JP2021080572A - Fabrication method of oiling-agent-adhesion-protein crimped fiber - Google Patents

Abstract

To provide a method of effectively fabricating an oiling-agent-adhesion-protein crimped fiber.SOLUTION: A fabrication method of an oiling-agent-adhesion-protein crimped fiber, according to the present invention includes a step of bringing a protein fiber into contact with an oiling-agent dispersion containing an aqueous solvent and an oiling agent dispersed in the aqueous solvent so that the oiling agent adheres to the protein fiber, and crimping the protein fiber, the protein fiber containing modified fibroin.SELECTED DRAWING: Figure 5

Description

The present invention relates to a method for producing an oil-adhered protein crimped fiber.

Unlike synthetic fibers, protein fibers are biodegradable and have low production and processing energy. Therefore, demand for protein fibers is expected to increase in various fields in response to the growing awareness of environmental conservation in recent years. It has been.

As natural protein fibers, crimp filaments such as silk and non-crimp staples such as wool are known. The former has a supple texture, and the latter has a soft feeling and heat retention. , Each has its own characteristics.

Recently, attempts have been made to process protein fibers and apply them to a wider range of applications. For example, Patent Documents 1 and 2 describe a method of mechanically crimping a silk filament, which is a kind of natural protein-free crimping fiber, to obtain a silk crimping filament, and a predetermined method for obtaining such a silk crimping filament. A method of manufacturing silk crimped staples by cutting them to a length has been proposed. The crimped protein filaments and protein staples are used, for example, in the former for producing non-woven fabrics and the latter for producing non-woven fabrics and spun yarns.

By the way, in general, when a spun yarn is obtained, for the purpose of improving the passability in the process, raw material fibers such as crimped staples are used, for example, for preventing chargeability, for reducing friction, or for flexibility. A treatment is performed to attach an oiling agent for application. Further, when the non-woven fabric is obtained, a treatment of attaching an oil agent for imparting functionality such as water repellency to the raw material fibers such as crimp filaments is performed.

Japanese Unexamined Patent Publication No. 2006-207069 Japanese Unexamined Patent Publication No. 2003-105661

When producing spun yarn, non-woven fabric, etc. from non-crimped protein fibers such as silk, the crimping process and the process for adhering an oil agent to the raw material fibers must be performed separately. Therefore, it is unavoidable that the number of steps is increased and the productivity is inferior as compared with the case of producing spun yarn, non-woven fabric and the like from natural protein crimp fibers such as wool.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for efficiently producing oil-adhered protein crimped fibers.

In the process of diligent studies to solve the above problems, the inventor has found that protein fibers containing modified fibroin are crimped (crimped) by contact with water. The present invention has been completed based on the above findings of the inventor and the like, and relates to, for example, the following inventions.
[1]
The protein fiber is modified by including a step of bringing the protein fiber into contact with an aqueous solvent and an oil agent dispersion containing an oil agent dispersed in the aqueous solvent to attach the oil agent to the protein fiber and crimp the protein fiber. A method for producing an oil-adhered protein crimped fiber containing fibroin.
[2]
The production method according to [1], wherein the shrinkage rate of the protein fiber defined by the following formula is more than 7%.
Shrinkage rate = {1- (length of protein fiber after contact with aqueous solvent / length of protein fiber before contact with aqueous solvent)} × 100 (%)
[3]
The production method according to [1] or [2], wherein the modified fibroin is a modified spider silk fibroin.
[4]
The production method according to any one of [1] to [3], wherein the temperature of the aqueous solvent is 10 ° C. or higher and lower than 100 ° C.
[5]
The production method according to any one of [1] to [4], further comprising a step of drying the protein fiber that has been crimped and to which the oil agent is attached.
[6]
The production method according to any one of [1] to [5], wherein the aqueous solvent further contains a volatile solvent.

According to the method according to the present invention, the crimping process and the oil agent adhering treatment can be performed at once in one step, so that the oil agent adhering protein crimped fiber can be efficiently produced. Therefore, according to the method according to the present invention, it is possible to improve the productivity of spun yarns, non-woven fabrics and the like made from protein crimped fibers.

It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is explanatory drawing which shows typically an example of the spinning apparatus for manufacturing a protein filament. It is a photograph of the oil-adhered protein crimped staple in the example. It is a photograph of a protein-free staple in a reference example.

The oil-adhered protein crimped fiber according to the present invention is produced by a method including a step of contacting the protein fiber with the oil-based dispersion, adhering the oil to the protein fiber, and crimping the protein fiber (oil-based dispersion treatment step). can do. The protein fiber contains modified fibroin as a main component, and may be a filament (long fiber) or a staple (short fiber). The protein fiber may contain components and impurities other than modified fibroin, such as proteins other than modified fibroin.

Protein fibers have shrinkage rates of more than 7%, 15% or more, 25% or more, 32% or more, 40% or more, 48% or more, 56% or more, 64% or more when contacted with an aqueous solvent described later. Alternatively, it may be 72% or more. The shrinkage rate is usually 80% or less. Here, the shrinkage rate of protein fibers is defined by the following equation.
Shrinkage rate = {1- (length of protein fiber after contact with aqueous solvent / length of protein fiber before contact with aqueous solvent)} × 100 (%)

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

As used herein, the term "modified fibroin" means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of the naturally occurring fibroin, or may be a fibroin having the same amino acid sequence as the naturally occurring fibroin. “Naturally derived fibroin” as used herein is also represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m − (A) _n motif. It is a protein containing a domain sequence to be used.

As long as the "modified fibroin" has the amino acid sequence specified in the present embodiment, the amino acid sequence of naturally occurring fibroin may be used as it is, and the "modified fibroin" depends on the amino acid sequence of naturally occurring fibroin. The amino acid sequence may be modified (for example, the amino acid sequence is modified by modifying the gene sequence of cloned naturally occurring fibroin), or artificially designed regardless of naturally occurring fibroin. And a synthesized one (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

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

The modified fibroin is, for example, modifying the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. Can be obtained at. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Energy, 100, 448 (1983).

