with the characteristic sequence of fibroin, and m is small. In this case (that is, when the domain sequence is short), it affects the calculation result of the GPGXX motif content rate, and is for eliminating this effect. When the “GPGXX motif” is located at the C-terminal of REP, it is treated as a “GPGXX motif” even if “XX” is “AA”.

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

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

In the present specification, “hydrophobicity of REP” is a value calculated by the following method. Formula A: [(A) _n Motif-REP] _m , or Formula B: [(A) _n Motif-REP] _m- (A) In the fibroin containing the domain sequence represented by the _n motif, the most C-terminal side In all REPs contained in the sequence (the sequence corresponding to “region A” in FIG. 1) in which the sequence from the located (A) _n motif to the C terminus of the domain sequence is excluded from the domain sequence, each amino acid in that region The total hydrophobicity index of residues is designated as e, and the sequence from the (A) _n motif located closest to the C terminus to the C terminus of the domain sequence is removed from the domain sequence, and the entire REP except the (A) _n motif is removed. The hydrophobicity of REP is calculated as e/f, where f is the total number of amino acid residues. In the calculation of the hydrophobicity of REP, the reason why "the sequence in which the sequence from the (A) _n motif located at the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence" is the target is the same as the above-mentioned reason. It is the same.

The domain sequence may have an amino acid sequence corresponding to the insertion of 1 or more and less than 30 lysine residues in REP, as compared to naturally occurring fibroin. That is, the total number of lysine residues corresponding to insertion into REP is 1 or more, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, Alternatively, it may be 22 or more, and 24 or less, 22 or less, or 18 or less. The number of lysine residues per REP in the domain sequence may be, for example, 1 to 10, may be 1 to 6, may be 1 to 3, and may be 1.

The total number of lysine residues in the modified fibroin according to this embodiment may be 1 or more and less than 30, 1 or more and 24 or less, or 1 or more and 18 or less, or 2 or more and 18 or less.

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

Although the molecular weight of the modified fibroin according to the present embodiment is not particularly limited, it may be, for example, 10 kDa or more and 700 kDa or less. The molecular weight of the modified fibroin according to the present embodiment may be, for example, 20 kDa or higher, 30 kDa or higher, 40 kDa or higher, 50 kDa or higher, 60 kDa or higher, 70 kDa or higher, 80 kDa or higher, 90 kDa or higher, or 100 kDa or higher, 600 kDa or lower, 500 kDa or lower, 500 kDa or lower. , 400 kDa or less, 300 kDa or less, or 200 kDa or less.

As a more specific example of the modified fibroin according to the present embodiment, (i) a 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 or SEQ ID NO: 5, Or (ii) modified fibroin containing the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7 can be mentioned.

The amino acid sequence (PRT1) shown in SEQ ID NO: 1 has one lysine residue at each REP located at the most N-terminal side and C-terminal side of the domain sequence with respect to the amino acid sequence shown in SEQ ID NO: 8 (PRT8). It was inserted at some places.

The amino acid sequence shown in SEQ ID NO: 2 (PRT2) is different from the amino acid sequence shown in SEQ ID NO: 8 (PRT8) in the REPs located at the 1st and 2nd positions from the N-terminal side and the C-terminal side of the domain sequence, respectively. A lysine residue is inserted at each position.

The amino acid sequence (PRT3) shown in SEQ ID NO: 3 is in the REP located at the 1st to 4th positions from the N-terminal side and the C-terminal side of the domain sequence, respectively, with respect to the amino acid sequence shown in SEQ ID NO: 8 (PRT8). A lysine residue is inserted at each position.

The amino acid sequence (PRT4) shown in SEQ ID NO: 4 is in the REP located at the 1st to 8th positions from the N-terminal side and the C-terminal side of the domain sequence, respectively, with respect to the amino acid sequence shown in SEQ ID NO: 8 (PRT8). A lysine residue is inserted at each position.

The amino acid sequence represented by SEQ ID NO: 5 (PRT5) has one position each in the REPs located at positions 1 to 12 from the N-terminal side of the domain sequence with respect to the amino acid sequence represented by SEQ ID NO: 8 (PRT8). A lysine residue is inserted, and one lysine residue is inserted into each of the REPs located at positions 1 to 7 and 9 to 10 from the C-terminal side, and the REP located at the 8th position from the C-terminal side. In which two lysine residues were inserted.

The amino acid sequence represented by SEQ ID NO: 6 (PRT6) has 6 positions in the REP located at the first position from the N-terminal side and the C-terminal side of the domain sequence with respect to the amino acid sequence represented by SEQ ID NO: 9 (PRT9). A lysine residue is inserted into each of the REPs, and three lysine residues are inserted into the REP located at the second position from each of the N-terminal side and the C-terminal side of the domain sequence.

