JP7174983B2 - Spinning stock solution, fibroin fiber and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a spinning dope, a fibroin fiber, and a method for producing the same. The present invention also relates to products containing the fibroin fibers described above.

In various industrial fields, fibroin fiber is attracting attention as a new material with high utility value. As fibroin fibers, regenerated silk fibroin fibers and spider silk fibroin fibers are conventionally known. Many wet spinning methods and dry-wet spinning methods have been reported as methods for producing these fibroin fibers. However, in the above manufacturing method, a large amount of liquid must be used in the coagulation bath and the washing bath, and there is a limit to improvement in productivity.

As a dry spinning method, for example, a method of forming fibers by discharging a dope prepared by dissolving refined silk fibroin in a mixed organic solvent of calcium chloride and formic acid from a syringe into the air has been reported (non-patent Reference 1).

Xiaoxiao Yue et al, Materials Letters, 128, 2014, pp. 175-178

In the production method disclosed in Non-Patent Document 1, in order to obtain a predetermined strength, after forming the fiber, a step of impregnating it in a liquid such as water or a lower alcohol, and a step of drying the liquid attached to the fiber. and other complicated post-processes were required. Therefore, the number of steps is unavoidably increased, the cost is heavy, and the productivity is low, which is industrially disadvantageous.

An object of the present invention is to provide a spinning dope that is useful for producing fibroin fibers having a predetermined strength with excellent productivity. Another object of the present invention is to provide a fibroin fiber having excellent productivity and a predetermined strength, and a method for producing the fibroin fiber with excellent productivity.

The inventors of the present invention have made extensive studies to solve the above-described problems of the prior art. As a result, by spinning a spinning stock solution containing fibroin, a surfactant, and an organic solvent by a dry spinning method, a predetermined strength can be obtained without going through the process of impregnating the fiber in the liquid and the processes associated therewith. It was found that fibroin fibers were obtained. The present invention is based on this new finding.

Specifically, the present invention relates to, for example, the following inventions.
[1]
A spinning dope comprising fibroin, a surfactant, and an organic solvent,
The fibroin concentration is 5 to 40% by weight based on the total amount of the spinning dope,
A spinning dope, wherein the concentration of the surfactant is 0.1 to 10% by weight based on the total amount of the spinning dope.
[2]
The spinning stock solution according to [1], wherein the fibroin is spider silk fibroin.
[3]
The organic solvent is at least one selected from the group consisting of hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), and formic acid. The spinning stock solution according to [1] or [2].
[4]
The spinning dope according to any one of [1] to [3], wherein the surfactant is a nonionic surfactant.
[5]
SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 41 43, or SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, The spinning dope according to any one of [1] to [4], comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:43.
[6]
The spinning dope according to any one of [1] to [5], wherein the nonionic surfactant contains polyoxyethylene alkyl ether.
[7]
A fibroin fiber comprising fibroin and a surfactant,
A fibroin fiber, wherein the content of the surfactant is 0.001 to 50% by weight based on the content of the fibroin.
[8]
The fibroin fiber according to [7], wherein the fibroin is spider silk fibroin.
[9]
A product containing the fibroin fiber according to [7] or [8],
A product selected from the group consisting of filaments, staples, yarns, yarns, filaments, cotton, paper, wovens, knits, braids, and nonwovens, and equivalents thereof.
[10]
A method for producing a fibroin fiber, comprising:
A step of preparing the spinning stock solution according to any one of [1] to [6];
a step of extruding the spinning stock solution from a spinneret into the air, followed by heating to evaporate the organic solvent to form fibroin fibers;
A method for producing a fibroin fiber, comprising:
[11]
The method for producing a fibroin fiber according to [10], further comprising a step of hot drawing the fibroin fiber.
[12]
The method for producing a fibroin fiber according to [11], wherein the draw ratio in the dry heat drawing step is 2 to 30 times.

According to the present invention, it is possible to provide a spinning dope that is useful for producing fibroin fibers having a predetermined strength with excellent productivity. Moreover, according to the present invention, fibroin fibers having a predetermined strength can be produced with superior productivity.

1 is a schematic diagram showing an example of a fibroin domain sequence. FIG. 1 is a schematic diagram showing an example of a fibroin domain sequence. FIG. 1 is a schematic diagram showing an example of a fibroin domain sequence. FIG.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[Spinning dope]
The spinning dope according to the present embodiment essentially contains a surfactant, and further contains fibroin and an organic solvent. The spinning dope according to the present embodiment can also be said to be a spinning dope obtained by dissolving fibroin and a surfactant in an organic solvent.

The organic solvent contained in the spinning dope may be any solvent as long as it can dissolve fibroin. Examples of organic solvents include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), and formic acid. These organic solvents may contain water. These organic solvents may be used singly or in combination of two or more. The organic solvent is preferably one selected from hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA) and formic acid, or a mixed solvent of two or more.

The surfactant contained in the spinning dope may be either an ionic surfactant or a nonionic surfactant. Ionic surfactants include cationic surfactants, anionic surfactants, and amphoteric surfactants. The surfactant contained in the spinning dope may be appropriately selected depending on the type of fibroin, the properties of the organic solvent, and the like. By including a surfactant in the spinning dope, a fibroin fiber with better mechanical properties can be obtained.

Examples of cationic surfactants include alkyltrimethylammonium chlorides such as octyltrimethylammonium chloride, decyltrimethylammonium chloride, lauryltrimethylammonium chloride, cetyltrimethylammonium chloride and stearyltrimethylammonium chloride, lauryltrimethylammonium bromide, and cetyltrimethyl bromide. ammonium and alkyltrimethylammonium bromides, such as stearyltrimethylammonium bromide; Alkyl quaternary ammonium salt-type cationic surfactants, benzalkonium chloride, benzalkonium bromide, quaternary ammonium salts such as ethylsulfate lanolin fatty acid aminopropylethyldimethylammonium, cetyltrimethylammonium saccharin, and stearyltrimethylammonium saccharin and alkylpyridinium salt-type cationic surfactants such as butylpyridinium chloride, dodecylpyridinium chloride and cetylpyridinium chloride.