Naturally-derived fibroin is a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} Yes, specifically, for example, fibroin produced by insects or arachnids.

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

More specific examples of insect-produced fibroin include, for example, the silk moth fibroin L chain (GenBank accession number M76430 (nucleic acid sequence) and AAA27840.1 (amino acid sequence)).

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

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleic acid sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleic acid sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank sequence No. AAC4011) (Nucleotide sequence) ), U37520 (nucleic acid sequence)), major amplifier spidroin 1 [derived from Latrodictus protein] (GenBank accession number ABR68856 (nucleic acid sequence), EF595246 (nucleic acid sequence)), dragline silk protein (derived from nuclear protein) Numbers AAL32472 (nucleic acid sequence), AF441245 (nucleic acid sequence)), major aminos protein 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (nucleotide sequence), AJ973155 (nucleic acid sequence)), and major protein (GenBank accession number CAM32249.1 (nucleic acid sequence), AM490169 (nucleic acid sequence)), minor amplify silk protein 1 [Nephila proteins] (GenBank accession number AAC14589.1 (nucleotide sequence)), minor amplifier clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nefilengys cruisetata] (GenBank accession) The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

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

The modified fibroin may be modified silk fibroin (modified amino acid sequence of silk protein produced by silkworm), or modified spider silk fibroin (modified amino acid sequence of spider silk protein produced by spiders). It may be). Among them, modified spider silk fibroin is preferably used.

Specific examples of modified fibroin include modified fibroin derived from a large spider canal bookmark thread protein produced in a large bottle of spider, modified fibroin with a reduced content of glycine residues, and (A) _n motifs. Examples thereof include modified fibroin having a reduced content, modified fibroin having a reduced content of glycine residue, and (A) _{modified fibroin having a reduced content of n motifs.}

Examples of the modified fibroin derived from the large spit tube bookmark thread protein produced in the large bottle-shaped gland of the spider include a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. For the modified fibroin derived from the large spider canal bookmarker thread protein produced in the spider's large bottle-shaped gland, n is preferably an integer of 3 to 20 in Formula 1, more preferably an integer of 4 to 20, and 8 to 20. Integers are even more preferred, integers from 10 to 20 are even more preferred, integers from 4 to 16 are even more preferred, integers from 8 to 16 are particularly preferred, and integers from 10 to 16 are most preferred. In the modified fibroin derived from the large spider thread tube bookmark thread protein produced in the large bottle-shaped gland of the spider, the number of amino acid residues constituting REP is preferably 10 to 200 residues in the formula 1. It is more preferably ~ 150 residues, even more preferably 20-100 residues, and even more preferably 20-75 residues. The modified fibroin derived from the alanine canal bookmark thread protein produced in the large bottle-shaped gland of the spider is a glycine residue contained in the amino acid sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The total number of serine residues and alanine residues is preferably 40% or more, more preferably 60% or more, still more preferably 70% or more, based on the total number of amino acid residues. ..

The modified fibroin derived from the large spitting tube bookmarker thread protein produced in the large bottle-shaped gland of the spider contains the unit of the amino acid sequence represented by _{the formula 1: [(A) n} motif-REP] _{m and has a C-terminal.} The sequence may be a polypeptide having an amino acid sequence shown in any of SEQ ID NOs: 14 to 16 or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 14 to 16.

The amino acid sequence shown in SEQ ID NO: 14 is the same as the amino acid sequence consisting of 50 residues of the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 15 is the sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C-terminal of the amino acid sequence shown in SEQ ID NO: 14, and the amino acid sequence shown in SEQ ID NO: 16 was obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 14. It has the same amino acid sequence.

As a more specific example of the modified fibroin derived from the large spit tube bookmarker thread protein produced in the large bottle-shaped gland of the spider, the amino acid sequence shown in (1-i) SEQ ID NO: 17 or the (1-ii) sequence. Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 17. The sequence identity is preferably 95% or more.

The amino acid sequence shown by SEQ ID NO: 17 is the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 18) consisting of the starting codon, His10 tag and the HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal. The 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue. The amino acid sequence at the C-terminal of the amino acid sequence shown in SEQ ID NO: 17 is the same as the amino acid sequence shown in SEQ ID NO: 16.

The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO: 17.

A modified fibroin having a reduced content of glycine residues has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin. It can be said that the modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP substituted with another amino acid residue as compared with naturally occurring fibroin.

The modified fibroin having a reduced content of glycine residue has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X) as compared with naturally occurring fibroin. Indicates an amino acid residue other than glycine.) Corresponds to the fact that at least one or more glycine residues in the motif sequence are replaced with another amino acid residue in at least one motif sequence selected from. It may have an amino acid sequence.

In the modified fibroin having a reduced content of glycine residue, the proportion of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more of the total motif sequence. ..

The modified fibroin having a reduced content of glycine residue contains _{a domain sequence represented by the formula 1: [(A) n} motif-REP] _m , and is located most C-terminal to the above domain sequence (A). ) Total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence from the _{n motif to the C-terminal of the above domain sequence. Let} z be, and let w be the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the above domain sequence. It may have an amino acid sequence in which / w is 30% or more, 40% or more, 50% or more, or 50.9% or more. (A) The _{number of alanine residues with respect 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. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The modified fibroin having a reduced content of glycine residue is obtained by substituting one glycine residue of the GGX motif with another amino acid residue to increase the content ratio of the amino acid sequence consisting of XGX. preferable. In the modified fibroin having a reduced content of glycine residue, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, and more preferably 10% or less. It is even more preferably 6% 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.