The amino acid sequence represented by SEQ ID NO: 7 (PRT7) is in the REP located at positions 1 to 9 from the N-terminal side and the C-terminal side of the domain sequence, respectively, with respect to the amino acid sequence represented by SEQ ID NO: 9 (PRT9). A lysine residue is inserted at each position.

The amino acid sequence represented by SEQ ID NO: 21 (PRT21) is an amino acid sequence represented by SEQ ID NO: 9 with three lysine residues inserted in REP located at the most C-terminal side of the domain sequence. ..

The modified fibroin (i) may consist of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. The modified fibroin of (ii) may consist of the amino acid sequence shown by SEQ ID NO: 6 or SEQ ID NO: 7.

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

As the tag sequence, for example, an affinity tag utilizing specific affinity (binding property, affinity) with another molecule can be mentioned. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are lined up and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of modified fibroin by chelating metal chromatography is performed. Can be used for. Specific examples of the tag sequence include, for example, the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18 (amino acid sequence containing His tag). The tag sequence may include lysine residues. Specific examples of the tag sequence containing a lysine residue include the amino acid sequence represented by SEQ ID NO: 19. The number of lysine residues contained in the tag sequence may be 1-5, 2-4, or 3.

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

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

Further, a tag sequence 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 is cleaved can be recovered.

As a more specific example of the modified fibroin containing a tag sequence, (iii) SEQ ID NO: 10 (PRT10), SEQ ID NO: 11 (PRT11), SEQ ID NO: 12 (PRT12), SEQ ID NO: 13 (PRT13), or SEQ ID NO: 14 ( The modified fibroin containing the amino acid sequence shown by PRT14), or the modified fibroin containing the amino acid sequence shown by (iv) sequence number 15 (PRT15), sequence number 16 (PRT16), or sequence number 20 can be mentioned.

The modified fibroin of (iii) may consist of the amino acid sequence shown by SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

GPGXX motif content rate of modified fibroin containing the amino acid sequence shown in SEQ ID NO: 10 (PRT10), SEQ ID NO: 11 (PRT11), SEQ ID NO: 12 (PRT12), SEQ ID NO: 13 (PRT13), or SEQ ID NO: 14 (PRT14) Is about 40%, and each is 10% or more.

The modified fibroin of (iv) may consist of the amino acid sequence shown by SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 19.

The modified fibroin containing the amino acid sequence of SEQ ID NO: 15 (PRT15), SEQ ID NO: 16 (PRT16), or SEQ ID NO: 20 each has a GPGXX motif content of 10% or more.

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

(Nucleic acid)
The nucleic acid according to one embodiment encodes the modified fibroin. Specific examples of the nucleic acid include 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, or SEQ ID NO: 7, or an amino acid sequence thereof. A nucleic acid encoding a modified fibroin or the like in which the amino acid sequence (tag sequence) represented by SEQ ID NO: 17 or SEQ ID NO: 18 is bound to either or both of the N-terminus and C-terminus is included.

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

The "stringent conditions" refer to conditions under which so-called specific hybrid is formed and non-specific hybrid is not formed. The "stringent conditions" may be low stringent conditions, medium stringent conditions, or high stringent conditions. Low stringency conditions mean that hybridization will occur only if there is at least 85% identity between the sequences, eg, 5×SSC containing 0.5% SDS at 42° C. The conditions for hybridizing are mentioned. The moderately stringent condition means that the hybridization occurs only when at least 90% identity exists between the sequences. For example, 5×SSC containing 0.5% SDS is used at 50° C. The conditions for hybridizing are mentioned. Highly stringent conditions mean that hybridization occurs only when at least 95% identity exists between the sequences, for example, using 5×SSC containing 0.5% SDS at 60° C. The conditions for hybridizing are mentioned.

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

The host according to one embodiment is transformed with the above expression vector. As the host, any of prokaryote and eukaryote such as yeast, filamentous fungus, insect cell, animal cell and plant cell can be preferably used.

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

When a prokaryote such as a bacterium is used as a host, the expression vector according to one embodiment is capable of autonomous replication in a prokaryote, and at the same time, a promoter, a ribosome binding sequence, a nucleic acid and a transcription termination sequence according to one embodiment. It is preferable that the vector contains A gene that controls the promoter may be included.

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

Examples of microorganisms belonging to the genus Escherichia include 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 No. 47076), Escherichia coli. 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia W1103, Escherichia W3 ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue and the like.