Examples of anionic surfactants include carboxylic acid-type anionic surfactants such as sodium laurate, sodium myristate, sodium palmitate and sodium stearate, sodium 1-hexanesulfonate, sodium 1-octanesulfonate, 1- Sulfonic acid type anionic surfactants such as sodium decane sulfonate and sodium octylbenzene sulfonate, sulfate type anionic surfactants such as sodium lauryl sulfate, sodium myristyl sulfate, sodium laureth sulfate and ammonium lauryl sulfate, and lauryl phosphate and Phosphate ester type anionic surfactants such as sodium lauryl phosphate are included.

Examples of amphoteric surfactants include alkyl betaine type amphoteric surfactants such as stearyldimethylaminoacetate betaine and dodecylaminomethyldimethylsulfopropyl betaine, 2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine and the like. and amine oxide type amphoteric surfactants such as lauryldimethylamine N-oxide and oleyldimethylamine N-oxide.

Nonionic surfactants include, for example, polyoxyethylene alkyl ether, polyoxyethylene phenyl ether, polyoxyethylene alkyl phenyl ether, polyoxypropylene alkyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene alkyl Ether-type nonionic surfactants such as ethers, polyoxyalkylene alkenyl ethers and polyoxyethylene castor oil; agents, and polyoxyethylene alkyl esters, polyoxyalkylene glycol rosinate esters, polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, Polyoxyethylene fatty acid esters such as polyoxyethylene sorbitan monooleate and polyoxyethylene sorbitan trioleate), polyoxyethylene sorbitol fatty acid esters, polyoxyethylene propylene glycol fatty acid esters and polyoxyethylene glycerin fatty acid ester boric acid esters Examples include ester-ether type nonionic surfactants, aminoether type nonionic surfactants such as polyoxyethylene alkylamine, and alkanolamide type nonionic surfactants such as fatty acid monoethanolamide and fatty acid diethanolamide. Polyoxyethylene fatty acid esters include saturated fatty acid alkyl ester molecules, monounsaturated fatty acid alkyl ester molecules, diunsaturated fatty acid alkyl ester molecules, and polyunsaturated fatty acid alkyl ester molecules (i.e., three or more carbon-carbon double bonds may be a fatty acid alkyl ester molecule having a

Surfactants may be used singly or in combination of two or more. Commercially available surfactants can be used.

When the organic solvent is formic acid, the surfactant preferably contains a nonionic surfactant (nonionic surfactant), more preferably polyoxyethylene alkyl ether or polyoxyethylene fatty acid ester, More preferably, it contains polyoxyethylene alkyl ethers or polyoxyethylene alkyl esters.

Polyoxyethylene alkyl ethers are represented by the formula: R ₁ —O(CH ₂ CH ₂ O) _m —R ₂ (R ₁ =C _n H _2n+1 , R ₂ =H). Examples of polyoxyethylene alkyl ethers include polyethylene glycol bis(3-aminopropyl) ether, polyoxyethylene nonylphenyl ether, polyethylene glycol mono-4-nonylphenyl ether, polyoxyethylene octylphenyl ether, and polyoxyethylene monododecyl. ether, polyoxyethylene cetyl ether, polyoxyethylene monooleyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl cetyl ether, polyoxyethylene octyldodecyl ether, polyoxyethylene docosyl ether, and the like.

Polyoxyethylene alkyl esters are represented by the formula: R ₃ —OCO(CH ₂ CH ₂ O) _m —R ₄ (R ₃ =C _n H _2n+1 , R ₄ =H). Examples of polyoxyethylene alkyl esters include polyoxyethylene monolaurate, polyoxyethylene monostearate, polyoxyethylene monooleate, and polyoxyethylene distearate.

Modified fibroin (Met-PRT410, Met-PRT468, Met-PRT799, PRT410, PRT468 and PRT799), or SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:42, SEQ ID NO:35, SEQ ID NO:36, Modified fibroin (Met-PRT888, Met-PRT965, Met-PRT889, Met-PRT916, Met-PRT918, Met -PRT699, Met-PRT698, Met-PRT699, PRT888, PRT965, PRT889, PRT916, PRT918, PRT699, PRT698 and PRT699), the surfactant preferably contains a nonionic surfactant, poly It more preferably contains oxyethylene alkyl ethers or polyoxyethylene alkyl esters, and even more preferably contains polyoxyethylene alkyl ethers. Further, as the polyoxyethylene alkyl ether, n of the alkyl group R ₁ in the formula: R ₁ —O(CH ₂ CH ₂ O) _m —R ₂ (R ₁ =C _n H _2n+1 , R ₂ =H) is It is preferably 4 to 22, more preferably 12 to 22, even more preferably 18 to 20. In the formula, m is preferably 4-50, more preferably 10-40, even more preferably 10-25. Specifically, for example, polyoxyethylene nonylphenyl ether, polyoxyethylene octylphenyl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene oleyl cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene lauryl ethers, and polyoxyethylene docosyl ethers. As the polyoxyethylene alkyl ester, n of the alkyl group R ₃ in the formula: R ₃ —OCO(CH ₂ CH ₂ O) _m —R ₄ (R ₃ =C _n H _2n+1 , R ₄ =H) is 4 to It is preferably 22, more preferably 12-22, even more preferably 18-20. In the formula, m is preferably 4-50, more preferably 10-40, even more preferably 10-25. Specific examples include polyethylene glycol monolaurate and polyethylene glycol monostearate.