The method of calculating z / w will be described in more detail. First, in the fibroin (modified fibroin or naturally-derived fibroin) containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m, it is located most on the C-terminal side from the domain sequence (A) _n. The amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminal 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. Further, for example, when X (center X) contained in two XGX exists as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used). is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 ( _{excluding the (A) n} motif located most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

In the modified fibroin in which the content of glycine residue is reduced, z / w is preferably 50.9% or more, more preferably 56.1% or more, and 58.7% or more. Is even more preferable, 70% or more is even more preferable, and 80% or more is even more preferable. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

A modified fibroin with a reduced glycine residue content encodes another amino acid residue, for example, by substituting at least part of the nucleotide sequence encoding the glycine residue from the cloned naturally occurring fibroin gene sequence. It can be obtained by modifying it so as to. 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 replaced so that z / w is 50.9% or more. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, in addition to the modification corresponding to the substitution of 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. , Insertion and / or modification of the amino acid sequence corresponding to the addition may be performed.

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

As a more specific example of the modified fibroin having a reduced content of glycine residue, (2-i) the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (2-i). ii) Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 3 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 1 corresponding to naturally occurring fibroin. _{In the amino acid sequence shown in SEQ ID NO: 4, every two (A) n} motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 3, and further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 10, two alanine residues are inserted on the C-terminal side _{of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and some glutamine (Q) residues are further added.} It is substituted with a serine (S) residue and a part of the amino acid on the N-terminal side is deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 12 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above four times.

The value of z / w in the amino acid sequence shown in SEQ ID NO: 1 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence shown in SEQ ID NO: 12 are 58.7%, respectively. It is 70.1%, 66.1% and 70.0%. The values 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 NOs: 1, 3, 4, 10 and 12 are 15.0% and 15. It is 0%, 93.4%, 92.7% and 89.3%.

The modified fibroin of (2-i) may consist of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

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

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography). Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence including His tag sequence and hinge sequence).

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

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

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

As a more specific example of the modified fibroin containing the tag sequence, (2-iii) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or the amino acid sequence shown by SEQ ID NO: 13 or (2-iv) SEQ ID NO: 8 , SEQ ID NO: 9, SEQ ID NO: 11 or modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13.

The amino acid sequences shown in SEQ ID NOs: 6, 7, 8, 9, 11 and 13 are the amino acids shown in SEQ ID NO: 5 at the N-terminal of the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 10 and 12, respectively. A sequence (including a His tag sequence and a hinge sequence) is added.

The modified fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

The modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (2-iv) is also a protein containing the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

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

(A) modified fibroin content of _n motifs has been reduced, the domain sequence is compared to the naturally occurring fibroin, having an amino acid sequence reduced the content of (A) _n motif. It can be said that the domain sequence of the modified fibroin has an amino acid sequence corresponding to the deletion of _{at least one or more (A) n motifs as compared with the naturally occurring fibroin.}

Modified fibroin content is reduced in (A) _n motif, it may have an amino acid sequence corresponding to the naturally occurring fibroin (A) _n motif was deleted 10-40% missing.

(A) The _{modified fibroin having a reduced content of n motifs has a domain sequence of at least 1 to 3 (A) n} motifs from the N-terminal side to the C-terminal side as compared with naturally occurring fibroin. It may have an amino acid sequence corresponding to the deletion of one (A) _{n motif for each.}

(A) The _{modified fibroin having a reduced content of n motifs has a domain sequence of at least two consecutive n} motifs from the N-terminal side to the C-terminal side as compared with naturally occurring fibroin. And the deletion of one (A) _n motif may have an amino acid sequence corresponding to being repeated in this order.

(A) modified fibroin content of _n motifs has been reduced, the domain sequence, amino acids corresponding to at least the N-terminal side 2 every other towards the C-terminal side (A) _n motifs lacking It may have a sequence.

(A) The _{modified fibroin having a reduced content of n-} motif contains a domain sequence represented by the formula 1: [(A) _n- motif-REP] _m , which is adjacent from the N-terminal side to the C-terminal side. When the number of amino acid residues of REP of two matching [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the amino acid residue of the other REP The maximum value of the total value of the sum of the number of amino acid residues of _{two adjacent [(A) n} motif-REP] units having a radix ratio of 1.8 to 11.3 is x, and the total of the domain sequences. When the number of amino acid residues is y, 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 with respect 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. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The calculation method of x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence consists of (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 an arrangement called n motifs.

Two adjacent [(A) _n motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, _{there may be a [(A) n} motif-REP] unit that is not selected. In FIG. 1, pattern 1 (comparison between the first REP and the second REP and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. 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.} The comparison is performed by finding the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with a smaller number of amino acid residues is set to 1, the fifth REP The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 1, when the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is 1.8 to 11.3 [(A) _n motif-REP] unit set. Shown by solid line. Hereinafter, such a ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 _{. The set of [(A) n} motif-REP] units is indicated by a broken line. Indicated.

In each pattern, add up the total number of amino acid residues of the two adjacent [(A) _n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of _{(A) n motif.} is there.). Then, the total values added are compared, and the total value (maximum value of the total value) of the pattern in which the total value is maximized is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.

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

(A) In _{the modified fibroin in which the content of the n} motif is reduced, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more. It is even more preferably 70% or more, even more preferably 75% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be, for example, 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x. / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.

(A) A _{plurality of modified fibroins having a reduced content of n} motifs are present in the domain sequence. (A) When _{at least 7 of the n} motifs are modified fibroins composed only of alanine residues, x / y is , 46.4% or more, more preferably 50% or more, further preferably 55% or more, further preferably 60% or more, and even more preferably 70% or more. Even more preferably, it is particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be 100% or less.

