WO2019066006A1 - Procédé de fabrication de fil torsadé, procédé de fabrication de fil fausse torsion et procédé fausse torsion - Google Patents

Procédé de fabrication de fil torsadé, procédé de fabrication de fil fausse torsion et procédé fausse torsion Download PDF

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WO2019066006A1
WO2019066006A1 PCT/JP2018/036377 JP2018036377W WO2019066006A1 WO 2019066006 A1 WO2019066006 A1 WO 2019066006A1 JP 2018036377 W JP2018036377 W JP 2018036377W WO 2019066006 A1 WO2019066006 A1 WO 2019066006A1
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amino acid
seq
sequence
yarn
fibroin
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PCT/JP2018/036377
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Japanese (ja)
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政隆 梶
隆平 遠藤
聡 宮口
瑞季 五十嵐
尚仁 西門
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Ｓｐｉｂｅｒ株式会社
小島プレス工業株式会社
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Priority to JP2019545156A priority Critical patent/JPWO2019066006A1/ja
Publication of WO2019066006A1 publication Critical patent/WO2019066006A1/fr

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    • 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
    • D02G1/02—Producing crimped or curled fibres, filaments, yarns, or threads, giving them latent characteristics by twisting, fixing the twist and backtwisting, i.e. by imparting false twist

Definitions

  • the present invention relates to a method of producing a twisted yarn, a method of producing a false twist yarn, and a method of twisting a yarn.
  • Thermoplastic fibers such as nylon and polyester fibers are sometimes subjected to twisting in order to improve their physical properties and texture.
  • heat treatment is performed after twisting the fibers in order to solidify the twisted state.
  • Patent Document 1 discloses that a yarn made of polylactic acid fiber and a yarn made of other thermoplastic fibers are twisted together, and the tensile strength of the yarn made of polylactic acid fiber is 0.80 cN / dtex or more, a tensile elongation There is disclosed a plied yarn characterized in that the degree is 20% or more and the number of twists is 30 to 200 T / m.
  • a strong twist in one direction is applied to a wild cocoon yarn, a composition is deformed by wet stretching, and then a refining dyeing is applied to thermally fix the twist of the strong twist by the wet heat and untwist.
  • a method of producing a silk thread having bulkiness and stretchability characterized by
  • the present invention has been made in view of such circumstances, and it is possible to more simply obtain a yarn containing fibroin, such as twisted yarn and false twisted yarn, to which twist is imparted, and false twisted yarn,
  • the purpose is to provide a manufacturing method of Another object of the present invention is to provide a yarn twisting method capable of providing twist more easily to a yarn containing fibroin.
  • the present invention relates to, for example, the following inventions.
  • twist can be more easily applied to fibroin fibers.
  • FIG. 1 is a schematic view showing the domain sequence of modified fibroin.
  • FIG. 2 is a diagram showing the distribution of the value of z / w (%) of naturally occurring fibroin.
  • FIG. 3 shows the distribution of x / y (%) values of naturally occurring fibroin.
  • FIG. 4 is a schematic view showing an example of the domain sequence of the modified fibroin.
  • FIG. 5 is a schematic view showing an example of the domain sequence of the modified fibroin.
  • FIG. 6 is a schematic view showing an example of a spinning apparatus for producing modified fibroin fibers.
  • FIG. 7 is a schematic view showing an example of a false twist device for producing a false twist yarn.
  • the method for producing a twisted yarn according to the first embodiment includes a twisting step of twisting a yarn containing a modified fibroin fiber, and a heating step of heating the yarn while maintaining the twist of the yarn.
  • the twisting step and the heating step may be independent of each other or may be performed simultaneously.
  • twisting process adds twist to the modified fibroin fiber.
  • known means can be used, and for example, a twisting machine can be used.
  • twisting machine twisting machines corresponding to various known types of twisting yarn (up type, down type, special cross bar type, air cross bar type, rewider, etc.), for example, Italian type twisting machine, double twister, ring twisting machine, composite A twisting machine, an interlace, a span winder etc. are mentioned.
  • the twisting direction may be S twist (right twist) or Z twist (left twist).
  • the twist number is preferably 200 T / m or more, more preferably 600 T / m or more, and may be 800 T / m or more.
  • the number of twists may be, for example, 1200 T / m or less, or 1000 T / m or less.
  • the twist number means how many times the yarn has been rotated (twisted) per 1 m of yarn.
  • the twist number can usually be adjusted by changing the set value of the twisting machine.
  • the modified fibroin according to this 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 that contains.
  • 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.
  • An N-terminal sequence and a C-terminal sequence are typically, but not limited to, regions having no repeat of the amino acid motif characteristic of fibroin, and consist of about 100 amino acids.
  • modified fibroin means artificially produced fibroin (artificial fibroin).
  • the modified fibroin may be fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or fibroin whose amino acid sequence is identical to that of naturally occurring fibroin.
  • “naturally-derived fibroin” 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
  • the “modified fibroin” may be one obtained by directly using the amino acid sequence of naturally occurring fibroin, or one obtained by modifying the amino acid sequence based on the amino acid sequence of naturally occurring fibroin (eg, cloned natural origin)
  • the amino acid sequence may be modified by modifying the gene sequence of fibroin), or artificially designed and synthesized without relying on naturally occurring fibroin (eg, a nucleic acid encoding the designed amino acid sequence) It may be one having a desired amino acid sequence by chemical synthesis).
  • domain sequence refers to a crystal region specific to fibroin (typically corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically the REP of the amino acid sequence).
  • Amino acid sequence which corresponds to the following formula 1: [(A) n motif -REP] m, or the amino acid represented by formula 2: [(A) n motif-REP] m- (A) n motif Means sequence.
  • the (A) n motif indicates an amino acid sequence mainly comprising an alanine residue, 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, 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 it consists only of alanine residues).
  • At least seven of the (A) n motifs present in the domain sequence may consist of only alanine residues.
  • REP represents an amino acid sequence composed of 2 to 200 amino acid residues.
  • the REP may be an amino acid sequence composed of 10 to 200 amino acid residues.
  • m is an integer of 2 to 300, and may be an integer of 10 to 300.
  • the plurality of (A) n motifs may be identical to each other or different from each other.
  • the plurality of REPs may be identical amino acid sequences to each other or different amino acid sequences.
  • the modified fibroin according to the present embodiment is, for example, an amino acid corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to the cloned gene sequence of naturally derived fibroin. It can be obtained by modifying the sequence. Substitutions, deletions, insertions and / or additions of amino acid residues can be carried out by methods 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 occurring fibroin is a protein comprising a domain sequence represented by Formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif -REP] m- (A) n Motif Specifically, for example, fibroin produced by insects or spiders can be mentioned.
  • fibroin produced by insects include Bombyx mori (Bombyx mori), Quwaco (Bombyx mandarina), pemphigus (Antheraea yamamai), moth (Anteraea pernyi), moth (Eriogyna pyretorum), moth (Pilosamia Cynthia ricini) ), Silk proteins produced by silkworms such as silkworms (Samia cynthia), chestnut beetles (Caligura japonica), tussah silkworms (Antheraea mylitta), and muga silkworms (Antheraea assama), and larvae of the hornets (Vespa simillima xanthoptera) Hornet silk protein is mentioned.
