WO2019066006A1 - Twisted thread manufacturing method, false-twisted thread manufacturing method, and thread twisting method - Google Patents

Twisted thread manufacturing method, false-twisted thread manufacturing method, and thread twisting method 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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French (fr)
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/en
Publication of WO2019066006A1 publication Critical patent/WO2019066006A1/en

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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.

Abstract

According to an aspect of the present disclosure, provided is a twisted thread manufacturing method comprising: a twisting step for twisting a thread including modified fibroin fibers; and a heating step for heating said thread while maintaining the twisting of said thread.

Description

撚糸の製造方法、仮撚り糸の製造方法、及び糸の撚り加工方法Method of producing twisted yarn, method of producing false twisted yarn, and method of twisting yarn
　本発明は、撚糸の製造方法、仮撚り糸の製造方法、及び糸の撚り加工方法に関する。 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.
　ナイロン、ポリエステル繊維等の熱可塑性繊維は、その物性、風合いなどの改善を目的として、撚糸加工が施されることがある。一般に、撚糸加工においては、撚り状態を固体するために、繊維に撚りを加えた後、熱処理が行われる。 Thermoplastic fibers such as nylon and polyester fibers are sometimes subjected to twisting in order to improve their physical properties and texture. In general, in twisting, heat treatment is performed after twisting the fibers in order to solidify the twisted state.
　例えば、特許文献１には、ポリ乳酸繊維からなる糸と他の熱可塑性繊維からなる糸とが撚り合わされてなり、前記ポリ乳酸繊維からなる糸の引張強度が０．８０ｃＮ／ｄｔｅｘ以上、引張伸度が２０％以上であり、かつ、撚数が３０～２００Ｔ／ｍであることを特徴とする合撚糸が開示されている。 For example, 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.
　一方、フィブロイン繊維（例えば、絹糸）等では、同様の方法で、繊維に撚りを付与することは容易ではなかった。そこで、例えば、特許文献２には、生糸に、絹繊維のセリシンを不溶化させる改質剤を含浸させた後、加撚加工を行い、次いで撚りを固定する熱処理を温度１２０～１４０℃で１０～３０分行なった後、加撚と逆方向へ撚る解撚を行ない、次いで０．６～３．０ｇ／ｌのタンパク質分解酵素に浸漬して精練し、絹糸に含まれるセリシンを取り除くことを特徴とする捲縮性を有する絹糸の製造方法が提案されている。また、特許文献３には、野蚕糸に一方向の強撚を施し、湿式延伸処理して組成変形させ、次いで精錬染色を施してその湿熱により強撚の旋回を熱固定して解撚することを特徴とする嵩高性及び伸縮性を有する絹糸の製法が提案されている。 On the other hand, in fibroin fibers (for example, silk yarn) etc., it was not easy to apply twist to the fibers by the same method. Therefore, for example, in Patent Document 2, after the raw yarn is impregnated with a modifier that insolubilizes sericin of silk fiber, twist processing is performed, and then heat treatment for fixing the twist is performed at a temperature of 120 to 140 ° C. After 30 minutes, twisting and untwisting in the opposite direction are carried out, and then it is dipped in 0.6 to 3.0 g / l of proteolytic enzyme for scouring to remove sericin contained in the silk thread. A process for producing silk yarn having crimpability has been proposed. In addition, according to Patent Document 3, 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
特開２００８－２３１５８３号公報Unexamined-Japanese-Patent No. 2008-231583 特開２０１６－０２３３８９号公報JP, 2016-023389, A 特開昭５９－１５７３３７号公報JP-A-59-157337
　しかしながら、特許文献２、３のような方法では、改質剤の添加、精錬染色を行うなど、工程数の増加に伴い製造工程が煩雑化するため、撚りが付与されたフィブロインを含む糸をより効率的に製造する方法が求められている。 However, in methods such as Patent Documents 2 and 3, since the manufacturing process becomes complicated as the number of processes increases, such as addition of a modifier and refining dyeing, a yarn containing fibroin to which twist is imparted is made more There is a need for an efficient method of manufacturing.