The concentration of the surfactant contained in the spinning stock solution is not particularly limited, and may be appropriately selected according to the type of fibroin, the concentration of fibroin, the type of organic solvent, and the like. For example, when the total amount of the spinning stock solution is 100% by weight, the concentration of the surfactant may be 0.1% by weight to 10% by weight, or 0.1% by weight to 5.0% by weight, may be from 0.2 wt% to 5.0 wt%, may be from 0.3 wt% to 5.0 wt%, may be from 0.4 wt% to 5.0 wt%, and may be 0.4 wt% to 5.0 wt%; 5% by weight to 5.0% by weight is preferable, 0.8% by weight to 3.0% by weight is more preferable, 1.0% by weight to 3.0% by weight is even more preferable, and 1.0% by weight to 2.0% by weight is more preferable. 0% by weight is particularly preferred. If the concentration of the surfactant is less than 0.1% by weight, the effect of improving the strength and elongation of fibroin fibers cannot be sufficiently obtained. If the concentration of the surfactant is 10% by weight, a sufficient effect can be obtained. If the concentration exceeds 10% by weight, the manufacturing cost increases, which is not preferable.

The concentration of fibroin in the spinning stock solution is preferably 5 to 40% by weight, more preferably 10 to 35% by weight, and 12 to 30% by weight when the total amount of the spinning stock solution is 100% by weight. is more preferred, and 15 to 25% by weight is particularly preferred. If the fibroin concentration is less than 5% by weight, it becomes difficult to sufficiently improve the stress and elongation of the fibroin fiber even if the above surfactant is added. If the concentration of fibroin exceeds 40% by weight, the viscosity of the spinning stock solution is significantly increased or poor dissolution occurs.

The viscosity of the spinning stock solution is not particularly limited as long as it is a viscosity that allows dry spinning, but from the viewpoint of productivity, it is preferably 10,000 to 100,000 mPa·sec at 25 ° C., and 20,000 to 100,000 mPa. · sec is more preferable, 20,000 to 80,000 mPa·sec is more preferable, and 30,000 to 50,000 mPa·sec is particularly preferable. The viscosity of the spinning stock solution can be measured, for example, using a product name "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

From the viewpoint of performing dry spinning, the spinning stock solution preferably does not contain a metal element. As a result, the quality of the finally obtained fibroin fiber is stabilized, and even when an oil agent or the like is applied when the fiber is post-processed, the generation of static electricity is further suppressed, and the processability is improved.

The method for preparing the spinning dope is not particularly limited, and the fibroin, surfactant and organic solvent may be mixed in any order. The fibroin may be dissolved after the surfactant and the organic solvent are mixed, or the surfactant may be mixed after the fibroin is dissolved in the organic solvent.

The spinning dope may be stirred or shaken for some time to facilitate dissolution. At that time, if necessary, the spinning dope may be heated to a temperature at which the fibroin and organic solvent used can be dissolved. The spinning dope may be heated to, for example, 30° C. or higher, 40° C. or higher, 50° C. or higher, 60° C. or higher, 70° C. or higher, 80° C. or higher, or 90° C. or higher. The upper limit of the heating temperature is, for example, the boiling point of the organic solvent or less.

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

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

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

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically corresponding to the REP of the amino acid sequence). ) and is represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif means an array. Here, (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and n is 2-27. n can be an integer from 2-20, 4-27, 4-20, 8-20, 10-20, 4-16, 8-16, or 10-16. In addition, (A) the ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed only of alanine residues. REP indicates an amino acid sequence composed of 2-200 amino acid residues. REP may be an amino acid sequence composed of 10-200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

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

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

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

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

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

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

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

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

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

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

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

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

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

As a more specific example of the first modified fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4, or (1-ii) having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4 A modified fibroin comprising an amino acid sequence having a Preferably, the sequence identity is 95% or greater.

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

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

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

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

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

The second modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the domain sequence, the (A) _n motif located most C-terminal to the domain sequence Let z be the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus of the domain sequence When w is the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence, z/w is 30% or more, It may have an amino acid sequence that is 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

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

A method for calculating z/w will be described in more detail. First, in a fibroin (modified fibroin or naturally occurring fibroin) containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , the (A) _n located on the most C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50×3=150. In addition, for example, when there is an X (central X) contained in two XGX, as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues be). w is the total number of amino acid residues contained in the domain sequence, excluding the sequence from the (A) _n motif positioned most on the C-terminal side to the C-terminus of the domain sequence. For example, for the domain sequence shown in Figure 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the most C-terminal (A) _n motif). Then z/w (%) can be calculated by dividing z by w.

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

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

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

More specific examples of the second modified fibroin include (2-i) the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (2-ii) SEQ ID NO: 6, sequence Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in No. 7, SEQ ID No. 8 or SEQ ID No. 9 can be mentioned.

The modified fibroin of (2-i) will be explained. The amino acid sequence shown by SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence shown by SEQ ID NO: 10 corresponding to naturally occurring fibroin with GQX. The amino acid sequence shown by SEQ ID NO: 7 is obtained by deleting every two (A) _n motifs from the amino acid sequence shown by SEQ ID NO: 6 from the N-terminal side to the C-terminal side, and furthermore, in front of the C-terminal sequence. [(A) _n motif-REP] is inserted into the . The amino acid sequence shown in SEQ ID NO: 8 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a partial glutamine (Q) residue. It is obtained by substituting serine (S) residues and deleting some amino acids on the N-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). A His tag is added to the C-terminus of a sequence consisting of 4 repeats.

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

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

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

The modified fibroin of (2-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and XGX contained in REP ( where X represents an amino acid residue other than glycine). is preferably 50.9% or more.

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

Examples of tag sequences include affinity tags that utilize specific affinity with other molecules. A specific example of the affinity tag is a histidine tag (His tag). His-tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has the property of specifically binding to metal ions such as nickel, so isolation of modified fibroin by metal chelating metal chromatography can be used for A specific example of the tag sequence is the amino acid sequence represented by SEQ ID NO: 12 (an amino acid sequence containing a His tag sequence and a hinge sequence).

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

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

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

More specific examples of the second modified fibroin containing the tag sequence include (2-iii) the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (2-iv) Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15 can be mentioned.

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

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

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

(2-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, and XGX contained in REP ( where X represents an amino acid residue other than glycine). is preferably 50.9% or more.