(A) modified fibroin content of _n motif is reduced, for example, encoding a cloned naturally occurring fibroin gene sequences, as x / y is more than 64.2% of the (A) _n motif It can be obtained by deleting one or more of the sequences. Further, for example, an amino acid sequence corresponding to the deletion _{of one or more (A) n} motifs so that x / y is 64.2% or more is designed and designed 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. _{In each case, in addition to the modification corresponding to the deletion of (A) n} motif from the amino acid sequence of naturally occurring fibroin, further substitution, deletion, insertion and / or addition of one or more amino acid residues. The amino acid sequence corresponding to the above may be modified.

(A) As _{a more specific example of the modified fibroin having a reduced content of n} motif, (3-i) the amino acid sequence shown by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 or (3-i) 3-ii) Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin of (3-i) will be described. The amino acid sequence shown in SEQ ID NO: 2 is obtained by deleting _{every other (A) n} motif from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 1, which corresponds to naturally occurring fibroin. Further, one [(A) _n motif-REP] is inserted before the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 4 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 2. In the amino acid sequence shown in SEQ ID NO: 10, two alanine residues are inserted on the C-terminal side _{of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and some glutamine (Q) residues are further added.} It is substituted with a serine (S) residue and a part of the amino acid on the N-terminal side is deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 12 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above four times.

The value of x / y in the jagged ratio of 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 1 (corresponding to naturally occurring fibroin) is 15.0%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 4 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 10 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 12 is 89.3%. The values of z / w in the amino acid sequences shown in SEQ ID NOs: 1, 2, 4, 10 and 12 are 46.8%, 56.2%, 70.1%, 66.1% and 70.0%, respectively. is there.

The modified fibroin of (3-i) may consist of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by _{the 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 represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _{The amino acid residue of two adjacent [(A) n} motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3) When the maximum value of the total value obtained by adding the radix is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

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

As a more specific example of the modified fibroin containing the tag sequence, (3-iii) SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or the amino acid sequence shown by SEQ ID NO: 13 or (2-iv) SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11 or modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13.

The amino acid sequences shown in SEQ ID NOs: 6, 7, 8, 9, 11 and 13 are the amino acids shown in SEQ ID NO: 5 at the N-terminal of the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 10 and 12, respectively. A sequence (including a His tag sequence and a hinge sequence) is added.

The modified fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

The modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The modified fibroin of (3-iv) is also a protein containing the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _Let x be the maximum value of the total value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units in which the ratio of the number of amino acid residues in REP is 1.8 to 11.3. , When the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

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

The content of glycine residues, and (A) modified fibroin content of _n motifs has been reduced, the domain sequence is compared to the naturally occurring fibroin was reduced content of (A) _n motif In addition, it has an amino acid sequence with a reduced content of glycine residues. The domain sequence of the modified fibroin lacks at least one or more (A) _n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with an amino acid residue. That is, it is a modified fibroin having the characteristics of the modified fibroin in which the content of the glycine residue is reduced and the modified fibroin in which _{the content of (A) n motif is reduced.} Specific aspects and the like are as described in the modified fibroin in which the content of glycine residue is reduced and the modified fibroin in which the content of (A) _n motif is reduced.

As a more specific example of the modified fibroin in which the content of the glycine residue and the content of (A) _n motif are reduced, the amino acids shown in (4-i) SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are shown. Examples include modified fibroin comprising a sequence, (4-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12. Specific embodiments of the modified fibroin containing the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are as described above.

The modified fibroin according to the other embodiment has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a high hydrophobicity index as compared with naturally occurring fibroin, and / Or it may have an amino acid sequence containing a region having a large hydrophobicity index locally, which corresponds to the insertion of one or more amino acid residues having a large hydrophobicity index into the REP.

The region having a locally large hydrophobicity index is preferably composed of consecutive 2 to 4 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). It is more preferably a residue.

In the modified fibroin according to the present embodiment, one or more amino acid residues in REP were replaced with amino acid residues having a large hydrophobicity index as compared with naturally occurring fibroin, and / or 1 in REP. Or, in addition to modifications corresponding to the insertion of multiple amino acid residues with a large hydrophobicity index, further substitution, deletion, insertion and / of one or more amino acid residues as compared to naturally occurring fibroin. Alternatively, there may be a modification of the amino acid sequence corresponding to the addition.

The modified fibroin according to the present embodiment has, for example, hydrophobicity of one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in REP from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting an amino acid residue (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. Also, for example, one or more hydrophilic amino acid residues in the REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in the 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 either case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acids in the REP. In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The modified fibroin according to still another embodiment _{contains the domain sequence represented by the formula 1: [(A) n} motif-REP] _{m , and the (A) n} motif located closest to the C-terminal side of the above domain sequence. The total number of amino acid residues contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs included in the sequence excluding the sequence up to the C-terminal from the above domain sequence. Is p, and p is the total number of amino acid residues contained in the sequence obtained by excluding the sequence from _{the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. It may have an amino acid sequence in which / q is 6.2% or more.

For the hydrophobicity index of amino acid residues, a known index (Hydrotherapy index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic protein, B. 105-132) is used. Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m. (Hereinafter referred to as "sequence A") is used. First, in all the REPs contained in the sequence A, the average value of the hydrophobicity index of consecutive four amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the total 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 consecutive 4 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 hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index", it should be included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Further, the total number of amino acid residues contained in the sequence A is q.

For example, when 20 consecutive "4 consecutive amino acid residues having an average hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of .6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are duplicated by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues is present. A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 2, p is 7 × 4 = because there are seven “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, 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 (excluding the (A) _n motif existing at the end of the C-terminal side). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 2, 28/170 = 16.47%.

In the modified fibroin according to the present embodiment, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and further preferably 20% or more. Is even more preferable, and 30% or more is even more preferable. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

The modified fibroin according to the present embodiment is, for example, one or a plurality of hydrophilic amino acid residues (for example, hydrophobic) in the REP so that the amino acid sequence of the cloned naturally occurring fibroin is subjected to the above-mentioned p / q condition. Replacing an amino acid residue with a negative sex index) with a hydrophobic amino acid residue (eg, an amino acid residue with a positive hydrophobicity index) and / or one or more hydrophobic amino acid residues in the REP. Can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. It can also be obtained, for example, by designing an amino acid sequence satisfying the above p / q condition from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP were replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring fibroin, and / or one or more amino acid residues in the REP. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. ..

