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WO2019151432A1 - Procédé de préparation d'une fibre protéinique frisée d'adhérence à l'huile - Google Patents

Procédé de préparation d'une fibre protéinique frisée d'adhérence à l'huile Download PDF

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Publication number
WO2019151432A1
WO2019151432A1 PCT/JP2019/003469 JP2019003469W WO2019151432A1 WO 2019151432 A1 WO2019151432 A1 WO 2019151432A1 JP 2019003469 W JP2019003469 W JP 2019003469W WO 2019151432 A1 WO2019151432 A1 WO 2019151432A1
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amino acid
seq
sequence
protein
acid sequence
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Japanese (ja)
Inventor
皓斗 佐藤
佑之介 安部
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Spiber Inc
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Spiber Inc
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    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
    • D—TEXTILES; PAPER
    • D02—YARNS; MECHANICAL FINISHING OF YARNS OR ROPES; WARPING OR BEAMING
    • D02G—CRIMPING OR CURLING FIBRES, FILAMENTS, THREADS, OR YARNS; YARNS OR THREADS
    • D02G1/00—Producing crimped or curled fibres, filaments, yarns, or threads, giving them latent characteristics
    • D—TEXTILES; PAPER
    • D02—YARNS; MECHANICAL FINISHING OF YARNS OR ROPES; WARPING OR BEAMING
    • D02G—CRIMPING OR CURLING FIBRES, FILAMENTS, THREADS, OR YARNS; YARNS OR THREADS
    • D02G3/00—Yarns or threads, e.g. fancy yarns; Processes or apparatus for the production thereof, not otherwise provided for
    • D02G3/02—Yarns or threads characterised by the material or by the materials from which they are made

Definitions

  • the present invention relates to a method for producing an oil-attached protein crimped fiber.
  • Protein fibers unlike synthetic fibers, are biodegradable and require less energy for production and processing. As a result, the demand for various fields is expected to increase in response to the recent increase in environmental conservation awareness. It is.
  • Known natural protein fibers include crimped filaments such as silk and non-crimped staples such as wool, the former having a supple texture, the latter having a soft feeling and heat retention, etc. , Each has its own characteristics.
  • Patent Documents 1 and 2 describe a method of obtaining a silk crimped filament by mechanically crimping a silk filament, which is a kind of natural protein-free crimped fiber, A method for producing a silk crimped staple by cutting into a length has been proposed.
  • the crimped protein filaments and protein staples are used, for example, in the former for the production of nonwoven fabrics and for the latter in the production of nonwoven fabrics and spun yarns.
  • the raw material fiber such as crimped staple
  • the raw material fiber such as crimped staple
  • the process which makes the oil agent for provision adhere is performed.
  • the process which makes the oil agent for providing functionalities, such as water repellency, for example, adhere to raw material fibers, such as a crimped filament is performed.
  • This invention is made
  • the inventor has found that the protein fiber containing the modified fibroin is crimped (shrinks) by contact with water in the course of intensive studies to solve the above-described problems.
  • the present invention has been completed based on the above findings of the inventors, and relates to, for example, the following inventions.
  • the protein fiber is modified by bringing the protein fiber into contact with an oil dispersion containing an aqueous solvent and an oil agent dispersed in the aqueous solvent, causing the oil agent to adhere to the protein fiber and crimping the protein fiber.
  • the manufacturing method of the oil agent adhesion protein crimp fiber containing a fibroin The manufacturing method of the oil agent adhesion protein crimp fiber containing a fibroin.
  • the method of the present invention it is possible to perform the crimping process and the oil agent attaching process all at once, so that the oil agent attaching protein crimp fiber can be efficiently produced. Therefore, according to the method of the present invention, it is possible to improve the productivity of spun yarn and nonwoven fabric made from protein crimped fibers.
  • the oil-attached protein crimped fiber according to the present invention is produced by a method comprising a step of bringing a protein fiber into contact with an oil dispersion to attach the oil to the protein fiber and crimping the protein fiber (oil agent 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 other than the modified fibroin and impurities such as a protein other than the modified fibroin.
  • Protein fibers have a shrinkage ratio (shrinkage ratio after drying) of more than 7%, 15% or more, 25% or more, 32% or more, 40% or more when brought into contact with an aqueous solvent described later after spinning and then dried. 48% or more, 56% or more, 64% or more, or 72% or more.
  • the shrinkage after drying is usually 80% or less.
  • the protein fiber has a shrinkage ratio (shrinkage ratio when wet) of, for example, 2% or more when it is wetted by contacting with an aqueous solvent (for example, water having a boiling point below) after spinning. It may be 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, or 6% or more. Also good.
  • the upper limit of the shrinkage rate when wet is not particularly limited, but 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3 % Or less.
  • the modified fibroin according to the present embodiment has a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein containing.
  • an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either one 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 these, but are typically regions having no amino acid motif repeat characteristic of fibroin and consisting of about 100 amino acids.
  • modified fibroin means an artificially produced fibroin (artificial fibroin).
  • the modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin or may be the same as the amino acid sequence of naturally occurring fibroin.
  • Natural fibroin as used herein is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif.
  • a protein comprising a domain sequence to be processed.
  • the amino acid sequence of naturally-occurring fibroin may be used as it is, and it depends on the amino acid sequence of naturally-occurring fibroin.
  • the amino acid sequence may be modified (for example, the amino acid sequence may be modified by modifying the gene sequence of a naturally-derived fibroin that has been cloned), or it may be artificially designed without relying on the naturally-occurring fibroin. And those synthesized (for example, those having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).