Examples of the microorganism belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus bolsterensis, Brevibacillus centroporus Brevibacillus formosas, Brevibacillus invocatuus, Brevibacillus latyrosporus, Brevibacillus rimnofilus, Brevibacillus parabrevis. Brevibacillus reuszeli, Brevibacillus thermolver, Brevibacillus Brevis 47 (FERM BP-1223), Brevibacillus Brevis 47K (FERM BP-2308), Brevibacillus Brevis 47-5 (FERM BP-1664), Brevi Bacillus brevis 47-5Q (JCM8975), Brevibacillus choshinensis HPD31 (FERM BP-1087), Brevibacillus choshinensis HPD31-S (FERM BP-6623), Brevibacillus choshinensis HPD31-OK (FERM BP- 4573), Brevibacillus choshinensis SP3 strain (manufactured by Takara) and the like.

Examples of the microorganism belonging to the genus Serratia include Serratia liquefaciens ATCC14460, Serratia entomophila, Serratia ficaria, Serratia firatia, and Serratia foncola. (Serratia grimesii), Serratia proteamaculans, Serratia odorifera, Serratia primicaa, etc., Serratia primuciab, Serratia primuciab, and Serratia primata.

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

Examples of the microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphilum ATCC15354 and the like.

Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC14067) ATCC13826, ATCC14067, Brevibacterium inmariophyllum. (Brevibacterium immariophilum) ATCC14068, Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC13869) ATCC13665, ATCC13869, Brevibacterium roseum ATCC13825, Brevibacterium saccharolyticum (Brevibacterium bacterium ATCC13825) Thiogenitalis ATCC19240, Brevibacterium album ATCC15111, Brevibacterium cerinum ATCC15112 and the like.

Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC 6871, ATCC 6872, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum bacterium 140, Corynebacterium glutamicum bacterium, CCCC 140CC. Corynebacterium acetoacidophilum ATCC13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanolyticum ATCC21511, Corynebacterium carnae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13032, ATCC13032, ATCC13032, ATCC13090, ATCC13032, ATCC13090 Corynebacterium meracecola ATCC17965, Corynebacterium thermoaminogenes AJ12340 (FERMBP-1539), Corynebacterium herculis ATCC13868, etc. can be mentioned.

As microorganisms belonging to the genus Pseudomonas, for example, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas fluoresceum, Pseudomonas fluorsceum, Pseudomonas fluorsceum (Pseudomonas sp.) D-0110 and the like.

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

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

Examples of the vector (hereinafter, simply referred to as “vector”) into which the nucleic acid according to one embodiment is introduced include pBTrp2, pBTac1, pBTac2 (all are commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), and pSE280. (Manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (JP 58-110600 A), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [EscherrJSpRJSpRJS]. )), pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), JP-A-60-221091. Gazette], pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol. , 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), and the like.

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

As a specific example of a vector suitable for a microorganism belonging to the genus Brevibacillus, pUB110 known as Bacillus subtilis vector, or pHY500 (JP-A-2-31682), pNY700 (JP-A-4-27891), pHY4831 (J Bacteriol., 1987, 1239-1245), pNU200 (Shigezo Utaka, Journal of Japan Society of Agricultural Chemistry 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211. (J. Biochem., 1992, 112:488-491), pNU211R2L5 (JP-A-7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58:525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177: 745-749), pHT210 (JP-A-6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42: 358-363), or a microorganism belonging to the genus Escherichia coli and Brevibacillus. And pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569), which is a shuttle vector with the above.

The promoter is not limited as long as it functions in the host cell. Examples thereof include promoters derived from Escherichia coli or phages such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. In addition, artificially designed and modified promoters such as a promoter in which two Ptrp are connected in series (Ptrp×2), tac promoter, lacT7 promoter, let I promoter, and the like 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 initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). In the expression vector, a transcription termination sequence is not necessarily required for expression of the nucleic acid, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

Examples of eukaryotic hosts include yeast, filamentous fungi (mold etc.) and insect cells.

As yeast, for example, Saccharomyces genus, Schizosaccharomyces genus, Kluyveromyces genus, Trichospora genus (Trichospida), Schwanniomyces (Schyciano, Schywanes), , Yarrowia genus, Hansenula genus and the like. More specifically, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces marxianus (Kluyveromyces marxianus), Trichosporon pullulans (Trichosporon pullulans), Shiwaniomaisesu - Albius (Schwanniomyces alluvius), Schwanniomyces occidentalis, Candida utilis metachia, Pichia pastoris Pichia pastoris (Pichia pastoris) (Pichia polymorpha), Pichia stititis (Pichia stititis), Yarrowia lipolytica (Yarrowia lipolytica), Hansenula polymorpha (Hansenula polymorpha) etc. can be mentioned.