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

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

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

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

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

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

A third modified fibroin comprises a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , with two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -REP] unit sequentially compares the number of amino acid residues of REP, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11. When the maximum value of the total sum of the numbers of amino acid residues of two adjacent [(A) _n motif-REP] units equal to 3 is x, and the total number of amino acid residues of the domain sequence is y Furthermore, it may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

A method for calculating x/y will be described in more detail with reference to FIG. FIG. 1 shows the domain sequence of fibroin with the N-terminal and C-terminal sequences removed. The domain sequence is, from the N-terminal side (left side), (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n Motif-third REP (10 amino acid residues)-(A) _n motif-fourth REP (20 amino acid residues)-(A) _n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called _n motif.

Two adjacent [(A) _n -motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, unselected [(A) _n motif-REP] units may be present. In FIG. 1, pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP and the fourth REP), pattern 2 (comparison of the first REP and the second REP, and comparison of the fourth REP and the fifth REP), pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP and the fifth REP), pattern 4 (comparison of the first REP and A second REP comparison) was shown. Note that there are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two adjacent [(A) _n motif-REP] units selected is compared. Comparison is carried out by determining the ratio of the number of amino acid residues to the number of amino acid residues of the other. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the second REP is The ratio of amino acid residue numbers is 100/50=2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with fewer amino acid residues is set to 1, the fifth REP is 30/20=1.5.

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

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

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

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

When the third modified fibroin is a modified fibroin in which at least 7 of the (A) _n motifs present in the domain sequence are composed only of alanine residues, x/y is 46.4% or more. is preferably 50% or more, more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and 80% or more It is particularly preferred to have The upper limit of x/y is not particularly limited as long as it is 100% or less.

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

More specific examples of the third modified fibroin include (3-i) the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (3-ii) SEQ ID NO: 18, sequence Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in No. 7, SEQ ID No. 8 or SEQ ID No. 9 can be mentioned.

The modified fibroin of (3-i) will be explained. The amino acid sequence represented by SEQ ID NO: 18 is obtained by deleting every two (A) _n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 10 corresponding to naturally occurring fibroin. , and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence shown by SEQ ID NO: 7 is obtained by substituting all GGX in REP of the amino acid sequence shown by SEQ ID NO: 18 with GQX. The amino acid sequence shown in SEQ ID NO: 8 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a partial glutamine (Q) residue. It is obtained by substituting serine (S) residues and deleting some amino acids on the N-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). A His tag is added to the C-terminus of a sequence consisting of 4 repeats.

The x/y value of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) is 15.0% at the jagged ratio of 1:1.8 to 11.3. The x/y values of the amino acid sequence shown by SEQ ID NO: 18 and the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 9 is 89.3%. The z/w values in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.0%, respectively. 1% and 70.0%.

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

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

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and from the N-terminal side to the C-terminal side , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Amino acid residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (Giza ratio is 1:1.8 to 11.3) It is preferable that x/y is 64.2% or more, where x is the maximum sum of the cardinal numbers and y is the total number of amino acid residues in the domain sequence.

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

More specific examples of the third modified fibroin containing the tag sequence include (3-iii) the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (2-iv) sequence Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in No. 17, SEQ ID No. 11, SEQ ID No. 14 or SEQ ID No. 15 can be mentioned.

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

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

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

(3-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, and , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Let x be the maximum value of the total sum of the amino acid residue numbers of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3. , x/y is preferably 64.2% or more, where y is the total number of amino acid residues in the domain sequence.

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

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

More specific examples of the fourth modified fibroin include (4-i) an amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, (4-ii) SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 9 can be mentioned. Specific embodiments of the modified fibroin containing the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 are as described above.

More specific examples of the fourth modified fibroin containing the tag sequence include (4-i) the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (4-ii) SEQ ID NO: 11, sequence Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in No. 14 or SEQ ID No. 15 can be mentioned.

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

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

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

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

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

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

Hydropathy indexes of amino acid residues are known (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 157). 105-132). Specifically, the hydropathic index (hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

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

For example, if 20 “continuous 4 amino acid residues with an average hydrophobic index of 2.6 or more” are extracted (no overlap), the average hydrophobic index of 4 consecutive amino acid residues is 2 A region that is 0.6 or greater will contain 20 consecutive 4-amino acid residues (no overlap), and p is 20×4=80. Further, for example, when two "consecutive 4 amino acid residues having an average hydrophobicity index of 2.6 or more" exist overlapping by one amino acid residue, the hydrophobic index of the consecutive 4 amino acid residues A region with an average of 2.6 or more contains 7 amino acid residues (p=2×4−1=7, where “−1” is duplicate subtraction). For example, in the case of the domain sequence shown in FIG. 2, there are seven non-overlapping “continuous 4 amino acid residues with an average hydrophobicity index of 2.6 or more”, so p is 7 × 4 = 28. For example, in the case of the domain sequence shown in FIG. 2, q is 4+50+4+40+4+10+4+20+4+30=170 (not including the (A) _n motif present at the end of the C-terminal side). Then p/q (%) can be calculated by dividing p by q. In the case of FIG. 2, 28/170=16.47%.

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

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

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

Specific examples of the fifth modified fibroin include (5-i) the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or (5-ii) SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 21 can be mentioned.

The modified fibroin of (5-i) will be explained. The amino acid sequence represented by SEQ ID NO: 22 is obtained by deleting the amino acid sequence of consecutive alanine residues in the (A) _n motif of naturally occurring fibroin so that the number of consecutive alanine residues is five. . The amino acid sequence shown by SEQ ID NO: 19 is the amino acid sequence shown by SEQ ID NO: 22 with two amino acid sequences (VLI) each consisting of 3 amino acid residues inserted every other REP and shown by SEQ ID NO: 22. A part of amino acids on the C-terminal side is deleted so that the molecular weight is almost the same as that of the amino acid sequence that is used. The amino acid sequence shown by SEQ ID NO: 23 is the amino acid sequence shown by SEQ ID NO: 22, with two alanine residues inserted on the C-terminal side of each (A) _n motif, and a part of glutamine (Q) residues. A group is replaced with a serine (S) residue, and some amino acids on the C-terminal side are deleted so that the molecular weight is almost the same as that of the amino acid sequence shown in SEQ ID NO:22. The amino acid sequence shown by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 23. The amino acid sequence shown by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 23.