  • domain sequence refers to a fibroin-specific crystal region (typically corresponding to the (A) n motif in the amino acid sequence) and an amorphous region (typically in the REP of the amino acid sequence).
  • (A) n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27.
  • the number of amino acid residues of the n motif 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 .
  • the ratio of the number of alanine residues to the total number of amino acid residues in the (A) n motif may be 40% or more, such as 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues).
  • a plurality of (A) n motifs present in the domain sequence may be composed of at least seven alanine residues alone.
  • REP indicates an amino acid sequence composed 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.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin is, for example, a modification of the amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned natural 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-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.
  • Naturally-derived fibroin is a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif.
  • Specific examples include fibroin produced by insects or spiders.
  • fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandaraina), Tengea (Antheraea yamanai), ⁇ ⁇ (Antereaperanii), ⁇ ⁇ (Eriothyraminey) ), Silkworms produced by silkworms, such as Samia cythia, chestnut worms (Caligula japonica), Chuser moth (Antherea mylitta), Antheraea assama, and vespax (Vespaxia spp.) Hornet silk protein.
  • fibroin produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
  • Fibroin produced by spiders includes, for example, spiders belonging to the genus spider (Araneus spp.) Such as the spider spider, the spider spider, the red spider spider, and the bean spider, the genus spiders of the genus Araneus, the spider spider spider, the spider spider genus e Spiders, spiders such as spiders, spiders belonging to the genus Spider, spiders belonging to the genus Pronos, spiders belonging to the genus Trinofunda, such as Torinofundamas (genus Cyrtarachne) Spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Spider (Ordgarius genus), such as the spiders, the spiders, and the spiders belonging to the genus Ordgarius Spiders belonging to the genus Argiope, such as the genus Argiope, spiders belonging to the genus Arachnura, such as the white-tailed spider, spiders belonging to the
  • Spiders belonging to the genus Azumigumi (Menosira), spiders belonging to the genus Dyschiriognatha (genus Dyschiriognatha) such as the common spider spider, the black spider spider, the genus Spider genus belonging to the genus Spider belonging to the genus (L) and the genus Spider belonging to the genus Usd Produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops
  • Examples include spider silk protein.
  • the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.
  • spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spiroin 1 [derived from Nephila clavipes] (GenBank accession number 4) ), U37520 (base sequence)), major ampulate spidro n 1 [derived from Latroductus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidolin 2 [derived from Nephila clavata (GenBank accession number AAL32 base sequence 44 AAL32 base sequence amino acid 44, amino acid sequence 44 AAL47)
  • Naturally derived fibroin include fibroin whose sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidin, sample, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION from sequences including INV as DIVISION among the sequence information registered in NCBI GenBank. It can be confirmed by extracting a character string of a specific product from the sequence, CDS, and a sequence in which the specific character string is described from SOURCE to TISSUE TYPE.
  • the modified fibroin may be modified silk fibroin (modified silk protein amino acid sequence produced by silkworm), modified spider silk fibroin (modified spider silk protein amino acid sequence produced by spiders) Thing). Among them, modified spider silk fibroin is preferably used.
  • modified fibroin examples include a modified fibroin derived from a large sphincter bookmark silk protein produced in a spider large bottle gland, a modified fibroin with a reduced content of glycine residues, (A) an n motif Modified fibroin with reduced content, content of glycine residue, and (A) modified fibroin with reduced content of n motif.
  • Examples of the modified fibroin derived from the large sphincter bookmark silk protein produced in the spider large bottle gland include a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • (A) the number of amino acid residues of the n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20
  • An integer of 8 to 20 is more preferred, an integer of 10 to 20 is still more preferred, an integer of 4 to 16 is still more preferred, an integer of 8 to 16 is particularly preferred, and an integer of 10 to 16 is most preferred.
  • the number of amino acid residues constituting REP is preferably 10 to 200 residues. More preferably, it is ⁇ 150 residues, more preferably 20-100 residues, and even more preferably 20-75 residues.
  • a modified fibroin derived from the large sphincter bookmark silk protein produced in the spider large bottle gland is a glycine residue contained in the amino acid sequence represented by Formula 1: [(A) n motif-REP] m ,
  • the total number of residues 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 sphincter bookmark silk protein produced in the spider large bottle gland comprises a unit of an amino acid sequence represented by the formula 1: [(A) n motif-REP] m and has a C-terminal. It may be a polypeptide whose sequence is 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 amino acids at 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
  • the amino acid sequence shown in SEQ ID NO: 14 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus, and the amino acid sequence shown in SEQ ID NO: 16 is 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID NO: 14. It is identical to the amino acid sequence.
  • modified fibroin derived from a large sphincter bookmark silk protein produced in the spider large bottle-like gland
  • amino acid sequence represented by SEQ ID NO: 17, or (1-ii) sequence Mention may be made of modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence indicated by number 17. The sequence identity is preferably 95% or more.
  • the amino acid sequence represented by SEQ ID NO: 17 is an amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 18) consisting of a start codon, His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminus.
  • the 13th repeat region was increased to approximately double, and the translation was mutated to terminate at the 1154th amino acid residue.
  • the C-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO: 17 is identical to the amino acid sequence shown in SEQ ID NO: 16.
  • the modified fibroin (1-i) may be composed of the amino acid sequence represented by SEQ ID NO: 17.
  • the modified fibroin with a reduced content of glycine residues has an amino acid sequence with a reduced content of glycine residues in the domain sequence 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.
  • Modified fibroin with a reduced content of glycine residues has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, X Is an amino acid residue other than glycine.)