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) Is preferable, and valine (V), leucine (L) and isoleucine (I) are more preferable.

As another specific example of the modified fibroin, (5-i) the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23, or (5-ii) SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23. Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by.

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

The modified fibroin of (5-i) may consist of the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23.

The modified fibroin of (5-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by _{the 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 represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23, and is located most on the C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p and the total number of _{amino acid residues contained in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

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

As a more specific example of the modified fibroin comprising a tag sequence, the amino acid sequence set forth in (5-iii) SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, or (5-iv) SEQ ID NO: 24, SEQ ID NO: 25 or Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 26.

The amino acid sequences shown in SEQ ID NOs: 24, 25 and 26 include the amino acid sequence shown in SEQ ID NO: 5 (including His tag sequence and hinge sequence) at the N-terminal of the amino acid sequence shown in SEQ ID NOs: 20, 22 and 23, respectively. It is an added one.

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

The modified fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth 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 the domain sequence represented by _{the 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 represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and is located most on the C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p and the total number of _{amino acid residues contained in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

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

The modified fibroin according to still another embodiment has an amino acid sequence having a reduced content of glutamine residues as compared with naturally occurring fibroin.

The modified fibroin according to the present embodiment preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

When the modified fibroin according to the present embodiment contains a GPGXX motif in the REP, the content of the GPGXX motif is usually 1% or more, may be 5% or more, and is 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.

In the present specification, 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) _{Fibroin containing a domain sequence represented by n-} motif (modified fibroin or naturally derived) In (fibroin), the number of GPGXX motifs contained in the region in all REPs included in the sequence excluding the sequence from _{the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. Let s be the number obtained by multiplying the total number by 3 (that is, corresponding to the total number of G and P in the GPGXX motif), and the _{sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence is taken from the domain sequence. The GPGXX motif content is calculated as s / t, where t is the total number of amino acid residues in all REPs excluding (A) _{n motifs.}

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

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

The modified fibroin according to the present embodiment preferably has a glutamine residue content of 9% or less, more preferably 7% or less, further preferably 4% or less, and preferably 0%. Especially preferable.

In the present specification, the "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) _{fibroin containing a domain sequence represented by n} motif (modified fibroin or naturally derived fibroin) _{In fibroin), all the sequences from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 3). In the REP of, the total number of glutamine residues contained in the region is u, and the _{sequence from the (A) n} motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and (A) _n. The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif. In the calculation of the glutamine residue content, the _{reason why "the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

The modified fibroin according to the present embodiment has a domain sequence in which one or more glutamine residues in REP have been deleted or replaced with other amino acid residues as compared with naturally occurring fibroin. It may have a corresponding amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than the glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

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

In the modified fibroin according to the present embodiment, the hydrophobicity of REP is preferably −0.8 or more, more preferably −0.7 or more, further preferably 0 or more, and 0. It is even more preferably 3 or more, and particularly preferably 0.4 or more. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

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

The modified fibroin according to the present embodiment has a domain sequence lacking one or more glutamine residues in REP as compared with naturally occurring fibroin, and / or one or more glutamines in REP. In addition to the modification corresponding to the substitution of the residue with another amino acid residue, there is a modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. May be good.

The modified fibroin according to the present embodiment may, for example, delete one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence and / or one or more glutamine residues in REP. Can be obtained by substituting with another amino acid residue. Also, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to this and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As a more specific example of the modified fibroin according to the present invention, (6-i) the amino acid 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: 38 or SEQ ID NO: 39. 90% or more of the modified fibroin, including the sequence, or the amino acid sequence set forth in (6-ii) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQ ID NO: 39. Modified fibroins containing amino acid sequences having sequence identity can be mentioned.

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

The amino acid sequence (Met-PRT410) shown in SEQ ID NO: 4 is based on the nucleotide sequence and amino acid sequence of Nephila clavipes (GenBank accession number: P4684.1, GI: 11744415), which is a naturally occurring fibroin, (A) _n. The amino acid sequence in which the alanine residues are continuous in the motif is modified to improve the productivity, such as increasing the number of consecutive alanine residues to five. On the other hand, since Met-PRT410 has not been modified for the glutamine residue (Q), the glutamine residue content is about the same as the glutamine residue content of naturally occurring fibroin.

The amino acid sequence (M_PRT888) shown in SEQ ID NO: 27 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with VL.

In the amino acid sequence (M_PRT965) shown in SEQ ID NO: 28, all QQs in Met-PRT410 (SEQ ID NO: 4) are replaced with TS, and the remaining Qs are replaced with A.

The amino acid sequence (M_PRT889) shown in SEQ ID NO: 29 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with VL and substituting the remaining Qs with I.

In the amino acid sequence (M_PRT916) shown in SEQ ID NO: 30, all QQs in Met-PRT410 (SEQ ID NO: 4) are replaced with VI, and the remaining Qs are replaced with L.

In the amino acid sequence (M_PRT918) shown in SEQ ID NO: 31, all QQs in Met-PRT410 (SEQ ID NO: 4) are replaced with VF, and the remaining Qs are replaced with I.

_{The amino acid sequence (M_PRT525) shown in SEQ ID NO: 37 has two alanine residues inserted in a region (A 5} ) in which alanine residues are continuous with respect to Met-PRT410 (SEQ ID NO: 4), and has a molecular weight of Met-PRT410. Two domain sequences on the C-terminal side were deleted, and 13 glutamine residues (Q) were replaced with serine residues (S) or proline residues (P) so as to be substantially the same as.