  • insect-produced fibroin include, for example, silkworm fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
  • the fibroins produced by the spiders include, for example, spiders belonging to the genus Araneus such as spider spiders, spider spiders, spider spiders, blue spider spiders, and spider spiders, spider spiders (genus Neoscona) such as spider spiders, spider spiders, spider spiders and spiders , Spiders belonging to the genus Pronus (Pronus), such as Torino Fundamas, spiders belonging to the genus Torino Fundama (Cyrtarachne) such as Torino Fundamas, and Otorino Fundames, such as spiders such as Togegumo and Tibusegumo Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius, such as the spiders belonging to the genus Gasteracantha and those belonging to the genus Ordgarius A spider belonging to the genus Angiope (Argiope), a spider belonging to the genus Angiope, a spider
  • spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC 47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 derived from Nephila clavipes (genbank accession number AAC 04504 (amino acid sequence) ), U37520 (base sequence)), major ampullate spidro n 1 [Latrodectus hesperus derived] (GenBank accession No.
  • ABR68856 amino acid sequence
  • EF 595246 base sequence
  • dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession No. AAL 32 472 (amino acid sequence), AF 441 245 (base sequence ), Major ampullate spidroin 1 [from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ 973 155 (base sequence)), and major ampullate spidroin 2 [Euprosthenops australi (GenBank Accession No. CAM 32249.
  • Naturally derived fibroin further include fibroin whose sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidroin, ampullate, fibroin, “silk and polypeptide”, or “silk and protein” are described as keywords among sequences including INV as DIVISION among sequence information registered in NCBI GenBank.
  • the sequence can be confirmed by extracting a specified product string from CDS, and a described sequence of a specific string from SOURCE to TISSUE TYPE.
  • the modified fibroin according to this embodiment may be a modified silk (silk) fibroin (a modified amino acid sequence of a silk protein produced by silkworm), or a modified spider silk fibroin (a spider silk protein produced by spiders)
  • the amino acid sequence may be modified).
  • modified spider silk fibroin is preferred.
  • a modified fibroin (first modified fibroin) derived from the large nasogastric silkworm silk protein produced in the large vein of the spider, a domain sequence with a reduced content of glycine residues (A) a modified fibroin (a third modified fibroin) having a domain sequence with a reduced content of n motif, a content of a glycine residue, and (A) n A modified fibroin having a reduced content of motif, a modified fibroin having a domain sequence including a region locally having a large hydrophobic index (a fifth modified fibroin), and a domain sequence having a reduced content of glutamine residue And modified fibroin (sixth modified fibroin).
  • the first modified fibroin includes a protein comprising a domain sequence represented by Formula 1: [(A) n Motif-REP] m .
  • the amino acid residue number of the (A) n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, and an integer of 10 to 20 Is even more preferred, the integer of 4 to 16 is even more preferred, the integer of 8 to 16 is particularly preferred, and the integer of 10 to 16 is most preferred.
  • the number of amino acid residues constituting the REP is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues More preferably, it is 20 to 75 residues.
  • the total number of residues of glycine, serine and alanine residues contained in the amino acid sequence represented by the formula 1: [(A) n motif-REP] m is an amino acid residue
  • the total number is preferably 40% or more, more preferably 60% or more, and still more preferably 70% or more.
  • the first modified fibroin comprises a unit of the amino acid sequence represented by the formula 1: [(A) n motif-REP] m , and the amino acid sequence whose C-terminal sequence is shown in any one of SEQ ID NOs: 1 to 3 or It may be a polypeptide which is an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.
  • the amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of 50 C-terminal amino acids of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is a sequence It is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in No. 1, and the amino acid sequence shown in SEQ ID NO: 3 has 29 residues removed from the C terminus of the amino acid sequence shown in SEQ ID NO. It is identical to the amino acid sequence.
  • the amino acid sequence represented by (1-i) SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) the amino acid sequence represented by SEQ ID NO: Mention may be made of modified fibroins which comprise amino acid sequences with% or more sequence identity.
  • the sequence identity is preferably 95% or more.
  • the amino acid sequence shown by SEQ ID NO: 4 is the first amino acid sequence of the amino acid sequence of ADF3 to which an amino acid sequence (SEQ ID NO: 5) consisting of an initiation codon, His10 tag and HRV3C protease (Human rhinovirus 3C protease) recognition site is added at the N terminus.
  • the 13th repeat region is about doubled and the translation is mutated to terminate at amino acid residue 1154.
  • the amino acid sequence at the C-terminus of the amino acid sequence shown in SEQ ID NO: 4 is identical to the amino acid sequence shown in SEQ ID NO: 3.
  • the modified fibroin of (1-i) may consist of the amino acid sequence shown by SEQ ID NO: 4.
  • the second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin.
  • the second modified fibroin can be said to have an amino acid sequence corresponding to the replacement of at least one glycine residue in REP with another amino acid residue as compared to naturally occurring fibroin .
  • GGX and GPGXX in REP (wherein G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine) in the second modified fibroin in comparison with the naturally derived fibroin in its domain sequence In which at least one glycine residue in at least one or more motif sequences is substituted with another amino acid residue.
  • the percentage of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more with respect to the entire motif sequence.
  • the second modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , and from the above domain sequence to the most C-terminally located (A) n motif from the above domain sequence
  • the alanine residue number 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 It is more preferred that there be 100%, meaning that it consists only of alanine residues.
  • the second modified fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by replacing one glycine residue of the GGX motif with another amino acid 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 still more preferably 10% or less. It is further more preferably 6% or less, still 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 calculation method of the content ratio (z / w) of the amino acid sequence consisting of XGX described below.
  • fibroin modified fibroin or naturally-derived fibroin
  • fibroin containing a domain sequence represented by the formula 1: [(A) n motif-REP] m , (A) n located most C-terminally from the domain sequence
  • An amino acid sequence consisting of XGX is extracted from all the REP contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence.
  • 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-terminus to the C-terminus of the domain sequence from the domain sequence.
  • z / w (%) can be calculated by dividing z by w.
  • z / w in naturally derived fibroin will be described.
  • 663 types of fibroin (of which 415 types of fibroin derived from spiders) were extracted.
  • z / w was calculated by the above-mentioned calculation method. The results are shown in FIG.
  • the horizontal axis of FIG. 2 indicates z / w (%) and the vertical axis indicates frequency.
  • z / w in all naturally occurring fibroin is less than 50.9% (highest, 50.86%).
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, and 70% or more It is further more preferred that the ratio is 80% or more.
  • the upper limit of z / w is not particularly limited, and may be, for example, 95% or less.
  • the second modified fibroin can be obtained, for example, by replacing at least a part of the nucleotide sequence encoding a glycine residue from the cloned gene sequence of naturally occurring fibroin to encode another amino acid residue You can get it.
  • a glycine residue to be modified one glycine residue in the GGX motif and the GPGXX motif may be selected, or z / w may be substituted so as to be 50.9% or more.
  • it can be obtained by designing an amino acid sequence satisfying the above embodiment from the amino acid sequence of naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues are further substituted or deleted.
  • the amino acid sequence may be modified corresponding to the insertion and / or addition.