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

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

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

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

More specific examples of the fifth modified fibroin containing a tag sequence include (5-iii) the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or (5-iv) SEQ ID NO: 24, sequence A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in No. 25 or SEQ ID No. 26 can be mentioned.

The amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 are added to the N-terminals of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, where the amino acid sequence shown in SEQ ID NO: 12 (His tag sequence and hinge sequence) are added.

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

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

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

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

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

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

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

As used herein, the "GPGXX motif content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif, the most C-terminal In all REPs contained in the sequence excluding the sequence from the located (A) _n motif to the C-terminus of the domain sequence from the domain sequence, triple the total number of GPGXX motifs contained in that region (i.e., (corresponding to the total number of G and P in the GPGXX motif) is s, the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and the (A) _n motif is further removed. The GPGXX motif content is calculated as s/t, where t is the total number of amino acid residues in all REPs removed.

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

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

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

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

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

The "other amino acid residue" may be an amino acid residue other than a glutamine residue, but preferably an amino acid residue with a higher hydrophobicity index than that of a glutamine residue. Hydrophobicity indexes of amino acid residues are shown in Table 1.

As shown in Table 1, amino acid residues having a higher hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H); can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

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

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

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

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

As a more specific example of the sixth modified fibroin, (6-i) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 42 (6-ii) a modified fibroin comprising the amino acid sequence shown, or (6-ii) represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 42 Modified fibroins can include amino acid sequences having greater than 90% sequence identity with the amino acid sequence.

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

The amino acid sequence (Met-PRT410) represented by SEQ ID NO: 7 is based on the base and amino acid sequences of naturally occurring fibroin Nephila clavipes (GenBank Accession Number: P46804.1, GI: 1174415), (A) _n Amino acid modifications were made to improve productivity, such as increasing the number of consecutive alanine residues to 5 in the amino acid sequence in which the alanine residues are consecutive in the motif. On the other hand, in Met-PRT410, since the glutamine residue (Q) is not modified, the glutamine residue content is comparable to that of naturally occurring fibroin.

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

The amino acid sequence (M_PRT965) shown in SEQ ID NO: 28 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with TS and the remaining Q with A.

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

The amino acid sequence (M_PRT916) shown in SEQ ID NO:30 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO:7) with VI and the remaining Q with L.

The amino acid sequence (M_PRT918) shown in SEQ ID NO:31 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO:7) with VF and the remaining Qs with I.

The amino acid sequence (M_PRT525) shown in SEQ ID NO: 34 is the same as Met-PRT410 (SEQ ID NO: 7), by inserting two alanine residues into the region (A ₅ ) where the alanine residues are continuous, and increasing the molecular weight of Met-PRT410. , two C-terminal domain sequences are deleted and 13 glutamine residues (Q) are replaced with serine residues (S) or proline residues (P) so as to be almost the same as .

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

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

The amino acid sequence shown by SEQ ID NO: 42 (Met-PRT966) is the amino acid sequence shown by SEQ ID NO: 9 (amino acid sequence before the amino acid sequence shown by SEQ ID NO: 12 is added to the N-terminus). and the remaining Q is replaced by I.

The amino acid sequences represented by SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33 and 42 all have a glutamine residue content of 9% or less. Yes (Table 2).

The modified fibroin of (6-i) consists of the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 42. There may be.

The modified fibroin of (6-ii) has an amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 42 and 90% or more contains amino acid sequences having the sequence identity of The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

More specific examples of the sixth modified fibroin containing a tag sequence include (6-iii) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or Modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 43, or (6-iv) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 41 A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 43 can be mentioned.

The amino acid sequences shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 43 are SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, respectively. , SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 42 The amino acid sequence represented by SEQ ID NO: 12 (including His tag sequence and hinge sequence) was added to the N-terminus It is a thing. Since the tag sequence was only added to the N-terminus, there was no change in the glutamine residue content, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 43 have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) consists of the amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 43. There may be.

The modified fibroin of (6-iv) has an amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 43 and 90% or more contains amino acid sequences having the sequence identity of The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

Among the features of the modified fibroin according to the present embodiment, the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin have , modified fibroin having at least two or more characteristics.

(Method for producing fibroin)
Fibroin (proteins) can be expressed, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. can be produced by

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

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

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

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

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

When a prokaryote is used as a host, examples of vectors into which fibroin-encoding nucleic acids are introduced include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2. (Japanese Unexamined Patent Application Publication No. 2002-238569).

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

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

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

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

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

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

Prokaryotes such as E. coli or eukaryotes such as yeast can be cultured under aerobic conditions such as shaking culture or deep aeration stir culture. The culture temperature is, for example, 15-40°C. Culture time is usually 16 hours to 7 days. The pH of the culture medium during cultivation is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.

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

Isolation and purification of the expressed fibroin can be performed by commonly used methods. For example, when the protein is expressed in a dissolved state in cells, the host cells are recovered by centrifugation after completion of the culture, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, or a mantongaurin. The host cells are disrupted with a homogenizer, a dyno mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method commonly used for protein isolation and purification, that is, a solvent extraction method, a salting-out method using ammonium sulfate, etc., a desalting method, an organic solvent Precipitation method, diethylaminoethyl (DEAE)-Sepharose, anion exchange chromatography method using resins such as DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.), positive chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia) Ion exchange chromatography, 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 A purified sample can be obtained by using the methods such as the above alone or in combination.

In addition, when fibroin forms an insoluble form in cells and is expressed, the host cells are similarly collected, disrupted, and centrifuged to collect an insoluble form of fibroin as a precipitate fraction. The collected insoluble fibroin can be solubilized with a protein denaturant. After this operation, a purified preparation of fibroin can be obtained by the same isolation and purification method as described above. When the protein is extracellularly secreted, the protein can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.