When yeast is used as a host cell, the expression vector is usually an origin of replication (if amplification in the host is required) and a selectable marker for growth of the vector in E. coli, a promoter for recombinant protein expression in yeast and It is preferred to include a terminator, as well as a selectable marker for yeast.

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

Examples of the vector when yeast is used as a host include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZB, and PGAPZB, pGAPZB, pGAPZB. Etc. can be mentioned.

The promoter is not limited as long as it can be expressed in yeast. For example, 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, Examples thereof include pGCW14 promoter, AOX1 promoter, MOX promoter and the like.

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

Examples of filamentous fungi include genus Acremonium, genus Aspergillus, genus Ustilago, genus Trichoderma, genus Neurospora, genus Fusarium, Fusarium. The genus Penicillium, the genus Myceliophtora, the genus Botryts, the genus Magnaporthe, the genus Mucor, the genus Metarhizus, the genus Metarhizus, the genus Monathus , And bacteria belonging to the genus Rhizomucor.

Specific examples of filamentous fungi include Acremonium alabamense, Acremonium cellulolyticus, Aspergillus aculaeus alusulaus sp. Aspergillus oryzae, Aspergillus salke, Aspergillus sojaje, Aspergillus twiggensir Aspergillus nigers, Aspergillus twiggens Parashichikusu (Aspergillus parasiticus), Aspergillus Fikumu (Fikyuumu) (Aspergillus ficuum), Aspergillus Fenikusu (Aspergillus phoeicus), Aspergillus Foechizusu (Fechidasu) (Aspergillus foetidus), Aspergillus Furabusu (Aspergillus flavus), Aspergillus fumigatus (Aspergillus fumigatus ), Aspergillus japonicus, Trichoderma viride, Trichoderma sperum, Trichoderma sperium, trichomes, Aspergillus japonicus, Aspergillus japonicus. Thermoascus, Sporotrichum, Sporotrichum cellulofilm, Talaromyces, Thielavia terrerosi, Thielavia terrestria, Thielavia terrestria, Thielavia terrestria, Chielavia terrestria, Thielavia terrestria, Thielavia terellatis ora crassa), Fusarium oxysporus, Fusarium graminearum, Fusarium venicerum nigiumensis numerus (Husicolium venicenum). (Penicillium camemberti), Penicillium Kanesensu (Penicillium canescens), Penicillium Emerusoni (Penicillium emersonii), Penicillium funiculosum (Penicillium funiculosum), Penicillium Gurizeorozeumu (Penicillium griseoroseum), Penicillium Papurogenamu (Penicillium purpurogenum), Penicillium Rokeforuchi (Penicillium roqueforti), Myceliophtaora thermophilum, Mucor ambiguus, Mucoa sillcine Royedes is Mucor corinelloides, Mucor cuirguis, Mucoa frugiris・Mucor inaequisporus, Mucor oblongiellipticus, Mucor racemusus, Mucor recurnusus, Mucor recurnusus, mucoa Mous (Mocor subtilismus), Ogataea polymorpha (Phanerochaete chrysosporium), Rhizomucor humorum (Rhizomucor humorum), Ogataea polymorpha (Phanerochaete chrysosporium) arrhizus) and the like.

When the host is a filamentous fungus, the promoter may be any of a gene related to glycolysis, a gene related to constitutive expression, an enzyme gene related to hydrolysis, etc. Specifically, amyB, glaA, agdA, glaB, TEF1 , XynF1 tannasegene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, catB, etc. can be mentioned.

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

Examples of insect cells include lepidopteran insect cells, and more specifically, insect cells derived from Spodoptera frugiperda such as Sf9 and Sf21, and nettle alba (Trichoplusia) such as High 5. ) Derived insect cells and the like.

Examples of the vector when insect cells are used as a host include baculovirus baculovirus such as Autographa californica nuclear polyhedrosis virus, which is a virus that infects night-thief moth insects. , A Laboratory Manual, WH Freeman and Company, New York (1992)).

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

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

The recombinant vector according to one embodiment preferably further contains a selectable marker gene for selecting transformants. For example, in Escherichia coli, as a selectable marker gene, resistance genes for various drugs such as tetracycline, ampicillin, kanamycin and the like can be used. A recessive selectable marker that can complement a gene mutation involved in auxotrophy can also be used. In yeast, a geneticin resistance gene can be used as a selectable marker gene, and a gene that complements a gene mutation involved in auxotrophy, LEU2, URA3, TRP1, HIS3, or another selectable marker can also be used. In filamentous fungi, niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6, 386-389, (1984)), sC (Gene,) as selectable marker genes. 84,329-334, (1989)), ptrA (BiosciBiotechnol Biochem, 64, 1416-1421, (2000)), pyrG (BiochemBiophys Res Commun, 112, 284-289, (1983)), amdS(Gene). 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 a marker gene selected from the group consisting of the hygromycin resistance gene (Gene, 57, 21-26 (1987)), a leucine-requiring complementary gene, and the like. When the host is an auxotrophic mutant, a wild-type gene that complements the auxotrophy can be used as a selectable marker gene.