  • G is a glycine residue
  • P is a proline residue
  • X is an amino acid residue other than glycine.
  • this corresponds to substitution of one glycine residue in at least one or more of the motif sequences with another amino acid residue. It may have an amino acid sequence.
  • the ratio of the motif sequence in which the above glycine residue is replaced with another amino acid residue may be 10% or more with respect to the total motif sequence.
  • the modified fibroin with a reduced content of glycine residues includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and is located on the most C-terminal side from the domain sequence (A )
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. More preferably, it is 100% (meaning that it is composed only of alanine residues).
  • the modified fibroin in which the content of glycine residues is reduced is that the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. preferable.
  • 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. More preferably, it is 6% or less, still more preferably 4% or less, still more preferably 2% or less.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.
  • a fibroin modified fibroin or naturally-occurring fibroin containing a domain sequence represented by Formula 1: [(A) n motif-REP] m , (A) n located closest to the C-terminal side from the domain sequence
  • An amino acid sequence consisting of XGX is extracted from all REPs included in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence.
  • z / w (%) can be calculated by dividing z by w.
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, and 58.7% or more. Is more preferably 70% or more, still more preferably 80% or more. Although there is no restriction
  • a modified fibroin with a reduced content of glycine residues encodes another amino acid residue by substituting at least a part of the base sequence encoding the glycine residue from the cloned gene sequence of naturally occurring fibroin. It can obtain by modifying so that. At this time, one glycine residue in GGX motif and GPGXX motif may be selected as a glycine residue to be modified, or substitution may be performed so that z / w is 50.9% or more.
  • an amino acid sequence satisfying the above-described aspect can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues are further substituted or deleted.
  • the amino acid sequence corresponding to the insertion and / or addition may be modified.
  • the other amino acid residue is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( M) hydrophobic amino acid residues such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and glutamic acid (E) residues are preferred, and valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) residue is more preferable, and glutamine (Q) residue is more preferable.
  • modified fibroin with a reduced content of glycine residues (2-i) the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or (2- ii)
  • SEQ ID NO: 3 amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or
  • 2- ii A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 can be mentioned.
  • the modified fibroin (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 3 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin.
  • the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence represented by SEQ ID NO: 3, in which every two (A) n motifs are deleted from the N-terminal side to the C-terminal side, and further before the C-terminal sequence.
  • One [(A) n motif-REP] is inserted into the.
  • the amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4.
  • the amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 4 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.
  • the value of z / w in the amino acid sequence represented by SEQ ID NO: 1 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%, 70.1%, 66.1% and 70.0%.
  • the value of x / y at the ratio of the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12 (described later) 1: 1.8 to 11.3 is: 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.
  • the modified fibroin (2-i) may be composed 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 (2-ii) includes 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 Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (2-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
  • tag sequences include affinity tags that use specific affinity (binding property, affinity) with other molecules.
  • affinity tag include a histidine tag (His tag).
  • His tag is a short peptide with about 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel. Therefore, the isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography) Can be used.
  • Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 5 (amino acid sequence including a His tag sequence and a hinge sequence).
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • an “epitope tag” using an antigen-antibody reaction can also be used.
  • a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound.
  • HA peptide sequence of hemagglutinin of influenza virus
  • myc tag peptide sequence of hemagglutinin of influenza virus
  • FLAG tag peptide sequence of hemagglutinin of influenza virus
  • a tag sequence that can be separated with a specific protease can also be used.
  • the modified fibroin from which the tag sequence has been separated can also be recovered.
  • modified fibroin containing a tag sequence (2-iii) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (2-iv) SEQ ID NO: 8,
  • a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 can be mentioned.
  • amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, respectively.
  • amino acid sequence represented by SEQ ID NO: 5 (including His tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 12.
  • the modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (2-iv) has an amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 with a sequence identity of 90% or more, and is contained in XREP ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set 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 to naturally occurring fibroin.
  • the modified fibroin in which the content of n motif is reduced may have an amino acid sequence corresponding to 10% to 40% deletion of (A) n motif from naturally occurring fibroin.
  • the modified fibroin with a reduced content of n motif has 1 to 3 (A) n motifs in which the domain sequence is at least from the N-terminal side to the C-terminal side compared to naturally occurring fibroin. Each may have an amino acid sequence corresponding to the deletion of one (A) n motif.
  • the domain sequence of the modified fibroin is at least two consecutive from the N-terminal side to the C-terminal side compared to the naturally derived fibroin (A) n motif And an amino acid sequence corresponding to the deletion of one (A) n motif 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 modified fibroin with a reduced content of n- motif contains a domain sequence represented by Formula 1: [(A) n- motif-REP] m , and is adjacent to the C-terminal side from the N-terminal side.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. More preferably, it is 100% (meaning that it is composed only of alanine residues).
  • FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin.
  • the domain sequence is from the N-terminal side (left side): (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called n motif.
  • FIG. 1 includes 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 4th REP and 5th REP), pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison), pattern 4 (first REP and Comparison of the second REP).
  • pattern 1 compare 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 4th REP and 5th REP
  • pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison
  • pattern 4 first REP and Comparison of the second REP
  • the number of amino acid residues of each REP in the two adjacent [(A) n motif-REP] units selected is compared.
  • each pattern the number of all amino acid residues of two adjacent [(A) n motif-REP] units indicated by solid lines is added (not only REP but also (A) the number of amino acid residues of the n motif. is there.). Then, the total value added is compared, and the total value (maximum value of the total value) of the pattern having the maximum total value is set as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
  • x / y (%) can be calculated by dividing x by the total number of amino acid residues y of the domain sequence.