  • the above 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 residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ), Hydrophilic amino acid residues such as lysine (K) residue and glutamic acid (E) residue are preferable, and valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine ( F) The residue and glutamine (Q) residue are more preferred, and glutamine (Q) residue is even more preferred.
  • SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT 525) or SEQ ID NO: 9 (Met)
  • the modified fibroin of (2-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 6 is one in which all GGX in the REP of the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin is replaced with GQX.
  • the amino acid sequence shown by SEQ ID NO: 7 is such that every other (A) n motif is deleted from the amino acid sequence shown by SEQ ID NO. [(A) n Motif-REP] is inserted into.
  • the amino acid sequence shown by SEQ ID NO: 8 inserts two alanine residues at the C-terminal side of each (A) n motif of the amino acid sequence shown by SEQ ID NO: 7, and further contains some glutamine (Q) residues.
  • SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 7 (however, several amino acid residues at the C-terminal side of the region are substituted).
  • a predetermined hinge sequence and a His tag sequence are added to the C terminus of the sequence repeated four times.
  • the value of z / w in the amino acid sequence shown in SEQ ID NO: 10 is 46.8%.
  • the value of x / y in the Giza ratio (described later) 1: 1.8 to 11.3 of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is 15.0%, 15.0%, 93.4%, 92.7%, 89.3% and 89.8%, respectively.
  • the modified fibroin of (2-i) may consist of the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (2-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (2-ii) is also a protein comprising a domain sequence represented by Formula 1: [(A) n Motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and However, when X represents the total number of amino acid residues of the amino acid sequence consisting of amino acid residues other than glycine) as z, and the total number of amino acid residues of REP in the above domain sequence as w, z / w Is preferably 50.9% or more.
  • the second modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This makes it possible to isolate, immobilize, detect, visualize, etc., the modified fibroin.
  • an affinity tag utilizing specific affinity (binding, affinity) with another molecule can be mentioned.
  • a histidine tag (His tag) can be mentioned as a specific example of an affinity tag.
  • the His tag is a short peptide consisting of 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel, so isolation of the modified fibroin by metalating metal chromatography It can be used to Specific examples of the tag sequence include, for example, the amino acid sequence shown in SEQ ID NO: 11 (His tag sequence and amino acid sequence including hinge sequence).
  • 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.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • epitope tags utilizing antigen-antibody reactions can also be used.
  • a peptide (epitope) showing antigenicity as a tag sequence an antibody against the epitope can be bound.
  • the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like.
  • tag sequence can be separated by a specific protease
  • modified fibroin from which the tag sequence has been separated can also be recovered by subjecting the protein adsorbed via the tag sequence to a protease treatment.
  • modified fibroin containing a tag sequence More specific examples of the modified fibroin containing a tag sequence are shown by (2-iii) SEQ ID NO: 12 (PRT 380), SEQ ID NO: 13 (PRT 410), SEQ ID NO: 14 (PRT 525) or SEQ ID NO: 15 (PRT 799) Mentioning a modified fibroin comprising an amino acid sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence of (2-iv) SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15 it can.
  • amino acid sequences represented by SEQ ID NO: 16 (PRT 313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are respectively SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9
  • amino acid sequence shown in SEQ ID NO: 11 (including His tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence shown.
  • the modified fibroin of (2-iii) may consist of the amino acid sequence shown by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and However, when X represents the total number of amino acid residues of the amino acid sequence consisting of amino acid residues other than glycine) as z, and the total number of amino acid residues of REP in the above domain sequence as w, z / w Is preferably 50.9% or more.
  • the second modified fibroin may comprise a secretion signal for releasing the protein produced in the recombinant protein production system outside the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the third modified fibroin has an amino acid sequence in which the content of the (A) n motif is reduced as compared to naturally occurring fibroin.
  • the domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to deletion of at least one or more (A) n motifs as compared to naturally occurring fibroin.
  • the third modified fibroin may have an amino acid sequence corresponding to 10-40% of the (A) n motif deleted from naturally occurring fibroin.
  • the third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal side toward the C-terminal one to three (A) n motif every one (A) n motif It may have an amino acid sequence corresponding to the deletion of
  • the third modified fibroin has a deletion of two consecutive (A) n motifs whose domain sequences are at least N-terminal to C-terminal as compared to naturally occurring fibroin, and one (A The amino acid sequence may correspond to the fact that the deletion of the n motif is repeated in this order.
  • the third modified fibroin may have an amino acid sequence corresponding to the deletion of (A) n motif every other two domain sequences from at least the N terminal side to the C terminal side .
  • the third modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , and two adjacent [(A) n motifs from the N terminal side toward the C terminal side
  • the ratio of the number of amino acid residues of the other REP is 1.8 to Assuming that the maximum value of the sum of the amino acid residue numbers of two adjacent [(A) n motif-REP] units to be 11.3 is x and the total amino acid residue number of the domain sequence 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.
  • the alanine residue number 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 It is more preferred that there be 100%, meaning that it consists only of alanine residues.
  • FIG. 1 shows domain sequences obtained by removing the N- and C-terminal sequences from the modified fibroin. From the N-terminal side (left side), the domain sequence is (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.
  • the number of amino acid residues of each REP in two adjacent selected [(A) n motif-REP] units is compared.
  • the comparison is carried out by determining the ratio of the number of amino acid residues of the other, assuming that the smaller number of amino acid residues is 1.
  • each pattern the numbers of all amino acid residues of two adjacent [(A) n motif-REP] units shown by the solid line are added (not only REP, but also the number of amino acid residues of (A) n motif is there.). Then, the summed total values are compared, and the total value (maximum value of the total values) of the patterns for which the total value is the largest is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the largest.
  • 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, still more preferably 70% or more It is more preferably 75% or more, still more preferably 80% or more.
  • x / y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, still more preferably 70% or more It is more preferably 75% or more, still more preferably 80% or more.
  • the upper limit of x / y may be, for example, 100% or less.
  • x / y is preferably 89.6% or more, and in the case of a Giza ratio of 1: 1.8 to 3.4, x It is preferable that / y is 77.1% or more, and when the Giza ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the Giza 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 Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, and 80% or more. Being particularly preferred.
  • the upper limit of x / y is not particularly limited, and may be 100% or less.
  • the horizontal axis of FIG. 3 indicates x / y (%) and the vertical axis indicates frequency.
  • x / y in naturally derived fibroin is less than 64.2% in all cases (highest, 64.14%).
  • the third modified fibroin deletes one or more of the sequences encoding the (A) n motif so that x / y is 64.2% or more from the cloned naturally-occurring fibroin gene sequence It can be obtained by Also, for example, from the amino acid sequence of naturally occurring fibroin, an amino acid sequence corresponding to deletion of one or more (A) n motifs so that x / y is 64.2% or more is designed and designed It can also be obtained by chemically synthesizing a nucleic acid encoding the above amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and / or added.
  • the amino acid sequence corresponding to the above may be modified.
  • the third modified fibroin (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT 525) or SEQ ID NO: 9 (Met)
  • the modified fibroin of (3-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 17 is different from the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin from every N terminal side toward C terminal side (A) n
  • the motif is deleted, and one [(A) n motif-REP] is inserted in front of the C-terminal sequence.