[Method for producing fibroin fiber]
The method for producing a fibroin fiber according to the present invention comprises spinning a fibroin fiber by a dry spinning method using the above-mentioned spinning dope according to the present invention. That is, the method for producing a fibroin fiber according to the present invention includes the steps of preparing the above-described spinning dope according to the present invention, and discharging the spinning dope from a spinneret into the air, followed by heating to vaporize the organic solvent. and a step of forming fibroin fibers (spinning step). The fibroin fibers obtained by each of the steps may be used as they are for any purpose. Alternatively, the fibroin fiber may be further subjected to a dry heat drawing step (dry heat drawing step) to obtain a fibroin fiber drawn at an arbitrary draw ratio, and the obtained fibroin fiber may be used for any application.

The production method according to the present embodiment may further include a step of filtering the stock solution for spinning before ejection (filtration step) and/or a step of defoaming the stock solution for spinning before ejection (defoaming step). .

The spinning step can be carried out, for example, by a known dry spinning method, except that the spinning dope according to the present invention is used as the spinning dope. The spinning process can also be performed by an electrospinning method (electrostatic spinning method), but from the viewpoint of simplification of the manufacturing process, it is preferably performed by a method other than the electrospinning method.

In the spinning process, the shape of the spinneret, the shape of the holes, the number of holes, etc. are not particularly limited, and can be appropriately selected according to the desired fiber diameter, the number of single yarns, and the like.

When the hole shape of the spinneret is circular, a hole diameter of 0.1 mm or more and 0.6 mm or less can be exemplified. It is preferable that the hole diameter is 0.1 mm or more because the pressure loss can be reduced and the facility cost can be suppressed. Further, when the pore diameter is 0.6 mm or less, the need for drawing operation for thinning the fiber diameter is reduced, and the possibility of drawing breakage occurring between discharge and take-up can be reduced, which is preferable.

The method of discharging the dope from the spinneret is not particularly limited, but for example, a method using a metering pump as a means for feeding the dope can be used. The discharge amount can be appropriately adjusted according to the production speed.

The temperature of the spinning dope when passing through the spinneret and the temperature of the spinneret are not particularly limited, and may be appropriately adjusted depending on the concentration and viscosity of the spinning dope used, the type of organic solvent, and the like. The temperature is preferably 30° C. to 100° C. from the viewpoint of preventing deterioration of fibroin. In addition, from the viewpoint of reducing the possibility of pressure increase due to volatilization of the organic solvent and clogging of the pipe due to solidification of the spinning dope, the upper limit of the temperature should be a temperature lower than the boiling point of the organic solvent used. is preferred. This improves process stability.

The undrawn yarn (or pre-drawn yarn) obtained in the spinning step can be made into drawn yarn through the drawing step. The drawing method includes, for example, dry heat drawing.

Before or after hot drawing, or before and after hot drawing, if necessary, to the undrawn yarn (or pre-drawn yarn) or drawn yarn for the purpose of imparting antistatic properties, lubricity, etc. An oil may be applied. The type and amount of the oil to be applied are not particularly limited, and can be appropriately adjusted in consideration of the use of the fibroin fiber, the handleability of the fibroin fiber, and the like.

In dry heat drawing, the undrawn yarn (or pre-drawn yarn) after drying is hot drawn. The heating temperature at this time is not particularly limited as long as it is a condition under which sufficient stretching and strength are exhibited. °C to 200 °C, 160 °C to 200 °C, or 160 °C to 180 °C. Moreover, the dry heat drawing process may be divided into multiple stages as necessary, and there is no particular problem. The device used for dry heat drawing includes a contact hot plate, a non-contact furnace, and the like, but is not particularly limited. Any device may be used as long as it can be stretched at a magnification of .

The draw ratio in the hot drawing step may be, for example, 2 to 30 times, 2 to 20 times, or 3 to 15 times the undrawn yarn (or pre-drawn yarn). , preferably 3 to 10 times, more preferably 4 to 8 times.

The fibroin fiber after dry heat drawing may be wound with a winder. As for the winding method using a winder, a known winder can be used, and winding conditions such as tension and contact pressure can be appropriately adjusted.

The fibroin fiber of the present invention obtained by the above method has a linear shape.

The fibroin fiber according to the present invention may be wound up with a winder after being further subjected to wet heat drawing treatment. The wet heat stretching can be carried out in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating. The temperature may be, for example, 40 to 200°C, 50 to 180°C, 50 to 150°C, preferably 75 to 90°C. In wet heat drawing, the fibroin fiber can be drawn, for example, 1 to 10 times, preferably 2 to 8 times, more preferably 2 to 4 times. Furthermore, by performing a wet heat drawing treatment, a higher stress (strength) can be imparted to the fibroin fiber. Moreover, this wet-heat drawing process may be performed in multiple stages as needed.

The lower limit of the final draw ratio is preferably more than 1 times, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times with respect to the undrawn yarn (or pre-drawn yarn). 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less. , 12 times or less, 11 times or less, and 10 times or less.

[Fibroin fiber]
A fibroin fiber according to the present invention contains fibroin and a surfactant. The lower limit of the surfactant content is 0.001% by weight or more, 0.01% by weight or more, 0.1% by weight or more, 0.2% by weight or more, and 0.3% by weight, based on the fibroin content. % by weight or more, 0.4% by weight or more, 0.5% by weight or more, 0.6% by weight or more, 0.7% by weight or more, 0.8% by weight or more, 0.9% by weight or more, 1% by weight or more , or 1.2 wt% or more, and the upper limit of the surfactant content is 1.5 wt% or less, 2.0 wt% or less, 2.0 wt% or less, based on the fibroin content % or less, 2.5 wt% or less, 3.0 wt% or less, 4.0 wt% or less, 5.0 wt% or less, 6.0 wt% or less, 7.0 wt% or less, 8.0 wt% Below, 9.0 wt% or less, 10 wt% or less, 10 wt% or less, 15 wt% or less, 20 wt% or less, 25 wt% or less, 30 wt% or less, 35 wt% or less, 40 wt% or less, 45 % by weight or less, or 50% by weight or less.