  • x / y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, It is still more preferably 70% or more, still more preferably 75% or more, and particularly preferably 80% or more.
  • x / y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, It is still more preferably 70% or more, still more preferably 75% or more, and particularly preferably 80% or more.
  • x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x / y / 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.
  • x / y is 46.4% or more, preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, and 70% or more. Even more preferable, 80% or more is particularly preferable.
  • x / y is 46.4% or more, preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, and 70% or more. Even more preferable, 80% or more is particularly preferable.
  • x / y is 46.4% or more, preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, and 70% or more. Even more preferable, 80% or more is particularly preferable.
  • (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.
  • an amino acid sequence corresponding to the deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and / or added.
  • the amino acid sequence corresponding to this may be modified.
  • the modified fibroin (3-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 2 has the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin deleted from the N-terminal side to the C-terminal side every two (A) n motifs Furthermore, one [(A) n motif-REP] is inserted in front of the C-terminal sequence.
  • the amino acid sequence shown in SEQ ID NO: 4 is obtained by substituting all GGX in REP of the amino acid sequence shown in SEQ ID NO: 2 with GQX.
  • the amino acid sequence shown in SEQ ID NO: 10 has two alanine residues inserted in the C-terminal side of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the N-terminal side is deleted so as to be almost the same as the molecular weight of SEQ ID NO: 4.
  • the amino acid sequence represented by SEQ ID NO: 12 is a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 9 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence in which a His tag is added to the C-terminal of the sequence repeated four times.
  • the value of x / y of the amino acid sequence represented by SEQ ID NO: 1 (corresponding to naturally-occurring fibroin) at a jagged ratio of 1: 1.8 to 11.3 is 15.0%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 4 is 93.4%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 10 is 92.7%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 12 is 89.3%.
  • the z / w values in the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12 are 46.8%, 56.2%, 70.1% and 66. respectively. 1% and 70.0%.
  • the modified fibroin (3-i) may be composed 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 (3-ii) includes 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 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 from the N-terminal side to the C-terminal side
  • 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
  • x / y is 64.2% or more, where x is the maximum total value of the total number of bases and y is the total number of amino acid residues in the domain sequence.
  • the above-described modified fibroin may contain the above-described tag sequence at one or both of the N-terminal and C-terminal.
  • modified fibroin containing a tag sequence As more specific examples of modified fibroin containing a tag sequence, (3-iii) SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, or (3-iv) SEQ ID NO: 7, A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 can be mentioned.
  • amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, respectively.
  • amino acid sequence represented by SEQ ID NO: 5 (including His tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 12.
  • the modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
  • the modified fibroin (3-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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 a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, and from the N-terminal side to the C-terminal side.
  • the other X is the maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3.
  • x / y is preferably 64.2% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the domain sequence of the modified fibroin is different from that of naturally occurring fibroin in addition to at least one or more glycine residues in REP. It can be said to have an amino acid sequence corresponding to substitution with an amino acid residue.
  • it is a modified fibroin having the characteristics of the modified fibroin in which the content of the glycine residue is reduced and (A) the modified fibroin in which the content of the n motif is reduced.
  • Specific embodiments and the like are as described in the modified fibroin in which the content of glycine residues is reduced and (A) the modified fibroin in which the content of n motif is reduced.
  • modified fibroin with reduced glycine residue content and (A) n- motif content (4-i) the amino acid represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12
  • modified fibroin comprising a sequence or (4-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12.
  • Specific embodiments of the modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12 are as described above.
  • the modified fibroin according to another embodiment has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin, and It may have an amino acid sequence including a region having a large hydrophobic index locally, corresponding to the insertion of one or more amino acid residues having a large hydrophobic index in REP.
  • the region where the hydrophobic index is locally large is preferably composed of 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). More preferably, it is a residue.
  • the modified fibroin according to the present embodiment has one or more amino acid residues in REP substituted with amino acid residues having a large hydrophobicity index and / or 1 in REP compared to naturally occurring fibroin.
  • one or more amino acid residues are substituted, deleted, inserted and / or compared with naturally occurring fibroin.
  • the modified fibroin according to the present embodiment for example, hydrophobicizes one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in REP from the gene sequence of naturally-derived fibroin that has been cloned. It can be obtained by substituting amino acid residues (for example, amino acid residues having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in REP.
  • hydrophilic amino acid residues for example, amino acid residues having a negative hydrophobicity index
  • one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to insertion of, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in 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 acids in REP
  • 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 another embodiment includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and (A) located at the most C-terminal side of the domain sequence from the n motif.
  • P, and (A) where the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence is q / Q may have an amino acid sequence of 6.2% or more.
  • hydrophobicity index of amino acid residues As for the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of bio.p. 7”. 105-132). Specifically, the hydrophobicity index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
  • a sequence obtained by removing the sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m to the most C-terminal side from the domain (A) n motif to the C terminus of the domain sequence. (Hereinafter referred to as “array A”).
  • array A the average value of the hydrophobicity index of four consecutive 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 the four consecutive amino acid residues by 4 (number of amino acid residues).
  • the average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of “four consecutive amino acid residues whose average value of hydrophobicity index is 2.6 or more”, 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.
  • the total number of amino acid residues contained in sequence A is q.
  • the average value of the hydrophobicity index of four consecutive amino acid residues is 2
  • p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and 20% or more. Even more preferably, it is still more preferably 30% or more.
  • the upper limit of p / q is not particularly limited, but may be 45% or less, for example.