  • the amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified fibroin.
  • the value of x / y in the Giza ratio 1: 1.8 to 11.3 of the amino acid sequence (corresponding to naturally occurring fibroin) represented by SEQ ID NO: 10 is 15.0%.
  • the amino acid sequence shown by SEQ ID NO: 17 and the value of x / y in the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%.
  • the value of x / y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%.
  • the value of x / y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%.
  • the values of z / w in the amino acid sequences shown by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.
  • the modified fibroin of (3-i) may consist of the amino acid sequence shown by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (3-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (3-ii) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and from N-terminal to C-terminal Sequentially comparing the number of amino acid residues of REP of two [(A) n motif-REP] units adjacent to each other, and assuming that the number of amino acid residues of REP having a small number of amino acid residues is 1, Amino acid residues of two adjacent [(A) n motif-REP] units in which the ratio of the number of amino acid residues of REP is 1.8 to 11.3 (the Giza ratio is 1: 1.8 to 11.3) It is preferable that x / y be 64.2% or more, where x is the maximum value of the sum total of the number of bases and x is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may contain the above-described tag sequence at either or both of the N-terminus and the C-terminus.
  • modified fibroin containing the tag sequence 3-iii) SEQ ID NO: 18 (PRT 399), SEQ ID NO: 13 (PRT 410), SEQ ID NO: 14 (PRT 525) or SEQ ID NO: 15 (PRT 799)
  • a modified fibroin can be mentioned, which comprises an amino acid sequence having 90% or more sequence identity with the sequence or (3-iv) the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 .
  • amino acid sequences shown by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 correspond to SEQ ID NO: 11 at the N-terminus of the amino acid sequences shown by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • the amino acid sequence (including His tag sequence and hinge sequence) is added.
  • the modified fibroin of (3-iii) may consist of the amino acid sequence shown by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the modified fibroin of (3-iv) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above 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: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and N-terminal to C-terminal Sequentially comparing the number of amino acid residues of REP of two [(A) n motif-REP] units adjacent to each other, and assuming that the number of amino acid residues of REP having a small number of amino acid residues is 1, The maximum value of the sum of the amino acid residue numbers of two adjacent [(A) n motif-REP] units in which the ratio of the amino acid residue number of REP is 1.8 to 11.3 is x.
  • x / y is 64.2% or more, where y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the fourth modified fibroin has an amino acid sequence in which the content of the glycine residue is reduced in addition to the content of the (A) n motif being reduced as compared to the naturally derived fibroin of the domain sequence. It is possessed.
  • the domain sequence of the fourth modified fibroin has at least one or more glycine residues in the REP in addition to the deletion of at least one or more (A) n motifs as compared to naturally occurring fibroin It can be said to have an amino acid sequence corresponding to substitution with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the features of both the second modified fibroin described above and the third modified fibroin. Specific embodiments and the like are as described for the second modified fibroin and the third modified fibroin.
  • modified fibroin As more specific examples of the fourth modified fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410) Or the amino acid sequence shown in SEQ ID NO: 14 (PRT 525) or SEQ ID NO: 15 (PRT 799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15
  • modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Specific embodiments of the modified fibroin comprising the amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.
  • the fifth modified fibroin is that its domain sequence has one or more amino acid residues in REP replaced with an amino acid residue having a large hydrophobicity index, as compared to naturally occurring fibroin, and / or REP It may have an amino acid sequence including a region having a locally large hydrophobicity index corresponding to insertion of one or more hydrophobicity index large amino acid residues therein.
  • the region locally having a large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
  • the amino acid residue having a large hydrophobicity index mentioned above is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) It is more preferable that it is a residue.
  • one or more amino acid residues in the REP are replaced with an amino acid residue having a large hydrophobicity index, as compared with naturally occurring fibroin, and / or one or more in the REP.
  • substitution, deletion, insertion and / or addition of one or more amino acid residues as compared with naturally occurring fibroin There may be amino acid sequence modifications corresponding to those described above.
  • the fifth modified fibroin is, for example, a hydrophobic amino acid residue remaining in one or more hydrophilic amino acid residues (for example, an amino acid residue having a negative hydrophobicity index) in REP from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting a group (for example, an amino acid residue whose hydrophobicity index is plus) and / or inserting one or more hydrophobic amino acid residues into the REP. Also, for example, from the amino acid sequence of naturally-derived fibroin, one or more hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid residues, and / or one or more hydrophobic amino acid residues in REP.
  • an amino acid sequence corresponding to the insertion of X can also be obtained by designing an amino acid sequence corresponding to the insertion of X, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in the REP are substituted with hydrophobic amino acid residues from the amino acid sequence of naturally derived fibroin, and / or one or more hydrophobic amino acids in the 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 fifth modified fibroin contains a domain sequence represented by the formula 1: [(A) n motif-REP] m , and from the (A) n motif located most at the C-terminal end to the C-terminus of the domain sequence
  • Let p be the total number of amino acid residues contained in a region in which 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 above sequence from the above domain sequence,
  • p / q is 6
  • hydrophobicity index of amino acid residues
  • known indices Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of a protein”, J. Mol. Biol., 157, pp. Use 105-132).
  • the hydrophobicity index (hydropathy index, hereinafter also referred to as "HI" of each amino acid is as shown in Table 1 below.
  • sequence A [(A) n motif-REP] m to the sequence from the (A) n motif located closest to the C terminal to the C terminus of the domain sequence (Hereinafter, referred to as "sequence A") is used.
  • sequence A the average value of the hydrophobicity index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the sum of HI of each amino acid residue contained in 4 consecutive amino acid residues by 4 (the number of amino acid residues).
  • the average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used to calculate an average of 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more is identified. Even if a certain amino acid residue corresponds to "a series of 4 amino acid residues in which the average value of the hydrophobicity index is 2.6 or more", the region is included as one amino acid residue become. And, the total number of amino acid residues contained in the region is p. In addition, the total number of amino acid residues contained in the sequence A is q.
  • the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2
  • p / q is preferably 6.2% or more, more preferably 7% or more, still more preferably 10% or more, and preferably 20% or more. Still more preferably, it is 30% or more.
  • the upper limit of p / q is not particularly limited, and may be, for example, 45% or less.
  • the fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP such that the amino acid sequence of the cloned naturally-derived fibroin satisfies the above p / q condition.
  • a hydrophobic amino acid residue eg, an amino acid residue with a positive hydrophobicity index
  • insertion of one or more hydrophobic amino acid residues into the REP By doing this, it can be obtained by locally modifying the amino acid sequence including the region having a large hydrophobicity index.
  • one or more amino acid residues in the REP are replaced with an amino acid residue having a large hydrophobicity index, and / or one or more amino acids in the REP as compared to naturally occurring fibroin, and / or
  • modification 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, and 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.
  • the fifth modified fibroin (5-i) an amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), Or (5-ii) a modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 19 consists of three amino acid residues for every REP, except for the domain sequence at the C-terminal end of the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410)
  • the amino acid sequence (VLI) is inserted in two places, and a part of glutamine (Q) residues is replaced with a serine (S) residue and a part of amino acids at the C-terminal side is deleted.