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

(Method for producing modified fibroin)
The modified fibroin according to the present embodiment can be produced by a method including a step of expressing the nucleic acid in a host transformed with the expression vector. As the expression method, in addition to direct expression, secretory production, fusion protein expression and the like can be performed according to the method described in Molecular Cloning 2nd edition. When expressed in yeast, animal cells, or insect cells, modified fibroin can be obtained as a sugar- or sugar-chain-added polypeptide.

Modified fibroin according to the present embodiment, for example, by culturing a host transformed with the expression vector in a culture medium, to produce and accumulate the modified fibroin according to the present embodiment in the culture medium, collected from the culture medium It can be manufactured. The method for culturing the above-mentioned host in the culture medium can be performed according to the method usually used for culturing the host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for the host contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and culture of the host. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently perform the above.

The carbon source may be any one that can be assimilated by the host, and examples thereof include glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolysates, and organic acids such as acetic acid and propionic acid. , And alcohols such as ethanol and propanol can be used.

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

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

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

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

As a culture medium for insect cells, a commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400, ExCell405 (both manufactured by JRH Biosciences), Grace'. s Insect Medium (Nature, 195, 788 (1962)) and the like can be used.

Cultivation of insect cells can be carried out, for example, under conditions of a culture medium pH of 6 to 7 and a culture temperature of 25 to 30° C. for a culture time of 1 to 5 days. If necessary, an antibiotic such as gentamicin may be added to the culture medium during the culture.

When the host is a plant cell, the transformed plant cell may be cultured as it is, or may be differentiated into a plant organ and cultured. As a medium for culturing the plant cells, a commonly used Murashige and Skoog (MS) medium, White (White) medium, or a medium in which a plant hormone such as auxin, cytokinin, etc. is added to these mediums is used. be able to.

Culturing of animal cells can be carried out for 3 to 60 days under the conditions of pH of culture medium of 5 to 9 and culture temperature of 20 to 40° C., for example. If necessary, an antibiotic such as kanamycin or hygromycin may be added to the medium during the culture.

As a method for producing a modified fibroin using a host transformed with the expression vector, a method for producing the modified fibroin in a host cell, a method for secreting it outside the host cell, and a method for producing it on the outer membrane of the host cell are provided. There is a way. 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 in the host cell or on the outer membrane of the host cell, the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Lowe et al. (Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Development., 4, 1288 (1990)), or by applying the methods described in JP-A-5-336963, International Publication No. 94/23021, and the like. The modified fibroin can be modified so as to be actively secreted outside the host cell. That is, the modified fibroin can be positively secreted outside the host cell by using a gene recombination technique to express the modified fibroin in a form in which a signal peptide is added to the polypeptide containing the active site.

The modified fibroin produced by the host transformed with the above expression vector can be isolated and purified by a method commonly used for protein isolation and purification. For example, when the modified fibroin is expressed in a lysed state in the cells, after the culture is completed, the host cells are recovered by centrifugation, suspended in an aqueous buffer solution, and then ultrasonically disrupted, French press, Mantongaurin. The host cells are crushed with a homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolation and purification of proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent is used. Precipitation method, anion-exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.), and cation using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography, hydrophobic chromatography using butyl sepharose, phenyl sepharose, etc. resins, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoretic methods such as isoelectric focusing The above method can be used alone or in combination to obtain a purified preparation.

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

When the modified fibroin is expressed by forming an insoluble substance in the cell, the host cell is similarly collected, crushed, and centrifuged to recover the insoluble substance of the modified fibroin as a precipitate fraction. The recovered insoluble body of modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified fibroin preparation can be obtained by the same isolation and purification method as described above.

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

(Contact process)
In the contacting step, the molded body precursor is brought into contact with water to obtain a protein molded body. The molded body precursor is a reaction mixture of a dope solution containing modified fibroin and a solvent, and a reactive agent having a reactive group capable of reacting with a protein to form a bond.

The molded body precursor is cured by being brought into contact with water. A protein molded product can be obtained as a cured product of the molded product precursor.

The temperature of the water to be brought into contact with the molded body precursor may be, for example, 10° C. or higher, 20° C. or higher, 90° C. or lower, and 70° C. or lower.