  • the modified fibroin according to this embodiment includes, for example, one or a plurality of hydrophilic amino acid residues (for example, hydrophobicity) in the REP so that the amino acid sequence of the naturally-derived fibroin thus cloned satisfies the above p / q condition.
  • hydrophilic amino acid residues for example, hydrophobicity
  • Substituting a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or one or more hydrophobic amino acid residues during REP Can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index.
  • an amino acid sequence satisfying the above p / q conditions can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index and / or one or more amino acid residues in REP.
  • modifications corresponding to 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 ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
  • modified fibroin (5-i) the amino acid sequence represented by 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 And a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by
  • the modified fibroin (5-i) will be described.
  • the amino acid sequence shown in SEQ ID NO: 19 is an amino acid sequence in which the alanine residues in the (A) n motif of (A) naturally derived fibroin are deleted so that the number of consecutive alanine residues is five.
  • the amino acid sequence represented by SEQ ID NO: 20 is inserted into the amino acid sequence represented by SEQ ID NO: 19 by two amino acid sequences (VLI) each consisting of 3 amino acid residues every other REP, and represented by SEQ ID NO: 19. A part of amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence is almost the same.
  • the amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two alanine residues at the C-terminal side of each (A) n motif with respect to the amino acid sequence represented by SEQ ID NO: 19, and further adding some glutamine (Q) residues. A group is substituted with a serine (S) residue, and a part of amino acids on the C-terminal side is deleted so as to be approximately the same as the molecular weight of the amino acid sequence represented by SEQ ID NO: 19.
  • the amino acid sequence represented by SEQ ID NO: 22 is obtained by inserting one amino acid sequence (VLI) consisting of 3 amino acid residues at every other REP to the amino acid sequence represented by SEQ ID NO: 21.
  • the amino acid sequence shown in SEQ ID NO: 23 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues into the amino acid sequence shown in SEQ ID NO: 21 every other REP.
  • the modified fibroin (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23.
  • the modified fibroin (5-ii) includes an amino acid sequence having 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.
  • the modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (5-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23, and is located at the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the above-mentioned modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal.
  • modified fibroin comprising a tag sequence
  • 5-iii the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, or (5-iv) SEQ ID NO: 24, SEQ ID NO: 25 or Mention may be made of modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 26.
  • amino acid sequences represented by SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 are the amino acid sequences represented by SEQ ID NO: 5 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 23, respectively (His tag). Including a sequence and a hinge sequence).
  • the modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
  • the modified fibroin (5-iv) includes an amino acid sequence having 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.
  • the modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (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 at the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the modified fibroin according to still another embodiment has an amino acid sequence in which the content of glutamine residues is reduced as compared with naturally occurring fibroin.
  • the modified fibroin according to this embodiment preferably includes at least one motif selected from a GGX motif and a GPGXX motif in the amino acid sequence of REP.
  • the content ratio of the GPGXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more.
  • the upper limit of GPGXX motif content rate 50% or less may be sufficient and 30% or less may be sufficient.
  • GPGXX motif content is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m- (A) fibroin (modified fibroin or naturally derived) containing a domain sequence represented by the n motif In fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the domain sequence (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
  • the number obtained by multiplying the total number by three is s, and is located at the most C-terminal side.
  • the sequence from the n motif to the C-terminal of the domain sequence is determined from the domain sequence.
  • the content ratio of the GPGXX motif is calculated as s / t, where t is the total number of amino acid residues of all REPs excluding the n motif. It is.
  • “A sequence located at the most C-terminal side (A) excluding the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence” (A)
  • the sequence from the n motif to the C terminus of the domain sequence ”(sequence corresponding to REP) may include a sequence that is not highly correlated with the sequence characteristic of fibroin, and m is small In this case (that is, when the domain sequence is short), the calculation result of the content ratio of the GPGXX motif is affected, so this influence is excluded.
  • the “GPGXX motif” is located at the C-terminus of REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.
  • FIG. 3 is a schematic diagram showing the domain sequence of the modified fibroin.
  • the modified fibroin according to this embodiment preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and preferably 0%. Particularly preferred.
  • the “glutamine residue content” is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m-
  • the total number of glutamine residues contained in the region is u
  • the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence
  • (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.
  • the reason why "A sequence located at the most C-terminal side (A) excluding the sequence from the n motif to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.
  • the domain sequence has one or more glutamine residues in REP deleted or substituted with other amino acid residues as compared to naturally occurring fibroin. It may have a corresponding amino acid sequence.
  • the “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. Table 1 shows the hydrophobicity index of amino acid residues.
  • amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can.
  • an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. More preferred is an amino acid residue selected from among isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
  • the hydrophobicity of REP is preferably ⁇ 0.8 or more, more preferably ⁇ 0.7 or more, still more preferably 0 or more, and It is still more preferable that it is 3 or more, and it is especially preferable that it is 0.4 or more.
  • the “hydrophobicity of REP” is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m-
  • the sum of the hydrophobicity index of each amino acid residue in the region is represented by v, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence, and ( 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.
  • t is the total number of amino acid residues of all REPs excluding the n motif.
  • the modified fibroin according to the present embodiment has a domain sequence in which one or more glutamine residues in REP are deleted compared to naturally-occurring fibroin and / or one or more glutamine in REP.
  • modifications corresponding to substitution of residues with other amino acid residues there are further alterations in amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues. Also good.
  • the modified fibroin according to the present embodiment includes, for example, deletion of one or more glutamine residues in REP from the cloned gene sequence of natural fibroin and / or one or more glutamine residues in REP. Can be obtained by substituting with other amino acid residues.