  • the amino acid sequence shown by SEQ ID NO: 20 is one obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues for every REP in addition to the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525). is there.
  • the amino acid sequence shown by SEQ ID NO: 21 is one obtained by inserting two amino acid sequences (VLI) consisting of three amino acid residues for every REP, to the amino acid sequence shown by SEQ ID NO: 8.
  • the modified fibroin of (5-i) may consist of the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) comprises an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 and is most C-terminally located (A) n Amino acids included in a region in which 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 from the motif to the C-terminus of the domain sequence from the domain sequence Assuming that the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C terminal to the C terminus of the domain sequence from the domain sequence is q. And p / q is preferably 6.2% or more.
  • the fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.
  • modified fibroin containing the tag sequence examples include (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv) A modified fibroin comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24).
  • amino acid sequences shown by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences shown by SEQ ID NO: 11 (His tag) at the N terminus of the amino acid sequences shown by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 respectively (Including sequence and hinge sequence).
  • the modified fibroin of (5-iii) may consist of the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) comprises an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) is also a protein comprising a domain sequence represented by the formula 1: [(A) n motif-REP] m .
  • the above sequence identity is preferably 95% or more.
  • the modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 and is most C-terminally located (A) n Amino acids included in a region in which 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 from the motif to the C-terminus of the domain sequence from the domain sequence Assuming that the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C terminal to the C terminus of the domain sequence from the domain sequence is q. And p / q is preferably 6.2% or more.
  • the fifth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues as compared to naturally occurring fibroin.
  • the sixth modified fibroin preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.
  • the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more.
  • the upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.
  • 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 containing a domain sequence represented by n motif (modified fibroin or naturally derived In fibroin)
  • the number of GPGXX motifs contained in the region of all REPs contained in the sequence excluding the sequence from the (A) n motif located most C-terminal to the C-terminus of the domain sequence from the domain sequence Let s be the number obtained by multiplying the total number by 3 (that is, the total number of G and P in the GPGXX motif) be s, and the sequence from the (A) n motif located closest to the C terminal to the C terminal of the domain sequence GPGXX motif content ratio is calculated as s / t, where t is the total number of amino acid residues of all REP excluding (A) n motif
  • GPGXX motif content “the sequence obtained by removing the sequence from the (A) n motif located at the most C terminal side to the C terminus of the domain sequence from the domain sequence” is “most C terminal side (A)
  • a sequence from the n motif to the C terminus of the domain sequence (sequence corresponding to REP) may contain a sequence with low correlation with the sequence characteristic of fibroin, and m is small If this is the case (that is, if the domain sequence is short), this affects the result of calculation of the GPGXX motif content, so this effect is eliminated.
  • GPGXX motif is located at the C-terminal of REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.
  • FIG. 5 is a schematic view showing the domain sequence of modified fibroin.
  • all the REPs are "the sequence from the (A) n motif located at the most C-terminal end to the C-terminal end of the domain sequence removed from the domain sequence" (the sequence shown in "region A” in FIG.
  • the sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%. .
  • glucose residue 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 containing a domain sequence represented by n motif (modified fibroin or naturally derived In fibroin), the sequence from the (A) n motif located closest to the C terminus to the C terminus of the domain sequence is all removed from the domain sequence (sequence corresponding to "region A" in Fig. 5).
  • the total number of glutamine residues contained in the area as u, except from the most located C-terminal side (a) sequence domain sequence from n motif to the C-terminal domain sequence, further (a) n
  • the glutamine residue content is calculated as u / t, where t is the total number of amino acid residues of all REPs excluding the motif.
  • the reason why “a 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” is the reason described above It is similar.
  • the sixth modified fibroin corresponds to deletion of one or more glutamine residues in the REP or substitution of another amino acid residue as compared to naturally occurring fibroin. It may have an amino acid sequence.
  • the “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a glutamine residue.
  • the hydrophobicity index of amino acid residues is as shown in Table 1.
  • amino acid residues having a larger hydrophobicity index than glutamine residues 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.
  • the amino acid residue is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). More preferably, it is an amino acid residue selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
  • the sixth modified fibroin preferably has a hydrophobicity of -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more, and 0.3 or more. It is further more preferable that the ratio be 0.4 or more, and particularly preferable.
  • the upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or 0.7 or less.
  • 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-
  • A) fibroin containing a domain sequence represented by n motif modified fibroin or naturally derived In fibroin
  • the sequence from the (A) n motif located closest to the C terminus to the C terminus of the domain sequence is all removed from the domain sequence (sequence corresponding to "region A" in Fig. 5).
  • the total of the hydrophobicity index of each amino acid residue in the region is v
  • the sequence from the (A) n motif located most C-terminal to the C-terminus of the domain sequence is removed from the domain sequence
  • the hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REP excluding n motif.
  • the reason for targeting “a sequence from the (A) n motif located closest to the C terminal to the C terminus of the domain sequence is excluded from the domain sequence” in the calculation of the hydrophobicity of REP is the reason described above and It is similar.
  • the sixth modified fibroin has its domain sequence deleted one or more glutamine residues in the REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in the REP
  • the modification corresponding to substitution of the amino acid with another amino acid residue there may be a modification of the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues.
  • the sixth modified fibroin may, for example, delete one or more glutamine residues in the REP from the cloned naturally occurring fibroin gene sequence and / or one or more glutamine residues in the REP It can be obtained by substitution of amino acid residues of Also, for example, one or more glutamine residues in REP are deleted from the amino acid sequence of naturally-derived fibroin, and / or one or more glutamine residues in REP are replaced with another amino acid residue. Particularly, it can be obtained by designing a corresponding amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met Modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 29 (Met-PRT 918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT 698) or SEQ ID NO: 32 (Met-PRT966), or (6-ii) 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 31 or SEQ ID NO: 32 Mention may be made of modified fibroin which comprises the amino acid sequence it has.
  • the modified fibroin of (6-i) will be described.
  • the amino acid sequence shown by SEQ ID NO: 25 is one in which all QQs in the amino acid sequence (Met-PRT410) shown by SEQ ID NO: 7 are replaced with VL.
  • the amino acid sequence shown by SEQ ID NO: 26 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with TS, and the remaining Q is replaced with A.
  • the amino acid sequence shown by SEQ ID NO: 27 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with VL, and the remaining Q is replaced with I.
  • the amino acid sequence shown by SEQ ID NO: 28 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with VI, and the remaining Q is replaced with L.
  • the amino acid sequence shown by SEQ ID NO: 29 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with VF, and the remaining Q is replaced with I.
  • the amino acid sequence shown by SEQ ID NO: 30 is one in which all QQs in the amino acid sequence (Met-PRT 525) shown by SEQ ID NO: 8 are replaced with VL.
  • the amino acid sequence shown by SEQ ID NO: 31 is one in which all QQs in the amino acid sequence shown by SEQ ID NO: 8 are replaced with VL, and the remaining Q is replaced with I.
  • amino acid sequence shown by SEQ ID NO: 32 is the same as the one shown in SEQ ID NO: 7 (Met-PRT410), in which the QQ in the double repeated sequence of the region of 20 domain sequences is replaced with VF, And the remaining Q is replaced by I.