　本発明は、このような事情に鑑みてなされたものであり、撚糸、仮撚り糸等の撚りが付与されたフィブロインを含む糸をより簡便に得ることが可能な、撚糸の製造方法、及び仮撚り糸の製造方法を提供することを目的とする。本発明はまた、フィブロインを含む糸に対して、より簡便に撚りを付与することが可能な糸の撚り加工方法を提供することを目的とする。 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.
　本発明は、例えば、以下の各発明に関する。
［１］
　改変フィブロイン繊維を含む糸を撚る加撚工程と、
　前記糸の撚りを維持した状態で、前記糸を加熱する加熱工程と、を備える、撚糸の製造方法。
［２］
　前記改変フィブロイン繊維が、改変クモ糸フィブロイン繊維である、［１］に記載の撚糸の製造方法。
［３］
　前記加熱工程が、５０℃超の雰囲気下で前記糸を加熱する工程である、［１］又は［２］に記載の撚糸の製造方法。
［４］
　前記加熱工程が、２４０℃以下の雰囲気下で前記糸を加熱する工程である、［１］～［３］のいずれかに記載の撚糸の製造方法。
［５］
　前記加撚工程が、前記糸の撚数が２００Ｔ／ｍ以上であるように前記糸を撚る工程である、［１］～［４］のいずれかに記載の撚糸の製造方法。
［６］
　改変フィブロイン繊維を含む糸を撚る加撚工程と、
　前記糸の撚りを維持した状態で、前記糸を加熱する加熱工程と、
　加熱された前記糸を解撚する解撚工程と、を備える、仮撚り糸の製造方法。
［７］
　前記改変フィブロイン繊維が、改変クモ糸フィブロイン繊維である、［６］に記載の仮撚り糸の製造方法。
［８］
　前記加熱工程が、５０℃超の雰囲気下で前記糸を加熱する工程である、［６］又は［７］に記載の仮撚り糸の製造方法。
［９］
　前記加熱工程が、２４０℃以下の雰囲気下で前記糸を加熱する工程である、［６］～［８］のいずれかに記載の仮撚り糸の製造方法。
［１０］
　前記加撚工程が、前記糸の撚数が８００Ｔ／ｍ以上であるように前記糸を撚る工程である、［６］～［９］のいずれかに記載の仮撚り糸の製造方法。
［１１］
　改変フィブロイン繊維を含む糸を撚ることと、
　前記糸の撚りを維持した状態で、前記糸を加熱することと、を含む、糸の撚り加工方法。 The present invention relates to, for example, the following inventions.
[1]
A twisting step of twisting a yarn containing the modified fibroin fiber,
And a heating step of heating the yarn while maintaining the yarn twist.
[2]
The method for producing a twisted yarn according to [1], wherein the modified fibroin fiber is a modified spider yarn fibroin fiber.
[3]
The method for producing a twisted yarn according to [1] or [2], wherein the heating step is a step of heating the yarn under an atmosphere of more than 50 ° C.
[4]
The method for producing a twisted yarn according to any one of [1] to [3], wherein the heating step is a step of heating the yarn under an atmosphere of 240 ° C. or less.
[5]
The method for producing a twisted yarn according to any one of [1] to [4], wherein the twisting step is a step of twisting the yarn such that the number of twists of the yarn is 200 T / m or more.
[6]
A twisting step of twisting a yarn containing the modified fibroin fiber,
A heating step of heating the yarn while maintaining the twist of the yarn;
And a twisting step of untwisting the heated yarn.
[7]
The method for producing a false twist yarn according to [6], wherein the modified fibroin fiber is a modified spider yarn fibroin fiber.
[8]
The method for producing a false twist yarn according to [6] or [7], wherein the heating step is a step of heating the yarn under an atmosphere of more than 50 ° C.
[9]
The method for producing a false twist yarn according to any one of [6] to [8], wherein the heating step is a step of heating the yarn under an atmosphere of 240 ° C. or lower.
[10]
The method for producing a false twist yarn according to any one of [6] to [9], wherein the twisting step is a step of twisting the yarn such that the number of twists of the yarn is 800 T / m or more.
[11]
Twisting a yarn containing the modified fibroin fiber,
Heating the yarn in a state in which the yarn is kept twisted.
　本発明によれば、撚糸、仮撚り糸等の撚りが付与されたフィブロインを含む糸をより簡便に得ることができる。また、本発明によれば、フィブロイン繊維に対してより簡便に撚りを付与することができる。 According to the present invention, it is possible to more simply obtain a yarn containing fibroin to which twist is imparted, such as twisted yarn and false twisted yarn. Moreover, according to the present invention, twist can be more easily applied to fibroin fibers.