  • one or more glutamine residues in REP are deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP are replaced with other amino acid residues.
  • it can also be obtained by designing a corresponding amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • modified fibroin As more specific examples 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 containing the sequence, or (6-ii) the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQ ID NO: 39 Mention may be made of modified fibroin comprising amino acid sequences having sequence identity.
  • the (6-i) modified fibroin will be described.
  • Amino acid sequence shown in SEQ ID NO: 4 is a fibroin naturally occurring Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415) based on the nucleotide sequence and amino acid sequence of, (A) n
  • the amino acid sequence in which the alanine residue in the motif is continued is modified with an amino acid to improve productivity, such as the number of consecutive alanine residues is five.
  • Met-PRT410 since Met-PRT410 has not altered the glutamine residue (Q), the glutamine residue content is comparable to the glutamine residue content of naturally occurring fibroin.
  • the amino acid sequence represented by SEQ ID NO: 27 (M_PRT888) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 4) with VL.
  • the amino acid sequence represented by SEQ ID NO: 28 (M_PRT965) is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with TS and substituting the remaining Q with A.
  • the amino acid sequence (M_PRT889) represented by SEQ ID NO: 29 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 4) with VL and replacing the remaining Q with I.
  • the amino acid sequence (M_PRT916) represented by SEQ ID NO: 30 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 4) with VI and replacing the remaining Q with L.
  • the amino acid sequence represented by SEQ ID NO: 31 is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 4) with VF and replacing the remaining Q with I.
  • the amino acid sequence (M_PRT525) represented by SEQ ID NO: 37 is obtained by inserting two alanine residues into a region (A 5 ) where alanine residues are continuous with respect to Met-PRT410 (SEQ ID NO: 4).
  • the two C-terminal domain sequences were deleted and 13 glutamine residues (Q) were replaced with serine residues (S) or proline residues (P) so that they were almost the same as those in FIG.
  • the amino acid sequence represented by SEQ ID NO: 38 (M_PRT699) is obtained by substituting VL for all QQs in M_PRT525 (SEQ ID NO: 37).
  • the amino acid sequence represented by SEQ ID NO: 39 is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 37) with VL and replacing the remaining Q with I.
  • amino acid sequences 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, and SEQ ID NO: 39 all have a glutamine residue content of 9% or less (Table 2). ).
  • the modified fibroin (6-i) may be composed of the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQ ID NO: 39. .
  • the modified fibroin of (6-ii) has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQ ID NO: 39.
  • the amino acid sequence having The modified fibroin of (6-ii) is also represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less.
  • the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.
  • modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
  • modified fibroin containing a tag sequence (6-iii) amino acids represented 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 or SEQ ID NO: 41 90% or more of the modified fibroin containing the sequence, or (6-iv) 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 Mention may be made of modified fibroin comprising amino acid sequences having sequence identity.
  • 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, SEQ ID NO: 30, respectively.
  • the amino acid sequence represented by SEQ ID NO: 5 (including His tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 31, SEQ ID NO: 38 and SEQ ID NO: 39.
  • the modified fibroin of (6-iii) may be composed of the amino acid sequence represented 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 or SEQ ID NO: 41. .
  • the modified fibroin (6-iv) has a sequence identity of 90% or more with the amino acid sequence represented 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 or SEQ ID NO: 41.
  • the 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.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less.
  • the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.
  • the aforementioned modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the modified fibroin according to any of the above embodiments 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.
  • the nucleic acid is produced by a method such as amplification by polymerase chain reaction (PCR), cloning, modification by genetic engineering techniques, or chemical synthesis. can do.
  • the method for chemically synthesizing nucleic acids is not particularly limited.
  • AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) is used based on the amino acid sequence information of proteins obtained from the NCBI web database.
  • a gene can be chemically synthesized by a method of linking oligonucleotides that are synthesized automatically by PCR or the like.
  • 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 terminus of the above amino acid sequence is synthesized. May be.
  • Regulatory sequences are sequences that control the expression of modified fibroin in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.), and can be appropriately selected depending on the type of host.
  • an inducible promoter that functions in the host cell and can induce expression of the modified fibroin may be used.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), 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 according to the type of host, such as a plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like.
  • a vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed is preferably used.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • microorganisms belonging to the genus Escherichia include Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like.
  • microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis.
  • microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • a prokaryotic host is used as a vector for introducing a nucleic acid encoding a modified fibroin
  • a prokaryotic host for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescript II, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238696) and the like can be mentioned.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.).
  • yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like.
  • Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma and the like.
  • examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEp13 (ATCC37115) and YEp24 (ATCC37051).
  • a method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • electroporation method electroporation method
  • spheroplast method protoplast method
  • lithium acetate method competent method, and the like.
  • a method for expressing a nucleic acid by a host transformed with an expression vector in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd edition, etc. .
  • 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 for culturing a host in a culture medium can be performed according to a method usually used for culturing a host.
  • the culture medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.
  • Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism.
  • Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid, etc.
  • Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source examples include ammonium salts of inorganic acids 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, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and agitation culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture 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.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter
  • indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter.
  • An acid or the like may be added to the medium.
  • Isolation and purification of the expressed modified fibroin can be performed by a commonly used method.
  • the host cell is recovered by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a Manton Gaurin.
  • the host cells are disrupted with a homogenizer, dynomill, or the like to obtain a cell-free extract.
  • a method usually used for protein isolation and purification that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent, etc.
  • Precipitation method anion exchange chromatography method using resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using resin such as S-Sepharose FF (manufactured by Pharmacia)
  • Electrophoresis methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing Using methods such as these alone or in combination, purification It is possible to obtain the goods.