The amino acid sequence (M_PRT699) shown in SEQ ID NO: 38 is obtained by substituting all QQs in M_PRT525 (SEQ ID NO: 37) with VL.

The amino acid sequence (M_PRT698) shown in SEQ ID NO: 39 is obtained by substituting all QQs in M_PRT525 (SEQ ID NO: 37) with VL and substituting the remaining Qs with I.

The amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 and SEQ ID NO: 39 all have a glutamine residue content of 9% or less (Table 2). ).

The modified fibroin of (6-i) may consist of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 17. ..

The modified fibroin of (6-ii) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQ ID NO: 39. It contains an amino acid sequence having. The modified fibroin of (6-ii) is also a domain represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

As a more specific example of the modified fibroin containing the tag sequence, (6-iii) the amino acid represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 20. 90% or more of the modified fibroin containing the sequence, or the amino acid sequence set forth in (6-iv) SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 20. Modified fibroins containing amino acid sequences having sequence identity can be mentioned.

The amino acid sequences shown by SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 and SEQ ID NO: 41 are SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively. , The amino acid sequence shown in SEQ ID NO: 5 (including His tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 38 and SEQ ID NO: 39. Since only the tag sequence was added to the N-terminal, there was no change in the glutamine residue content, and SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 and SEQ ID NO: 41. All of the amino acid sequences shown in (Table 3) have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 or SEQ ID NO: 41. ..

The modified fibroin (6-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 or SEQ ID NO: 41. It contains an amino acid sequence having. The modified fibroin of (6-iv) is also a domain represented by _{formula 1: [(A) n} motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

<Manufacturing method of modified fibroin>
The modified fibroin according to any of the above embodiments (hereinafter, may be simply referred to as “protein”) also includes, for example, a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing the nucleic acid in a host transformed with an expression vector having.

The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding natural fibroin and modifying it by a genetic engineering method, or a method of chemically synthesizing it. can do. The chemical synthesis method of 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 or the like, AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.) or the like. Genes can be chemically synthesized by ligating automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the modified fibroin, a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N-terminal of the above amino acid sequence is synthesized. You may.

The regulatory sequence is a sequence that controls the expression of modified fibroin in the host (for example, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, a vector containing a promoter at a position capable of autonomous replication in a host cell, integration into a host chromosome, and transcription of a nucleic acid encoding modified fibroin is preferably used.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia marcescens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus satirus and the like. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium, Ammonia Philum and the like. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium and Ammonia Genes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, as a vector into which a nucleic acid encoding modified fibroin is introduced, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, Examples thereof include pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569).

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

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

As a method for expressing 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 2nd Edition. ..

The modified 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 the method usually used for culturing the host.

When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host, and the host can be efficiently cultured. If so, either a natural medium or a synthetic medium may be used.

The carbon source may be any assimilated by the above-mentioned transforming microorganisms, for example, glucose, fructose, sucrose, carbohydrates such as molasses, starch and starch hydrolyzate containing them, acetic acid, propionic acid and the like. Organic acids and alcohols such as ethanol and propanol can be used. Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and their digests can be used. As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

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

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

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

When the modified fibroin is expressed by forming an insoluble matter in the cells, the insoluble matter of the modified fibroin is recovered as a precipitation fraction by similarly collecting the host cell, crushing the host cell, and centrifuging the cell. The insoluble form of the recovered modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above. When the protein is secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

<Method for producing protein (modified fibroin) fiber>
When the protein fiber is a protein filament, the protein filament can be produced by a known spinning method. That is, for example, when producing a protein filament containing modified fibroin as a main component, first, the modified fibroin produced according to the above-mentioned method is dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), and formic acid. Or, if necessary, it is added to a solvent such as hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution accelerator and dissolved to prepare a dope solution. Then, using this doping solution, a protein filament can be obtained by spinning by a known spinning method such as wet spinning, dry spinning, dry wet spinning, or melt spinning. Preferred spinning methods include wet spinning or dry-wet spinning.

FIG. 4 is an explanatory diagram schematically showing an example of a spinning device for producing a protein filament. The spinning device 10 shown in FIG. 4 is an example of a spinning device for dry / wet spinning, and includes an extrusion device 1, an undrawn yarn manufacturing device 2, a moist heat drawing device 3, and a drying device 4.

A spinning method using the spinning device 10 will be described. First, the doping liquid 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8. On a lab scale, a cylinder may be filled with a dope solution and extruded from a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied into the coagulation liquid 11 of the coagulation liquid tank 20 through the air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulant is formed. Next, the fibrous solidified body is supplied into the hot water 12 in the stretching bath 21 and stretched. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14. Then, the stretched fibrous coagulant is supplied to the drying device 4 and dried in the yarn path 22, and the protein filament 36 is obtained as the wound yarn body 5. 18a to 18g are thread guides.

The coagulation liquid 11 may be any solvent that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulant 11 may contain water as appropriate. The temperature of the coagulating liquid 11 is preferably 0 to 30 ° C. When a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm is used as the base 9, the extrusion speed is preferably 0.2 to 6.0 mL / hour, and 1.4 to 4.0 mL / hour per hole. More preferably, it is time. The distance through which the coagulated modified fibroin passes through the coagulation liquid 11 (substantially, the distance from the thread guide 18a to the thread guide 18b) may be as long as the solvent can be efficiently removed, for example, 200 to 200. It is 500 mm. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, preferably 1 to 3 m / min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulating liquid 11. The coagulation liquid tank 20 may be provided in multiple stages, and stretching may be performed in each stage or in a specific stage, if necessary.

As the stretching performed when obtaining the protein filament, for example, in addition to the pre-stretching performed in the coagulation liquid tank 20 and the moist heat stretching performed in the stretching bath 21, dry heat stretching is also adopted.