In the contact step, as a method of contacting the molded body precursor with water, for example, a method of adding water at room temperature to the molded body precursor, a method of immersing the molded body precursor in water, a molded body precursor Examples thereof include a method in which water is sprayed on the body in a state of normal temperature or heated steam or the like, and a method in which the molded body precursor is exposed to a high humidity environment filled with water vapor. Among these methods, a method of adding water at room temperature to the molded body precursor is preferable.

The contacting step may include, for example, drying the shaped body precursor after contacting with water. Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known contact equipment or non-contact equipment can be used. Further, the drying temperature is not limited to any temperature, for example, a temperature lower than the temperature at which the modified fibroin contained in the molded body precursor is decomposed or the molded body precursor is thermally damaged. Generally, the temperature is in the range of 20 to 150°C, and preferably the temperature is in the range of 50 to 100°C. When the temperature is in this range, the molded body is dried more quickly and efficiently without causing thermal damage to the molded body or decomposition of the modified fibroin contained in the molded body. The drying time is appropriately set according to the drying temperature and the like, and, for example, a time such that the influence of overdrying on the quality and physical properties of the molded body can be eliminated as much as possible is adopted.

The shape of the protein molded body may be, for example, a plate shape, a film, a sheet, a cone shape, a column shape, a spherical shape, a lump shape, or the like.

The manufacturing method according to the present embodiment may include a step of preparing a molded body precursor. That is, the manufacturing method according to the present embodiment includes, as a step of preparing the molded body precursor, for example, a step of preparing a dope solution containing modified fibroin and a solvent, and a step of reacting the dope solution and the reactant. May be.

Examples of the solvent in the dope solution include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and urea, guanidine, sodium dodecyl sulfate ( SDS), lithium bromide, calcium chloride, an aqueous solution containing lithium thiocyanate, and the like. These solvents may be used alone or in combination of two or more.

The content of the modified fibroin in the dope solution may be 15% by mass or more, 30% by mass or more, 40% by mass or more, or 50% by mass or more, based on the total mass of the dope solution. The content of the modified fibroin may be 70% by mass or less, 65% by mass or less, or 60% by mass or less based on the total mass of the dope solution from the viewpoint of the production efficiency of the dope solution.

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

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

You may heat at 30-90 degreeC at the time of manufacture of the dope liquid which concerns on this embodiment. The temperature at which it can be dissolved may be appropriately set depending on the solvent used, the type of modified fibroin, and the like. Shaking and stirring may be performed to promote dissolution.

The viscosity of the dope solution may be set to 100 to 15,000 cP (centipoise) at 25° C. or 35° C., 100 to 30,000 cP (centipoise) at 40° C., and the like. The viscosity of the spinning dope can be measured using, for example, a product name "EMS viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd.

The molded body precursor is a reaction mixture obtained by reacting a dope liquid with a reactant. The molded body precursor may contain a modified fibroin at least a part of which is crosslinked with a reaction agent, and a solvent.

Modified fibroin has a reactive functional group. Examples of the reactive functional group include an amino group, a hydroxyl group and a carboxyl group. The reactive group contained in the reactive agent may be a group capable of reacting with the reactive functional group contained in the modified fibroin to form a bond.

As the reactive group, the following formulas (A-1), (A-2), (A-3), (A-4), (A-5), (A-6), (A-7), It may be a group represented by (A-8), (A-9) or (A-10). The wavy line in each formula represents a bond of each group.

In formula (A-2), X ¹ represents an oxygen atom (O) or a sulfur atom (S). In formula (A-4), X ² represents a halogen atom, and may be, for example, a chlorine atom. X ³ represents a leaving group. X ³ may be, for example, a group represented by —N ₃ , —OR ¹ , or —OAr ¹ . R ¹ in X ³ may be, for example, a succinimide group. Ar ¹ in X ³ represents a substituted or unsubstituted aryl group. The substituted or unsubstituted aryl group is preferably a halogenated aryl group. The halogenated aryl group is preferably, for example, a pentafluorophenyl group. X ⁴ represents an alkyl group. The carbon number of the alkyl group in X ⁴ may be, for example, 1 to 6, or 1 to 3. X ⁴ may be, for example, a methyl group.

The number of reactive groups contained in the reactant may be, for example, 1 to 10, 2 to 5, or 2. The reactant may contain one kind of reactive group or may contain two or more kinds of reactive groups.

The reactive agent is a first reactive group capable of reacting with modified fibroin to form a bond, a second reactive group capable of reacting with modified fibroin to form a bond, and a first reactive group and a second reactive group. It may be a compound having a group (linking group) that links the reactive group of 2. The first reactive group and the second reactive group may be different reactive groups or the same reactive group.