  • the modified fibroin when expressed by forming an insoluble substance in the cell, the host cell is similarly collected, crushed, and centrifuged to collect the modified fibroin insoluble substance as a precipitate fraction.
  • the recovered insoluble form of modified fibroin can be solubilized with a protein denaturant.
  • a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.
  • the protein when secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture with a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the protein filament can be produced by a known spinning method. That is, for example, when a protein filament containing modified fibroin as a main component is produced, first, the modified fibroin produced according to the above-described method is converted into dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid. Or a solvent such as hexafluoroisopropanol (HFIP), together with an inorganic salt as a dissolution accelerator, if necessary, and dissolved to prepare a dope solution. Next, using this dope 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.
  • DMSO dimethyl sulfoxide
  • DMF N, N-dimethylformamide
  • HFIP hexafluoroisopropanol
  • a protein filament can be obtained by spinning by a known spinning method such as wet spinning
  • FIG. 4 is an explanatory view schematically showing an example of a spinning device for producing protein filaments.
  • a spinning device 10 shown in FIG. 4 is an example of a spinning device for dry and wet spinning, and includes an extrusion device 1, an undrawn yarn production device 2, a wet heat drawing device 3, and a drying device 4.
  • the dope solution 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8.
  • the dope solution may be filled into a cylinder and extruded from a nozzle using a syringe pump.
  • the extruded dope liquid 6 is supplied into the coagulating liquid 11 in the coagulating liquid tank 20 through the air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulated body is formed.
  • the fibrous solidified body is supplied into the hot water 12 in the drawing bath 21 and drawn.
  • the draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14.
  • the stretched fibrous solidified body is supplied to the drying device 4 and dried in the yarn path 22 to obtain the protein filament 36 as the wound body 5.
  • Reference numerals 18a to 18g denote 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 coagulation liquid 11 may appropriately contain water.
  • the temperature of the coagulation liquid 11 is preferably 0 to 30 ° C.
  • the extrusion speed is preferably 0.2 to 6.0 mL / hour per hole, and 1.4 to 4.0 mL / hour More preferably it is time.
  • the distance through which the coagulated modified fibroin passes through the coagulating liquid 11 may be long enough to efficiently remove the solvent. 500 mm.
  • the take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min.
  • the residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes.
  • stretching pre-stretching
  • the coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as necessary.
  • the stretching performed when obtaining the protein filament for example, the pre-stretching performed in the coagulating liquid tank 20 and the wet heat stretching performed in the stretching bath 21 are used, and dry heat stretching is also employed.
  • Wet heat stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating.
  • the temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C.
  • undrawn yarn or predrawn yarn
  • Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, or the like.
  • the temperature may be, for example, 140 ° C. to 270 ° C., and preferably 160 ° C. to 230 ° C.
  • an undrawn yarn or predrawn yarn
  • an undrawn yarn can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
  • Wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet 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 final draw ratio of the lower limit of the undrawn yarn (or predrawn yarn) is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times. Above, 7 times or more, 8 times or more, 9 times or more, and upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less 12 times or less, 11 times or less, and 10 times or less.
  • 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, 15000 m or more, or 20000 m or more.
  • the protein staple can be produced by cutting the protein filament produced by the above method.
  • the protein filament can be cut using any apparatus such as a table-type fiber cutter (s / NO.IT-160201-NP-300).
  • 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.
  • the protein fiber obtained by the above method is brought into contact with the oil agent dispersion to attach the oil agent to the protein fiber and crimp the protein fiber.
  • the protein fiber can be brought into contact with the oil dispersion by, for example, immersing the protein fiber in the oil dispersion.
  • the protein fiber in the present invention is shrunk and crimped by contact with water. Therefore, the protein fiber that has undergone the oil dispersion treatment process has crimps.
  • the amount of crimp is, for example, 10 to 100 pieces / 40 mm or 20 to 40 pieces / 40 mm.
  • the oil dispersion includes an aqueous solvent and an oil dispersed in the aqueous solvent.
  • An aqueous solvent is a liquid containing water.
  • the aqueous solvent may be water or a mixed solution of water and a hydrophilic solvent.
  • a 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.
  • 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 characteristics for smoothly performing each step in the production of the oil agent-attached protein crimped fiber and the product using the same, such as antistatic, friction reducing, flexibility imparting, water repellency imparting, etc. (That is, for passing through the process), or those that impart desired properties such as flexibility and antistatic properties to the oil-attached protein cocoon filaments (that is, for imparting functional properties or for finishing). May be.
  • the amount of the oil agent is not particularly limited, but is preferably 1 to 10% by mass and more preferably 2 to 5% by mass with respect to the total amount of the aqueous solvent and the oil agent.
  • the temperature of the oil dispersion may be, for example, 10 ° C. or more and less than 100 ° C., 10 to 60 ° C., 20 to 50 ° C., 30 to 40 ° C. Since the shrinkage ratio of the protein fiber changes depending on the temperature of the oil dispersion, the amount of crimp (degree of crimp) can be controlled by adjusting the temperature of the oil dispersion.
  • the time during which the oil dispersion is brought into contact with the protein fiber is not particularly limited, and may be 30 seconds or longer, 1 minute or longer, or 2 minutes or longer, and is preferably 10 minutes or shorter from the viewpoint of productivity. Contact of the oil dispersion with protein fibers may be performed under normal pressure or under reduced pressure (for example, vacuum).
  • the contraction rate of the protein fiber after being brought into contact with the oil dispersion may be 2% or more, for example, 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5 % Or more, 5% or more, 5.5% or more, or 6% or more.