Wet heat stretching can be performed in warm water, in a solution of warm water to which an organic solvent or the like is added, or in steam heating. The temperature may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C. In the moist heat drawing, the undrawn yarn (or the pre-drawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.

Dry heat stretching can be performed using an electric tube furnace, a dry heat plate, or the like. The temperature may be, for example, 140 ° C. to 270 ° C., preferably 160 ° C. to 230 ° C. In the dry heat drawing, the undrawn yarn (or the pre-drawn yarn) can be drawn 0.5 to 8 times, preferably 1 to 4 times.

Wet heat stretching and dry heat stretching may be performed individually, or they may be performed in multiple stages or in combination. That is, the first-stage stretching is performed by moist heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by moist heat stretching, the second stage stretching is performed by moist heat stretching, and the third stage stretching is performed by dry heat stretching. Wet heat stretching and dry heat stretching can be appropriately combined.

The lower limit of the final draw ratio is preferably more than 1 time, 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). It is any of the above, 7 times or more, 8 times or more, and 9 times or more, and the upper limit values are preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, and 13 times or less. , 12 times or less, 11 times or less, 10 times or less.

The length of the protein filament obtained by the above method can be appropriately adjusted depending on the spinning conditions. The length of the protein filament is not particularly limited, but may be, for example, over 1500 m, 10,000 m or more, 15,000 m or more, or 20,000 m or more.

When the protein fiber is a protein staple, the protein staple can be produced by cutting the protein filament produced by the above method. The protein filament can be cut using any device such as a tabletop fiber cutting machine (s / NO.IT-160201-NP-300). The protein filament may be cut after the oil dispersion treatment step or the drying step described later.

The length of the protein staple is not particularly limited, but may be 20 mm or more, 20 to 140 mm, 70 to 140 mm, or 20 to 70 mm.

<Oil dispersion liquid treatment process>
In the oil agent dispersion treatment step, for example, the protein fibers obtained by the above method are brought into contact with the oil agent dispersion to attach the oil agent to the protein fibers and crimp the protein fibers. The protein fiber can be brought into contact with the oil agent dispersion, for example, by immersing the protein fiber in the oil agent dispersion. The protein fibers in the present invention contract and are crimped when they come into contact with water. Therefore, the protein fibers that have undergone the oil dispersion treatment step have crimps. The amount of crimp is, for example, 10 to 100 pieces / 40 mm or 20 to 40 pieces / 40 mm.

The oil agent dispersion liquid contains an aqueous solvent and an oil agent dispersed in an aqueous solvent. The aqueous solvent is a liquid containing water. The aqueous solvent may be water or a mixed solution of water and a hydrophilic solvent. As the hydrophilic solvent, for example, a volatile solvent such as ethanol and methanol can be used. The aqueous solvent is preferably a mixed solution of water and ethanol. By using an aqueous solvent containing a volatile solvent, the protein fibers dry quickly, and the finally obtained oil-adhered protein crimped fibers have a softer texture. The ratio of water to the volatile solvent is not particularly limited, and for example, the water: volatile solvent may have a mass ratio of 10:90 to 90:10.

The oil agent is not particularly limited, and can be selected from known oil agents according to the properties imparted to the protein fiber. The oil agent imparts properties for smoothly carrying out each process in the manufacture of the oil-adhered protein crimped fiber and the product using the oil agent, such as for antistatic, friction-reducing, flexibility-imparting, and water-repellent-imparting. It may be one (that is, for passing through a process), or one that imparts desired properties such as flexibility and antistatic property to an oil-adhered protein crimped fiber (that is, for imparting functional properties or for finishing). You may. The amount of the oil agent is not particularly limited, but is preferably 1 to 10% by mass, more preferably 2 to 5% by mass, based on the total amount of the aqueous solvent and the oil agent.

The temperature of the oil dispersion may be, for example, 10 ° C. or higher and lower than 100 ° C., 10 to 60 ° C., 20 to 50 ° C., and 30 to 40 ° C. Since the shrinkage rate of protein fibers changes depending on the temperature of the oil dispersion, the amount of crimping (degree of crimping) can be controlled by adjusting the temperature of the oil dispersion. The time for contacting the oil dispersion with the protein fibers is not particularly limited, and may be 30 seconds or longer, 1 minute or longer, or 2 minutes or longer, and preferably 10 minutes or shorter from the viewpoint of productivity. The contact of the oil dispersion liquid with the protein fibers may be carried out under normal pressure or under reduced pressure (for example, vacuum).

The shrinkage rate of protein fibers after contact with the oil dispersion is more than 7%, 15% or more, 25% or more, 32% or more, 40% or more, 48% or more, 56% or more, 64% or more, or 72. It may be% or more. The shrinkage rate is usually 80% or less. Here, the shrinkage rate of protein fibers can be calculated by the following formula.
Shrinkage rate = {1- (length of protein fiber after contact with oil dispersion / length of protein fiber before contact with oil dispersion)} x 100 (%)

The crimping of the protein fiber due to contact with the oil agent dispersion liquid containing an aqueous solvent is preferably in a state where no tensile force is applied to the protein fiber (no tension is applied in the fiber axis direction), or a predetermined size. It is carried out with only added (tensioned by a predetermined amount in the fiber axis direction). By adjusting the tensile force applied to the protein fiber at that time, it is possible to control the degree of crimping. Examples of the method of adjusting the tensile force applied to the protein fiber include a method of adjusting the load applied to the filaments and staples by suspending weights of various weights on the protein fiber, and a method of relaxing the protein fiber. A method of fixing both ends in a slack state and changing the amount of slack in various ways, winding protein fibers around a wound body such as a paper tube or bobbin, and winding force at that time (tightening to the paper tube or bobbin) A method of appropriately changing the force) can be mentioned.