That is, the reactant may be a compound represented by the formula (B): A ¹ -Z-A ² . In formula (B), A ¹ represents a first reactive group, A ² represents a second reactive group, and Z represents a linking group. A ¹ and A ² may be the same reactive group or different reactive groups. A ¹ and A ² are preferably reactive groups of the same kind. A ¹ and A ² are each independently (A-1), (A-2), (A-4), (A-5), (A-6), (A-7), (A -8), a group represented by (A-9) or (A-10), and an aldehyde group (a group represented by formula (A-1)) is preferable. Z may be a divalent hydrocarbon group. Examples of Z include an alkylene group, a cycloalkylene group, and an arylene group. The carbon number of Z may be, for example, 1 to 20, 1 to 10, or 2 to 6.

Examples of the reactant include amine reactive chemical groups such as isocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, acid anhydride, fluorobenzene, carbonate, N-hydroxy ester, imide ester, epoxide, fluorophenyl ester and/or the like. Included are compounds having at least one of the alcohol-reactive chemical groups. That is, the reactive agent has a reactive group capable of reacting with a reactive group such as an amino group, a hydroxyl group and a carboxyl group to form a bond. The reactant is preferably an aldehyde compound, more preferably glutaraldehyde.

The reaction temperature may be, for example, 20 to 80°C, or 25 to 60°C. The reaction temperature may be constant or may be adjusted so as to increase in steps. The reaction time (time kept at the reaction temperature) may be, for example, 1 to 10 hours, 2 to 8 hours, or 3 to 6 hours.

The molded body precursor can be obtained, for example, by introducing a dope liquid and a reactive agent into the cavity and reacting the dope liquid with the reactive agent in the cavity. The dope solution and the reaction agent may be introduced separately or substantially at the same time. For example, the dope solution may be introduced in advance and then the reaction agent may be introduced, or the reaction agent may be introduced in advance and then the reaction agent may be introduced. The introduction of the reactant may be carried out by introducing an aqueous solution containing the reactant. The aqueous solution containing the reactant may be, for example, an aqueous solution containing the reactant in a concentration of 30 to 70% by mass or 40 to 60% by mass.

The shape of the cavity can be appropriately set according to the target shape of the protein molded body. The cavity may be a closed space or an open space. The open space may be, for example, the space inside the container. For example, the dope solution and the reaction agent can be reacted in a container (for example, a round bottom test tube).

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

[Production of modified fibroin]
(1) Preparation of Expression Vector Modified fibroin (PRT13) having the amino acid sequence shown by SEQ ID NO: 13 and modified fibroin (PRT20) having the amino acid sequence shown by SEQ ID NO: 20 were designed.

The amino acid sequence represented by SEQ ID NO: 13 (PRT13) is in the REP located at the 1st to 8th positions from the N-terminal side and the C-terminal side of the domain sequence, respectively, with respect to the amino acid sequence represented by SEQ ID NO: 22 (PRT22). A lysine residue is inserted at each position.

The amino acid sequence represented by SEQ ID NO: 20 (PRT20) is an amino acid sequence represented by SEQ ID NO: 23 (PRT23) with three lysine residues inserted at each of the N-terminal side and the C-terminal side.

A nucleic acid encoding the modified fibroin having the designed amino acid sequence was synthesized. An NdeI site was added to the 5'end and an EcoRI site was added downstream of the stop codon to the nucleic acid. This nucleic acid was cloned into a cloning vector (pUC118). Then, the same nucleic acid was digested with restriction enzymes NdeI and EcoRI and cut out, and then recombined into a protein expression vector pET-22b(+) to obtain an expression vector.

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

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

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

(3) Purification of protein Two hours after the addition of IPTG, the collected bacterial cells 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 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) at a concentration of 100 mg/mL, and the suspension was adjusted to 60°C. The mixture was stirred for 30 minutes with a stirrer and dissolved. After the dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation, the water was removed by a freeze dryer, and the freeze-dried powder was collected to obtain two types of powdered modified fibroin (PRT13 and PRT20).

<Example 1>
(Preparation of dope solution)
PRT13 was dissolved in formic acid to obtain a dope containing PRT13. This was designated as Dope Liquid 1. In the dope liquid 1, the addition amount (content) of the modified fibroin was set to 24% by mass with respect to the total amount of the dope liquid.