Since the fibroin fiber according to the present embodiment contains a surfactant in the spinning stock solution, the surfactant is attached to the surface of the fibroin fiber, is included in the fibroin fiber, or is attached to the surface of the fibroin fiber. may adhere to and be encapsulated in the fibers. Therefore, the surfactant adhering to the fibroin fiber surface and/or the surfactant included in the fibroin fiber may be removed as necessary. When removing the surfactant, the removal method is not particularly limited, but the fibroin fibers may be washed with water, hot water, or steam. It is preferable to wash the fibroin fibers with superheated steam from the viewpoint that drying treatment or the like is unnecessary.

The fibroin fiber according to this embodiment may be further chemically crosslinked between polypeptide molecules within the fibroin fiber. Functional groups that can be crosslinked include, for example, amino groups, carboxyl groups, thiol groups, and hydroxy groups. For example, the amino group of a lysine side chain contained in a polypeptide can be crosslinked with the carboxyl group of a glutamic acid or aspartic acid side chain with an amide bond through dehydration condensation. Crosslinking may be performed by performing a dehydration-condensation reaction under vacuum heating, or by using a dehydration-condensation agent such as carbodiimide.

Cross-linking between polypeptide molecules may be performed using a cross-linking agent such as carbodiimide or glutaraldehyde, or may be performed using an enzyme such as transglutaminase. Carbodiimide is represented by the general formula R ₁ N=C=NR ₂ (where R ₁ and R ₂ each independently represent an organic group including an alkyl group having 1 to 6 carbon atoms and a cycloalkyl group). is a compound. Specific examples of carbodiimides include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide , diisopropylcarbodiimide (DIC) and the like. Among these, EDC and DIC are preferable because they have a high ability to form an amide bond between polypeptide molecules and are easily crosslinked.

The cross-linking treatment is preferably carried out by imparting a cross-linking agent to the fibroin fibers and performing cross-linking by vacuum heat drying. The cross-linking agent may be applied to the fibroin fibers in its pure form, or diluted to a concentration of 0.005 to 10% by mass with a lower alcohol having 1 to 5 carbon atoms and a buffer solution, etc., and applied to the fibroin fibers. good. The cross-linking treatment is preferably carried out at a temperature of 20-45° C. for 3-42 hours. The cross-linking treatment can impart higher stress (strength) to the fibroin fibers.

〔product〕
The fibroin fiber according to this embodiment can be applied as a fiber or thread to fabrics such as woven fabrics, knitted fabrics, braided fabrics, and non-woven fabrics. It also finds application in high-strength applications such as ropes, surgical sutures, flexible fasteners for electrical components, and bioactive materials for implants (eg, artificial ligaments and aortic bands). These can be produced according to the method described in International Publication WO2013/065650 and the like. In addition, fibroin fibers according to the present embodiment can also be applied to long fibers, short fibers, spun yarns, filaments, paper, cotton, and equivalents thereof, which are disclosed in Japanese Patent Application Laid-Open No. 2009-505668 and Japanese Patent No. 5678283. It can be produced according to the method described in the publication, Japanese Patent No. 4638735, and the like.

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

[Evaluation method for fibroin fiber]
Each characteristic value of the fibroin fiber was evaluated by the following methods.

(Fiber diameter evaluation of fibroin fiber)
The fibroin fiber was cut perpendicular to the fibroin fiber axis, the cross section of the fiber was observed using ECLIPSE LV100ND manufactured by Nikon, and the fiber diameter was measured using image processing software (NIS-Elements D manufactured by Nikon).

(Evaluation of mechanical properties of fibroin fibers)
The test was conducted based on JIS L1013. Using an Instron 3345 series tensile tester, the strength (tensile strength) and elongation of fibroin fibers were measured under test conditions of a test length of 300 mm and a test speed of 30 mm/min.

[Production of modified fibroin]
(1) Preparation of expression vector Based on the nucleotide sequence and amino acid sequence of Nephila clavipes-derived fibroin (GenBank Accession No.: P46804.1, GI: 1174415), represented by SEQ ID NO: 15 and SEQ ID NO: 43 A modified fibroin having an amino acid sequence (hereinafter also referred to as "PRT799" and "PRT966", respectively) was designed. The amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence obtained by subjecting the amino acid sequence of fibroin derived from Nephila clavipes to substitution, insertion and deletion of amino acid residues for the purpose of improving productivity. , and the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 12 is added to the N-terminus. The amino acid sequence (PRT966) represented by SEQ ID NO: 43 is the amino acid sequence represented by SEQ ID NO: 9 (amino acid sequence before the amino acid sequence represented by SEQ ID NO: 12 is added to the N-terminus) for the purpose of improving hydrophobicity. All QQs in the fragment were replaced with VF, the remaining Qs were replaced with I, and the amino acid sequence (tag sequence and hinge sequence) of SEQ ID NO: 12 was added to the N-terminus.

Next, nucleic acids encoding PRT799 and PRT966 were synthesized. The nucleic acid was added with an NdeI site at the 5' end and an EcoRI site downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was treated with restriction enzymes NdeI and EcoRI, cut out, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Expression of Modified Fibroin Escherichia coli BLR (DE3) was transformed with the expression vector obtained in (1). The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin to an OD ₆₀₀ of 0.005. The temperature of the culture solution was kept at 30° C., and flask culture was performed until OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 5) to an OD ₆₀₀ of 0.05. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, 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 kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and culture was carried out for 20 hours. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce expression of the modified fibroin. After 20 hours from the addition of IPTG, the culture solution was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of the desired modified fibroin size band depending on the addition of IPTG. did.

(3) Purification of Modified Fibroin Cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The precipitate after washing 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 heated at 60°C. for 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed with a freeze dryer, and the freeze-dried powder was recovered to obtain modified fibroins (PRT799 and PRT966).