<Drying process>
The protein fibers may be dried after the oil dispersion is brought into contact with the protein fibers. The drying method is not particularly limited, and natural drying may be used, or the protein fibers may be forcibly dried using a drying facility. The crimping with the oil dispersion and the subsequent drying can be performed continuously. Specifically, for example, the protein fibers can be sent out from the bobbin, immersed in an oil dispersion, and then blown with hot air or sent out onto a hot roller for drying. The drying temperature is not particularly limited, and may be, for example, 20 to 150 ° C, 40 to 120 ° C, and 60 to 100 ° C.

As described above, in the method for producing an oil-adhered protein crimped fiber according to the present invention, the protein fiber shrinks and crimps when it comes into contact with water. Therefore, according to the method according to the present invention, by using the oil agent dispersion liquid containing an aqueous solvent, the protein fibers can be crimped and the oil agent can be attached to the protein fibers at the same time. It is possible to efficiently produce crimped fibers. Further, as described above, the protein fiber used in the method of the present invention shrinks when it comes into contact with water, but once it comes into contact with water and shrinks, the shrinkage rate when it comes into contact with water next time is remarkably reduced. descend. Since the oil-adhered protein crimped fiber produced by the method according to the present invention has already contracted in contact with water as described above, the oil-adhered protein crimped fiber is being stored or the oil-adhered protein crimped fiber is rolled. Even if the oil-adhered protein crimped fiber absorbs moisture again during the manufacturing process of the product using the crimped fiber, the shrinkage due to the moisture absorption can be reduced. Therefore, the oil-adhered protein crimped fiber and the product using the same. It is possible to reduce the change in the dimensions of.

The oil-adhered protein crimped fiber produced by the method according to the present invention can be used, for example, for producing a non-woven fabric and a composite (composite material with a base material such as resin). Further, when the oil-adhered protein crimped fiber is a staple, it can also be used for producing a spun yarn.

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

<Manufacturing of modified spider silk fibroin>
(1) Preparation of plasmid expression strain Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes, the amino acid sequence shown by SEQ ID NO: 13 was obtained. A modified fibroin having (hereinafter, also referred to as “PRT799”) was designed. The amino acid sequence shown in SEQ ID NO: 13 has an amino acid sequence in which amino acid residues are substituted, inserted or deleted for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes. Further, the amino acid sequence (tag sequence and hinge sequence) shown by SEQ ID NO: 5 is added to the N-terminal.

Next, a nucleic acid encoding PRT799 was synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into a protein expression vector pET-22b (+) to obtain an expression vector.

(2) Protein expression Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequence shown in SEQ ID NO: 13. 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 so that OD _{600 was 0.005.} The culture solution temperature was maintained at 30 ° C., _{and flask culture was carried out until the OD 600} reached 5, (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 5) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

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

(3) Purification of protein The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL and 30 at 60 ° C. Stir for 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 by a freeze-dryer, and the freeze-dried powder was recovered to obtain a modified spider silk fibroin "PRT799".

<Manufacturing of protein filament>
(1) Preparation of Dope Solution The above-mentioned modified fibroin (PRT799) was added to DMSO to a concentration of 24% by mass, and then LiCl was added as a dissolution accelerator to a concentration of 4.0% by mass. Then, using a shaker, the modified fibroin was dissolved over 3 hours to obtain a DMSO solution. Dust and bubbles in the obtained DMSO solution were removed to prepare a doping solution. The solution viscosity of the dope solution was 5000 cP (centipores) at 90 ° C.

(2) Spinning A protein filament was obtained by performing known dry-wet spinning using the doping solution obtained as described above and the spinning device 10 shown in FIG. The resulting protein filament was wound around a bobbin. Here, dry-wet spinning was performed under the following conditions.
Coagulant (methanol) temperature: 5-10 ° C
Stretching ratio: 4.52 times Drying temperature: 80 ° C

<Example>
A plurality of protein filaments obtained as described above were bundled and cut into a length of 40 mm with a tabletop fiber cutting machine to prepare protein staples. The protein staples were immersed in an oil dispersion at 20 ° C. for 1 minute to attach the oil to the protein staples and crimp the protein staples. As the oil agent dispersion liquid, a commercially available antistatic oil agent dispersed in water so as to have a concentration of 1% by mass was used. The shrinkage rate of the protein staples by immersing in the oil dispersion was 50%. The protein staples were then dried at 40 ° C. for 18 hours to give oil-adhered protein crimped staples. The obtained oil-adhered protein crimp staples are shown in FIG.

<Reference example>
A plurality of protein filaments obtained in the same manner as in Examples were bundled and cut to obtain non-crimped protein staples. The obtained non-crimped protein staples are shown in FIG.

1 ... Extruding device, 2 ... Undrawn yarn manufacturing device, 3 ... Moist heat drawing device, 4 ... Drying device, 6 ... Dope solution, 10 ... Spinning device, 20 ... Coagulation liquid tank, 21 ... Stretching bath, 36 ... Protein filament.

Claims (6)

It comprises a step of bringing a protein fiber into contact with an aqueous solvent and an oil agent dispersion containing an oil agent dispersed in the aqueous solvent to attach the oil agent to the protein fiber and crimp the protein fiber.
A method for producing an oil-adhered protein crimped fiber, wherein the protein fiber contains modified fibroin. The production method according to claim 1, wherein the shrinkage rate of the protein fiber defined by the following formula is more than 7%.
Shrinkage rate = {1- (length of protein fiber after contact with aqueous solvent / length of protein fiber before contact with aqueous solvent)} × 100 (%) The production method according to claim 1 or 2, wherein the modified fibroin is modified spider silk fibroin. The production method according to any one of claims 1 to 3, wherein the temperature of the aqueous solvent is 10 ° C. or higher and lower than 100 ° C. The production method according to any one of claims 1 to 4, further comprising a step of drying the protein fiber that has been crimped and to which the oil agent is attached. The production method according to any one of claims 1 to 5, wherein the aqueous solvent further contains a volatile solvent.

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