  • amino acid sequences shown by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 all have glutamine residue content of 9% or less Yes (Table 2).
  • the modified fibroin of (6-i) consists of the amino acid sequence shown by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32 It may be.
  • the modified fibroin of (6-ii) has 90% or more amino acid sequence shown by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32 Containing an amino acid sequence having the sequence identity of
  • the modified fibroin of (6-ii) 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 above sequence identity is preferably 95% or more.
  • the modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Moreover, it is preferable that the modified fibroin of (6-ii) has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This makes it possible to isolate, immobilize, detect, visualize, etc., the modified fibroin.
  • modified fibroin containing the tag sequence are (6-iii) SEQ ID NO: 33 (PRT 888), SEQ ID NO: 34 (PRT 965), SEQ ID NO: 35 (PRT 889), SEQ ID NO: 36 (PRT 916), SEQ ID NO: 37 (PRT 918), SEQ ID NO: 38 (PRT 699), SEQ ID NO: 39 (PRT 698) or modified fibroin comprising the amino acid sequence shown by SEQ ID NO: 40 (PRT 966), or (6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 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 shown in SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 are SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 respectively.
  • SEQ ID NO: 28 SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32
  • the amino acid sequence shown in SEQ ID NO: 11 was added to the N terminus of the amino acid sequence shown in It is a thing.
  • amino acid sequence shown by SEQ ID NO: 40 has a glutamine residue content of 9% or less (Table 3).
  • the modified fibroin of (6-iii) consists of the amino acid sequence shown by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40 It may be.
  • the modified fibroin of (6-iv) has 90% or more of the amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40 Containing an amino acid sequence having the sequence identity of
  • 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 above sequence identity is preferably 95% or more.
  • the modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. Moreover, it is preferable that the modified fibroin of (6-iv) has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of host.
  • the modified fibroin is at least two or more of the characteristics possessed by the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin It may be a modified fibroin having the characteristics of
  • the modified fibroin is expressed, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the target modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence.
  • a product produced by expression can be used.
  • the nucleic acid is produced by a method of amplifying and cloning by polymerase chain reaction (PCR) or the like using a gene encoding natural fibroin, and modifying by a genetic engineering method or a method of chemical synthesis. can do.
  • the chemical synthesis method of the nucleic acid is not particularly limited, and, for example, AKTA oligopilot plus 10/100 (manufactured by GE Healthcare Japan Co., Ltd.) based on the amino acid sequence information of fibroin obtained from the NCBI web database or the like.
  • a nucleic acid can be chemically synthesized by a method of ligating the oligonucleotide synthesized at step 1 by PCR or the like. At this time, in order to facilitate purification and confirmation of the modified fibroin, a nucleic acid encoding a protein consisting of an amino acid sequence having an amino acid sequence consisting of a start codon and a His10 tag added to the N terminus of the above amino acid sequence is synthesized It is also good.
  • the regulatory sequence is a sequence that controls the expression of a recombinant protein in a host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host.
  • a promoter an inducible promoter which functions in a host cell and is capable of inducible expression of the target altered fibroin may be used.
  • An inducible promoter is a promoter that can control transcription due to the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as temperature, osmotic pressure or an increase or decrease in pH value.
  • the type of expression vector may be a plasmid vector, a viral vector, a cosmid vector, a fosmid vector, an artificial chromosome vector or the like, and can be appropriately selected according to the type of host.
  • a vector capable of autonomous replication in a host cell or capable of integration into the host chromosome and containing a promoter at a position capable of transcribing a nucleic acid encoding a target protein is suitably used. .
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.
  • Preferred examples of the prokaryote include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
  • 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.
  • microorganisms belonging to the genus Serratia include Serratia liquofaciens and the like.
  • microorganism belonging to the genus Bacillus for example, Bacillus subtilis and the like
  • microorganism belonging to the genus Microbacterium include, for example, Microbacterium ammoniafilum and the like.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam and the like.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes and the like.
  • Pseudomonas for example, Pseudomonas putida etc. can be mentioned.
  • examples of a vector into which a target nucleic acid encoding a modified fibroin is introduced include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescript II, pSupex, pET22b and pCold. And pUB110, pNCO2 (Japanese Patent Application Laid-Open No. 2002-238569), and the like.
  • Eukaryotic hosts can include, for example, yeast and filamentous fungi (molds and the like).
  • yeast the yeast which belongs to Saccharomyces genus, Pichia genus, Schizosaccharomyces genus etc. can be mentioned, for example.
  • filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
  • examples of a vector into which a nucleic acid encoding a target modified fibroin is introduced include YEp13 (ATCC 37115), YEp24 (ATCC 37051) and the like.
  • any method of introducing DNA into the host cell can be used.
  • a method using calcium ion [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, secretion production, fusion protein expression and the like can be performed according to the method described in Molecular Cloning 2nd Edition, etc. .
  • the target modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting it from the culture medium. it can.
  • the method of culturing the host in a culture medium can be carried out according to a method usually used for culturing the host.
  • the culture medium for the host contains a carbon source, nitrogen source, inorganic salts and the like that can be used by the host, and the host can be cultured efficiently.
  • a natural medium or a synthetic medium may be used as long as the medium can be used.
  • the carbon source may be any source as long as the transformed host can assimilate, for example, glucose, fructose, sucrose, and molasses containing them, carbohydrates such as starch and starch hydrolysate, acetic acid and propionic acid And the like, and alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include, for example, ammonium, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate
  • other nitrogen-containing compounds such as peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • potassium monophosphate potassium monobasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
  • the culture of a prokaryote such as E. coli or a eukaryote such as yeast can be performed under aerobic conditions such as shake culture or submerged aeration culture, for example.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. Adjustment of the pH of the culture medium can be carried out using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia and the like.
  • Antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during culture.
  • an inducer may be added to the medium as needed.
  • indole acrylic An acid or the like may be added to the medium.
  • Isolation and purification of the desired modified fibroin produced and accumulated by the host can be performed by a commonly used method.
  • host cells are recovered by centrifugation and suspended in an aqueous buffer, and then sonicator, French press, Manton The host cells are disrupted by a Gaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified preparation can be obtained by a method usually used for protein isolation and purification.
  • the host cell when the modified fibroin forms an insoluble form in cells and is expressed, the host cell is similarly recovered and then disrupted and centrifuged to recover the insoluble form of the modified fibroin as a precipitate fraction. .
  • the insoluble form of the modified fibroin recovered can be solubilized with a protein denaturant.
  • a purified preparation of the modified fibroin can be obtained by the same isolation and purification method as described above.
  • the modified fibroin When the modified fibroin is extracellularly secreted, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method 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.
  • Methods commonly used for isolation and purification of proteins include solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents, diethylaminoethyl (DEAE) -sepharose, DIAION HPA-75
  • Anion exchange chromatography method using resin such as those manufactured by Kasei Chemical Co., Ltd., cation exchange chromatography method using resin such as S-Sepharose FF (manufactured by Pharmacia), resin such as butyl sepharose or phenyl sepharose
  • Methods such as hydrophobic chromatography, gel filtration using a molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, and the like can be mentioned. These methods may be used alone or in combination.
  • the fibers used in the twisting process include modified fibroin.