(Production of molded body precursor)
A predetermined amount of dope solution 1 was placed in a round bottom test tube. Next, an aqueous solution (GA aqueous solution) containing glutaraldehyde (GA) at a concentration of 50% by mass was added into the round-bottom test tube to react the dope solution with the GA aqueous solution. The GA aqueous solution was added under the condition that the temperature of the dope solution was 25°C. After the GA aqueous solution was added, the reaction temperature was maintained at 25°C, then the temperature was raised to 40°C, and the temperature was maintained at 40°C for 7500 seconds. Then, the reaction temperature was lowered to 25° C. and then raised to 60° C. FIG. 2 is a graph showing the relationship between reaction time, viscosity, and reaction temperature. The horizontal axis in FIG. 2 represents reaction time, and the vertical axis represents viscosity or reaction temperature. After the reaction, a molded body precursor was obtained as an elastic gel. As shown in FIG. 3, the molded body precursor was capable of reversible deformation (FIGS. 3(a), (b) and (c)).

(Production of molded body)
At room temperature (20 to 30° C.), 10 mL of water was added into the round-bottom test tube containing the molded body precursor to bring the molded body precursor into contact with water at room temperature. The molded body precursor was brought into contact with water and cured to obtain a protein molded body. FIG. 3D is a photograph showing the obtained protein molded body.

<Example 2>
(Preparation of dope solution)
PRT20 was added to 1 g of formic acid (2.57 g) and dissolved by stirring at room temperature for 1 hour or more. As a result, a dope liquid 2 containing PRT20 was obtained. In the dope liquid 2, the addition amount (content) of the modified fibroin was set to 28% by mass with respect to the total amount of the dope liquid.

(Production of molded body precursor)
The dope liquid 2 was put into a test tube and the viscosity was measured. To the dope solution 2, 10 molar equivalents of glutaraldehyde (GA) were added to the modified fibroin. The viscosity of the reaction mixture in which GA was added to the dope solution was measured (1 minute measurement). For the viscosity measurement, a 4.7 mm probe ball was used. The viscosity measurement was performed 5 times at 25°C. The temperature was raised to 60° C. and held for 5 minutes. At 60° C., the viscosity measurement was performed 5 times (1 minute measurement). This operation was performed 4 times. Gelation of the reaction mixture of dope and GA was confirmed using a probe. The viscosity of the dope solution 2 (without GA added) was 3500 cP at 25° C. and 1000 cP at 60° C. The measurement result of the viscosity of the reaction mixture is shown in FIG. Gelation occurred at the time indicated by the arrow in FIG.

(Production of molded body)
Water was added to the gelled reaction mixture, which was kept for 24 hours, the water was removed, and then water was added again. After 1 week, water was removed and the obtained reaction mixture was dried to obtain a molded product of Example 2. A photograph of the molded body of Example 2 is shown in FIG.

Claims (7)

A step of contacting a molded body precursor with water to obtain a protein molded body,
The molded body precursor is a reaction mixture of a dope solution containing modified fibroin and a solvent, and a reactive agent having a reactive group capable of reacting with a protein to form a bond,
The modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , or the formula 2: [(A) _n motif-REP] _m -(A) _n motif,
The method for producing a protein molded product, wherein the domain sequence has an amino acid sequence corresponding to insertion of a lysine residue in REP, as compared with naturally-occurring fibroin.
[In Formula 1 and Formula 2, the (A) _n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and the number of alanine residues is 80% of the total number of amino acid residues in the (A) _n motif. That is all. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m shows the integer of 10-300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences. ] The molded protein according to claim 1, wherein the domain sequence contains a GPGXX (where X represents an amino acid residue other than a glycine residue) motif in REP, and the GPGXX motif content is 10% or more. Manufacturing method. The domain sequence contains a glycine residue, a serine residue, or an alanine residue in REP, and at a position adjacent to the glycine residue, the serine residue, or the alanine residue in REP, The method for producing a protein molded product according to claim 1 or 2, which has an amino acid sequence corresponding to the insertion of a lysine residue. 4. The domain sequence according to claim 1, wherein the domain sequence has an amino acid sequence corresponding to insertion of the lysine residue at or near the center of the amino acid sequence in the REP, as compared with naturally-occurring fibroin. The method for producing a protein molded body according to any one of claims. The domain sequence contains a hydrophobic amino acid residue in REP, and has an amino acid sequence corresponding to the insertion of the lysine residue at a position adjacent to the hydrophobic amino acid residue in REP, The method for producing the protein molded product according to any one of claims 1 to 4. The method for producing a protein molded product according to any one of claims 1 to 5, wherein the naturally-derived fibroin is fibroin derived from insects or arachnids. The method for producing a protein molded product according to any one of claims 1 to 6, wherein the naturally-derived fibroin is a large bottle-shaped spider protein (MaSp) or a small bottle-shaped spider protein (MiSp) of spiders.