[Production and evaluation of fibroin fibers]
(Example 1)
25% by weight of the modified fibroin (PRT966) obtained as described above, 73.5% by weight of formic acid as an organic solvent, and 1.5% by weight of polyoxyethylene (20) stearyl ether as a surfactant were mixed. to prepare a spinning dope.

The prepared spinning dope was filtered through a metal filter with an opening of 1 μm, then defoamed, and discharged downward using a spinneret with a diameter of 0.2 mm. Next, the spinning dope discharged from the spinneret was heated in a cylindrical heating furnace at 100° C. for 30 seconds, and then taken up with a roller to obtain a fibroin fiber.

(Example 2)
Spinning was carried out in the same manner as in Example 1, except that the modified fibroin (PRT799) obtained as described above was used as the solute in the spinning stock solution, and the concentrations of formic acid and surfactant were changed as shown in Table 6. A stock solution was prepared. The prepared spinning dope was treated in the same manner as in Example 1 and discharged downward using a spinneret with a diameter of 0.2 mm. Next, the spinning dope discharged from the spinneret was heated in a cylindrical heating furnace at 100° C. for 30 seconds, taken up by rollers, and further hot-drawn 5 times to obtain a fibroin fiber.

(Comparative example 1)
A spinning dope was prepared in the same manner as in Example 1, except that the concentration of formic acid in the spinning dope was changed as shown in Table 6 and no surfactant was added. When the prepared spinning dope was treated in the same manner as in Example 1 and discharged from the spinneret in the same manner as in Example 1, the discharged spinning dope could not be stretched and fibers could not be formed.

(Comparative example 2)
A spinning dope was prepared in the same manner as in Example 2, except that the concentration of formic acid in the spinning dope was changed as shown in Table 6 and no surfactant was added. When the prepared spinning dope was treated in the same manner as in Example 1 and discharged from the spinneret in the same manner as in Example 1, the discharged spinning dope could not be stretched and fibers could not be formed.

As shown in Table 6, when the fibroin concentration in the spinning stock solution was 25 wt %, fibroin fibers could be obtained only when a surfactant-added spinning stock solution was used.

The fibroin fibers obtained in Example 2 were evaluated for fiber diameter and mechanical properties. The fiber diameter, mechanical properties and manufacturing conditions of the fibroin fibers were as shown in Table 7.

(Example 3)
A spinning dope was prepared in the same manner as in Example 2, except that the fibroin concentration, formic acid concentration, and surfactant concentration in the spinning dope were changed as shown in Table 7 to obtain fibroin fibers. The fiber diameter, mechanical properties and production conditions of the obtained fibroin fibers were as shown in Table 7.

(Example 4)
A spinning dope was prepared in the same manner as in Example 2, except that the fibroin concentration, formic acid concentration, and surfactant concentration in the spinning dope were changed as shown in Table 7, and the draw ratio was changed to 4 times. , to obtain fibroin fibers. The fiber diameter, mechanical properties and production conditions of the obtained fibroin fibers were as shown in Table 7.

(Example 5)
A spinning dope was prepared in the same manner as in Example 2, except that the fibroin concentration, formic acid concentration, and surfactant concentration in the spinning dope were changed as shown in Table 7 to obtain fibroin fibers. The fiber diameter, mechanical properties and production conditions of the obtained fibroin fibers were as shown in Table 7.

(Comparative Example 3)
A spinning dope was prepared in the same manner as in Example 2, except that the fibroin concentration and formic acid concentration in the spinning dope were changed as shown in Table 7, and no surfactant was added. The prepared spinning stock solution was treated in the same manner as in Example 2 and discharged from the spinneret in the same manner as in Example 2. Although fiberization was possible, the draw ratio in the drawing process could not be increased, resulting in The fibroin fibers obtained had poor mechanical properties (Table 7).

Claims (8)

A spinning dope comprising fibroin, a surfactant, and an organic solvent,
wherein the 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 fibroin concentration is 5 to 40% by weight based on the total amount of the spinning dope,
The concentration of the surfactant is 0.1 to 10% by weight based on the total amount of the spinning dope,
wherein the surfactant is a nonionic surfactant;for dry spinningSpinning stock solution.
[In formulas 1 and 2, (A) _n The motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) _n The ratio of the number of alanine residues to the total number of amino acid residues in the motif is 40% or more. REP indicates an amino acid sequence composed of 2-200 amino acid residues. m represents an integer from 2 to 300; Multiple (A) _n Motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences. ] The spinning dope according to claim 1, wherein the fibroin is spider silk fibroin. The organic solvent is at least one selected from the group consisting of hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), and formic acid. The spinning dope according to claim 1 or 2. SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 41 43, or SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, The spinning dope according to any one of claims 1 to 3, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:43. The spinning dope according to any one of claims 1 to 4, wherein the nonionic surfactant comprises a polyoxyethylene alkyl ether. A method for producing a fibroin fiber, comprising:
A step of preparing the spinning stock solution according to any one of claims 1 to 5;
a step of discharging the spinning stock solution from a spinneret into the air and then heating to evaporate the organic solvent to form fibroin fibers;
A method for producing a fibroin fiber, comprising: A method for producing a fibroin fiber, comprising:
A spinning dope comprising fibroin, a surfactant, and an organic solvent,
wherein the 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 fibroin concentration is 5 to 40% by weight based on the total amount of the spinning dope,
a step of preparing a spinning stock solution, wherein the concentration of the surfactant is 0.1 to 10% by weight based on the total amount of the spinning stock solution;
a step of discharging the spinning stock solution from a spinneret into the air and then heating it to evaporate the organic solvent to form fibroin fibers;
A method for producing a fibroin fiber, further comprising a step of hot drawing the fibroin fiber.
[In formulas 1 and 2, (A) _n The motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) _n The ratio of the number of alanine residues to the total number of amino acid residues in the motif is 40% or more. REP indicates an amino acid sequence composed of 2-200 amino acid residues. m represents an integer from 2 to 300; Multiple (A) _n Motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences. ] The method for producing a fibroin fiber according to claim 7 , wherein the draw ratio in the dry heat drawing step is 2 to 30 times.

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