  • the fiber is preferably a fiber composed of modified fibroin (hereinafter also referred to as modified fibroin fiber).
  • modified fibroin fibers are preferably fibers consisting of modified spider silk fibroin (modified spider silk fibroin fibers).
  • the fiber containing the modified fibroin can be produced by spinning a composition containing the modified fibroin by a known spinning method.
  • a method of preparing the modified fibroin fiber will be described by taking a fiber made of the modified fibroin as an example. That is, when producing the modified fibroin fiber, first, the modified fibroin produced according to the above-mentioned method can be dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), or hexafluoroisopronol (HFIP). And a solvent such as formic acid, if necessary, together with an inorganic salt as a dissolution accelerator, and dissolved to prepare a dope solution. Then, using this dope solution (spinning stock solution), it can be spun by a known spinning method such as wet spinning, dry spinning or dry-wet spinning to obtain a target modified fibroin fiber.
  • DMSO dimethyl sulfoxide
  • DMF N, N-dimethylformamide
  • FIG. 6 is a schematic view showing an example of a spinning apparatus for producing modified fibroin fibers.
  • the spinning device 10 shown in FIG. 6 is an example of a spinning device for dry-wet spinning, and comprises an extrusion device 1, a coagulation bath 20, a washing bath 21, and a drying device 4 in this order from the upstream side. .
  • the extrusion device 1 has a storage tank 7, in which a dope solution (spinning stock solution) 6 is stored.
  • Coagulation liquid 11 eg, methanol
  • the dope solution 6 is pushed out from a nozzle 9 provided by opening a air gap 19 between the dope solution 6 and the coagulating solution 11 by a gear pump 8 attached to the lower end of the storage tank 7.
  • the extruded dope 6 is supplied into the coagulating liquid 11 through the air gap 19.
  • the solvent is removed from the dope solution 6 in the coagulation solution 11 to coagulate the protein.
  • the coagulated modified fibroin is guided to the washing bath 21 and washed with the washing liquid 12 in the washing bath 21, and then to the drying device 4 by the first nip roller 13 and the second nip roller 14 installed in the washing bath 21. Sent. At this time, for example, when the rotational speed of the second nip roller 14 is set to be faster than the rotational speed of the first nip roller 13, a modified fibroin fiber 36 drawn at a magnification corresponding to the rotational speed ratio is obtained.
  • the modified fibroin fiber 36 drawn in the washing solution 12 is removed when it passes through the drying device 4 after leaving the inside of the washing bath 21 and then taken up by a winder. In this way, the modified fibroin fiber 36 is obtained by the spinning device 10 as the wound product 5 which is finally wound around the winder.
  • Reference numerals 18a to 18g denote yarn guides.
  • the coagulating solution 11 may be an organic solvent capable of extracting (desolving) the solvent from the dope 6 extruded from the nozzle 9.
  • organic solvents include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone.
  • the coagulation liquid 11 may contain water as appropriate.
  • the temperature of the coagulating solution 11 is preferably 0 to 30 ° C.
  • the distance the coagulated modified fibroin passes through in the coagulating liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) is such that the solvent can be removed efficiently. It is good if there is a length that can secure the stay time.
  • the residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, or may be 0.5 to 1 minute. Alternatively, stretching (pre-stretching) may be performed in the coagulating solution 11.
  • Mainly water can be used as the cleaning liquid 12.
  • the cleaning solution 12 may include those listed as agents for use in the coagulation solution 11.
  • the stretching performed in the washing bath 21 when obtaining the modified fibroin fiber may be so-called wet heat stretching performed in warm water, in a solution in which an organic solvent or the like is added to warm water, or the like.
  • the temperature of the wet heat drawing may be, for example, 0 to 90 ° C., preferably 20 to 70 ° C., and more preferably 30 to 60 ° C.
  • the draw ratio of the undrawn yarn (or pre-drawn yarn) in wet heat drawing may be, for example, 1 to 10 times or 2 to 8 times.
  • the modified fibroin fibers may be further drawn (so-called dry heat drawing) when passing through the drying device 4 in the present embodiment.
  • the lower limit of the draw ratio of the final modified fibroin fiber is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times that of the undrawn yarn (or pre-drawn yarn).
  • the upper limit is preferably at least 100 times, at most 80 times, at most 60 times, at most 40 times, at most 30 times, and is at least 6 times, at least 7 times, at least 8 times, at least 9 times. 20 times or less, 15 times or less, 14 times or less, 13 times or less, 12 times or less, 11 times or less, 10 times or less.
  • the yarn provided with twist in the twisting step is heated while maintaining the twist.
  • the method for maintaining the twist of the yarn is not particularly limited.
  • the whole may be restrained with a net or the like without slack.
  • the heating step may be performed in a state in which twisting is applied by a twisting machine (in-line).
  • the means for heating is not particularly limited, and a heater, an oven, a thermostat, or the like can be used.
  • the heating temperature is preferably more than 50 ° C., more preferably 100 ° C. or more, still more preferably 115 ° C. or more, still more preferably 130 ° C. or more. When the heating temperature is 50 ° C. or more, fixation of the twist applied to the yarn can be made more sufficient.
  • the heating temperature is preferably 240 ° C. or less, more preferably 180 ° C. or less, and still more preferably 150 ° C. or less, from the viewpoint of further suppressing the decomposition and the like of the modified fibroin fiber.
  • the heating step may be a step of heating the yarn in an atmosphere of more than 50 ° C.
  • the heating step may be a step of heating the yarn in an atmosphere of 240 ° C. or less.
  • the heating temperature may be constant or may be adjusted to gradually increase the temperature.
  • the heating temperature is preferably adjusted to be constant.
  • the heating temperature means the ambient temperature to which the yarn is exposed, and usually means the set temperature of the target yarn heating means (for example, a heater).
  • the heating time is appropriately changed depending on the state of the yarn, the heating temperature, etc.
  • a state (inline) in which twist is applied by a twisting machine it may be 0.1 seconds or more, or 1 second or more. It may be 3 seconds or more.
  • the heating time may be 0.5 hours or more, 1 hour or more, 3 hours or more, 6 hours or more It may be 12 hours or more.
  • twist can be more reliably applied to the yarn.
  • the heating time may also be, for example, 24 hours or less, or 18 hours or less from the viewpoint of sufficiently reducing the degradation of the modified fibroin fibers.
  • the twisted yarn obtained by the method of manufacturing a twisted yarn according to the first embodiment is such that the twist imparted in the twisting step is sufficiently maintained and fixed.
  • the twist number of the obtained twisted yarn can be, for example, 200 T / m or more, 600 T / m or more, or 800 T / m or more.
  • the method for producing a false twist yarn according to the second embodiment includes a twisting step of twisting a yarn containing a modified fibroin fiber, a heating step of heating the yarn while maintaining the twist of the yarn, and the heating step. And an untwisting step for untwisting the yarn.
  • the twisting step and the heating step may be steps independent of each other or may be simultaneously performed, preferably simultaneously.
  • the conditions and the like described in the method for manufacturing a twisted yarn can be applied to the portions common to the method for manufacturing a false twisted yarn according to the present embodiment and the method for manufacturing a twisted yarn according to the first embodiment.