  • the upper limit of the shrinkage ratio of the protein fiber after being brought into contact with the oil dispersion is not particularly limited, but 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, It may be 5% or less, 4% or less, or 3% or less.
  • the crimp of the protein fiber due to contact with the oil dispersion 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 in a state where it is added only (strained by a predetermined amount in the fiber axis direction). At that time, the degree of crimping can be controlled by adjusting the tensile force applied to the protein fiber. Examples of the method for adjusting the tensile force applied to the protein fiber include a method of adjusting a load applied to the filament or staple by suspending weights of various weights on the protein fiber, or relaxing the protein fiber.
  • the protein fiber After contacting the oil dispersion with the protein fiber, the protein fiber may be dried.
  • a drying method is not specifically limited, Natural drying may be sufficient and a protein fiber may be forcedly dried using drying equipment. Crimping with an oil dispersion and subsequent drying can be performed continuously. Specifically, for example, the protein fiber is fed out from the bobbin and immersed in the oil dispersion, and then dried by blowing hot air or feeding it onto a hot roller.
  • the drying temperature is not particularly limited, and may be, for example, 20 to 150 ° C., 40 to 120 ° C., 60 to 100 ° C.
  • the shrinkage of the protein fiber after drying may be 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% or more. .
  • the shrinkage after drying is usually 80% or less.
  • the protein fiber contracts and crimps when brought into contact with water. For this reason, according to the method according to the present invention, by using an oil dispersion containing an aqueous solvent, crimping of protein fibers and adhesion of oil to protein fibers can be performed simultaneously. Crimped fibers can be produced efficiently.
  • the protein fiber used in the method of the present invention shrinks when contacted with water, but once contacted with water and contracted, the contraction rate when contacted with water is markedly increased. descend.
  • the oil-attached protein crimped fiber produced by the method according to the present invention has already contracted in contact with water as described above, during storage of the oil-attached protein crimped fiber or oil-attached protein crimped fiber Even when the oil-attached protein crimped fiber absorbs moisture again during the manufacturing process of the product using the crimped fiber, the shrinkage due to moisture absorption can be reduced, and therefore the oil-attached protein crimped fiber and the product using the same The change of the dimension of can be reduced.
  • the oil agent-adhered protein crimped fiber produced by the method according to the present invention can be used, for example, for producing a nonwoven fabric and a composite (a composite material with a base material such as a resin). Moreover, when the oil agent-attached protein crimped fiber is a staple, it can also be used for producing a spun yarn.
  • ⁇ Manufacture of modified spider silk fibroin> (1) Preparation of plasmid expression strain Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession numbers: P46804.1, GI: 1174415) derived from Nephila clavipes, the amino acid sequence represented by SEQ ID NO: 13 A modified fibroin (hereinafter also referred to as “PRT799”) was designed.
  • the amino acid sequence represented by SEQ ID NO: 13 has an amino acid sequence in which substitution, insertion, and deletion of amino acid residues are performed for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes.
  • an amino acid sequence (tag sequence and hinge sequence) represented by SEQ ID NO: 5 is added to the N-terminus.
  • nucleic acid encoding PRT799 was synthesized.
  • the nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon.
  • the nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • the seed culture was added to a jar fermenter to which 500 mL of production medium (Table 5) was added so that the OD 600 was 0.05.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and cultured for 20 hours.
  • 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the modified fibroin.
  • the culture solution was centrifuged, and the cells were collected. Perform SDS-PAGE using cells prepared from the culture solution before and after IPTG addition, and confirm the expression of the desired modified fibroin by the appearance of the desired modified fibroin size band depending on the addition of IPTG did.
  • the washed precipitate is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 30 ° C. at 30 ° C. Stir with a stirrer for minutes to dissolve.
  • dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.).
  • the white aggregated protein obtained after dialysis was collected by centrifugation, water was removed with a freeze dryer, and the lyophilized powder was collected to obtain a modified spider silk fibroin “PRT799”.
  • Example> A plurality of protein filaments obtained as described above were bundled and cut into a length of 40 mm with a table-type fiber cutter to produce a protein staple.
  • the protein staple was immersed in an oil dispersion at 20 ° C. for 1 minute to attach the oil to the protein staple and crimp the protein staple.
  • As the oil dispersion a commercially available antistatic oil dispersed in water so as to have a concentration of 1% by mass was used.
  • the shrinkage ratio of the protein staple by being immersed in the oil dispersion was 50%.
  • the protein staple was then dried at 40 ° C. for 18 hours to obtain an oil-attached protein crimped staple.
  • the obtained oil-attached protein crimped staple is shown in FIG.
  • Uncrimped protein staples were obtained by bundling and cutting a plurality of protein filaments obtained in the same manner as in Examples. The resulting uncrimped protein staple is shown in FIG.

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Abstract

La présente invention vise à fournir un procédé de préparation efficace d'une fibre protéinique frisée d'adhérence à l'huile. Le procédé de préparation d'une fibre protéinique frisée d'adhérence à l'huile, selon la présente invention, comprend une étape consistant à mettre en contact une fibre protéinique avec une dispersion d'huile comprenant un solvant aqueux et de l'huile dispersée dans le solvant aqueux, à faire adhérer l'huile à la fibre protéinique et à crêper la fibre protéinique, la fibre protéinique comprenant une fibroïne modifiée.
PCT/JP2019/003469 2018-01-31 2019-01-31 Procédé de préparation d'une fibre protéinique frisée d'adhérence à l'huile Ceased WO2019151432A1 (fr)

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