  • the description is omitted, and parts different from the first embodiment will be described.
  • the twisting process adds twist to the modified fibroin fiber.
  • known means can be used, and for example, a false twister can be used.
  • a false twisting machine a pin false twisting machine, a friction false twisting machine, a belt false twisting machine etc. are mentioned, for example.
  • the twisting direction may be S twist (right twist) or Z twist (left twist).
  • the twist number is preferably 800 T / m or more, more preferably 1000 T / m or more, and may be 1200 T / m or more.
  • the number of twists in the twisting step may be, for example, 1800 T / m or less, or 1600 T / m or less.
  • the yarn subjected to the twisting step and the heating step is twisted in the opposite direction to the twisting step.
  • the number of twists in the untwisting step may be equal to the number of twists in the twisting step, or may be equal to or greater than the number of twists in the twisting step.
  • the number of twists in the untwisting process can be adjusted by changing the setting value of the false twisting machine.
  • the false twisting machine 100 as shown in FIG. 7 may be used for the manufacturing method of the false twist yarn which concerns on 2nd embodiment, for example.
  • FIG. 7 is a schematic view showing an example of a false twist device for producing a false twist yarn.
  • the false twisting machine 100 shown in FIG. 7 includes a delivery roller 40, a first feed roller 42, a first heater 50, a twisting unit 60, a second feed roller 44, a second heater 52, and a third feed.
  • the roller 46 and the winding roller 48 are provided in order from the upstream side.
  • the modified fibroin fiber 36 is fed out of the wound material 5 through the delivery roller 40 into the false twisting machine.
  • the fed out modified fibroin fibers 36 are heated in the first heater 50 and twisted at the top of the twisting section 60 while maintaining the temperature.
  • the twist applied to the yarn in the heated state fixes the twist applied to the yarn.
  • twisting is applied to the yarn in the direction opposite to the twisting direction added first. This untwists.
  • the untwisted yarn is fed to the second heater 52 by the second feed roller 44, and further wound around a paper tube through the third feed roller 46 and the winding roller, and finally, the target A false twist yarn is obtained as a wound product 70.
  • the yarn twisting method according to the third embodiment includes twisting a yarn containing modified fibroin fibers, and heating the yarn while maintaining the yarn twist.
  • the conditions for applying twist to the modified fibroin and the yarn containing the modified fibroin fiber, the heating conditions, and the like in the yarn twisting method according to the embodiment apply the conditions and the like described in the method for manufacturing the yarn according to the first embodiment. be able to.
  • the twisted yarn obtained by the method of manufacturing the twisted yarn according to the first embodiment and the false twist yarn obtained by the method of manufacturing the false twist yarn according to the second embodiment sufficient twist is imparted to the yarn and sufficiently fixed. It can be suitably used, for example, in composite fibers, textiles, apparel products, various knitting fabrics, and the like.
  • the yarn is heated in a state in which the yarn containing the modified fibroin fiber is given twist.
  • the present inventors speculate that the thermal contraction of the yarn containing the modified fibroin fiber causes plastic deformation in a twisted state, and the twist in that state is fixed.
  • amino acid sequence shown by SEQ ID NO: 15 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes Furthermore, the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 11 is added to the N-terminus.
  • nucleic acid encoding PRT799 was synthesized.
  • the NdeI site at the 5 'end and the EcoRI site downstream of the stop codon were added to the nucleic acid.
  • the nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was digested with NdeI and EcoRI, cut out, and then recombined into a protein expression vector pET-22b (+) to obtain an expression vector.
  • Protein expression E. coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequence shown by SEQ ID NO: 10.
  • the transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture broth was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that the OD 600 was 0.005.
  • the culture solution temperature was maintained at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours) to obtain a seed culture solution.
  • the seed culture solution was added to a jar fermenter to which 500 mL of 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 culture was controlled at a constant pH of 6.9. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the temperature of the culture solution was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1 M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target protein. Twenty hours after the addition of IPTG, the culture solution was centrifuged to recover the cells. SDS-PAGE is performed using cells prepared from the culture solution before IPTG addition and after IPTG addition, and expression of target spider silk fibroin is achieved by appearance of a target spider silk fibroin size band depending on IPTG addition It was confirmed.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • 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 at 60 ° C. Stir with a stirrer for a minute to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Pure Chemical Industries, Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, the water was removed by a lyophilizer, and the lyophilized powder was recovered to obtain spider silk fibroin "PRT 799".
  • 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • DMSO dimethylsulfoxide
  • Example 1 A plurality of monofilaments obtained above are bundled, and a yarn wound with S twist is applied to a bobbin with S twist at a setting of 606 T / m of twist using an Italy type base twisting machine. Obtained. The resulting yarn was about 160 denier. The wound body was fixed with a net together with the wound body so that the yarn was not unwound, housed in a constant temperature and humidity chamber at 60 ° C. and a relative humidity of 0% RH, and the yarn was heated for 6 hours. Thus, the target twisted yarn 1 was obtained.
  • the twisted yarn 1 was stored in a constant temperature and humidity chamber at 20 ° C. and a relative humidity of 65% RH for 1 hour. Thereafter, five twisted yarns of 1 to 90 cm in size were cut out of the paper tube, and their mass was measured. The mass of each of the five twisted yarns was averaged, and the value obtained by multiplying the obtained average value by 10000 was defined as the fineness. The results are shown in Table 6.
  • Example 6 A target twisted yarn was obtained in the same manner as in Example 1 except that the heating temperature and heating time described in Table 6 were used. The fineness, the number of twists, and the residual torque were measured in the same manner as in Example 1 for each of the obtained twisted yarns. The results are shown in Table 6.
  • Example 1 The yarn provided with twist before heating obtained in Example 1 was used as a sample in a comparative example. The fineness, the number of twists, and the residual torque were measured for the sample in the same manner as in Example 1. The results are shown in Table 6.
  • Example 8 to 11 A target false twist yarn was obtained in the same manner as in Example 7 except that the conditions described in Table 7 were changed. The fineness and the elongation of each of the obtained false-twisted yarns were measured in the same manner as in Example 7. The results are shown in Table 7. From the results of Table 7, it was found that elongation was improved by performing false twisting according to the method of the present invention. Moreover, the tendency for a higher improvement in elongation to be obtained was confirmed when the false twist yarn 1 was not reheated after being false-twisted.

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  • Engineering & Computer Science (AREA)
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  • Textile Engineering (AREA)
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  • Yarns And Mechanical Finishing Of Yarns Or Ropes (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Un aspect de la présente invention concerne un procédé de fabrication de fil torsadé comprenant : une étape de torsion pour torsader un fil comprenant des fibres de fibroïne modifiées ; et une étape de chauffage pour chauffer ledit fil tout en maintenant la torsion dudit fil.
PCT/JP2018/036377 2017-09-29 2018-09-28 Procédé de fabrication de fil torsadé, procédé de fabrication de fil fausse torsion et procédé fausse torsion WO2019066006A1 (fr)

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WO2019182040A1 (fr) * 2018-03-22 2019-09-26 株式会社島精機製作所 Procédé de crêpage de fibre de protéine, procédé de production de fibre de protéine, fibres de protéine, fil filé et produit textile

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