図１は、改変フィブロインのドメイン配列を示す模式図である。FIG. 1 is a schematic view showing the domain sequence of modified fibroin. 図２は、天然由来のフィブロインのｚ／ｗ（％）の値の分布を示す図である。FIG. 2 is a diagram showing the distribution of the value of z / w (%) of naturally occurring fibroin. 図３は、天然由来のフィブロインのｘ／ｙ（％）の値の分布を示す図である。FIG. 3 shows the distribution of x / y (%) values of naturally occurring fibroin. 図４は、改変フィブロインのドメイン配列の一例を示す模式図である。FIG. 4 is a schematic view showing an example of the domain sequence of the modified fibroin. 図５は、改変フィブロインのドメイン配列の一例を示す模式図である。FIG. 5 is a schematic view showing an example of the domain sequence of the modified fibroin. 図６は、改変フィブロイン繊維を製造するための紡糸装置の一例を示す概略図である。FIG. 6 is a schematic view showing an example of a spinning apparatus for producing modified fibroin fibers. 図７は、仮撚り糸を製造するための仮撚装置の一例を示す概略図である。FIG. 7 is a schematic view showing an example of a false twist device for producing a false twist yarn.
　以下、本発明の好適な実施形態について説明する。ただし、本発明は下記実施形態に何ら限定されるものではない。 Hereinafter, preferred embodiments of the present invention will be described. However, the present invention is not limited to the following embodiment.
＜撚糸の製造方法＞
　第一実施形態に係る撚糸の製造方法は、改変フィブロイン繊維を含む糸を撚る加撚工程と、糸の撚りを維持した状態で、糸を加熱する加熱工程と、を備える。加撚工程と、加熱工程と、は互いに独立した工程であってもよく、同時に行われる工程であってもよい。 <Production method of twisted 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.
　加撚工程は、改変フィブロイン繊維に撚りを加える。撚りを加える手段は、公知の手段を用いることができ、例えば、撚糸機などを使用できる。撚糸機としては、公知の各種の撚糸形式（アップ形式、ダウン形式、特殊桟形式、エア桟形式、リワイダ等）に対応した撚糸機、例えば、イタリー式撚糸機、ダブルツイスター、リング撚糸機、複合撚糸機、インタレス、パーンワインダ等が挙げられる。撚り方向は、Ｓ撚り（右撚）であってよく、Ｚ撚り（左撚）であってもよい。 The twisting process adds twist to the modified fibroin fiber. As means for applying twist, known means can be used, and for example, a twisting machine can be used. As the 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).
　撚数は、好ましくは２００Ｔ／ｍ以上であり、より好ましくは６００Ｔ／ｍ以上であり、また８００Ｔ／ｍ以上であってもよい。加撚工程における撚数を２００Ｔ／ｍ以上とすることにより、糸により効率的に撚りを付与することができる。また撚数は、例えば、１２００Ｔ／ｍ以下であってよく、または１０００Ｔ／ｍ以下であってもよい。撚数を調整することにより、いわゆる甘撚糸、中撚糸、強撚糸又は極強撚糸を調製することができる。ここで、撚数とは、糸１ｍあたりに糸が何回回転したか（撚りをかけたか）を意味する。撚数は、通常、撚糸機の設定値を変更することにより、調整することができる。 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. By setting the number of twists in the twisting process to 200 T / m or more, twist can be efficiently applied to the yarn. The number of twists may be, for example, 1200 T / m or less, or 1000 T / m or less. By adjusting the number of twists, it is possible to prepare so-called sweet-twisted yarns, medium-twisted yarns, strong-twisted yarns or extra-strong-twisted yarns. Here, 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.
＜改変フィブロイン＞
　本実施形態に係る改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。改変フィブロインは、ドメイン配列のＮ末端側及びＣ末端側のいずれか一方又は両方に更にアミノ酸配列（Ｎ末端配列及びＣ末端配列）が付加されていてもよい。Ｎ末端配列及びＣ末端配列は、これに限定されるものではないが、典型的には、フィブロインに特徴的なアミノ酸モチーフの反復を有さない領域であり、１００残基程度のアミノ酸からなる。 <Modified fibroin>
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.
　本明細書において「改変フィブロイン」とは、人為的に製造されたフィブロイン（人造フィブロイン）を意味する。改変フィブロインは、そのドメイン配列が、天然由来のフィブロインのアミノ酸配列とは異なるフィブロインであってもよく、天然由来のフィブロインのアミノ酸配列と同一であるフィブロインであってもよい。本明細書における「天然由来のフィブロイン」もまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。 As used herein, "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. In the present specification, “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
　「改変フィブロイン」は、天然由来のフィブロインのアミノ酸配列をそのまま利用したものであってもよく、天然由来のフィブロインのアミノ酸配列に依拠してそのアミノ酸配列を改変したもの（例えば、クローニングした天然由来のフィブロインの遺伝子配列を改変することによりアミノ酸配列を改変したもの）であってもよく、また天然由来のフィブロインに依らず人工的に設計及び合成したもの（例えば、設計したアミノ酸配列をコードする核酸を化学合成することにより所望のアミノ酸配列を有するもの）であってもよい。 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).
　本明細書において「ドメイン配列」とは、フィブロイン特有の結晶領域（典型的には、アミノ酸配列の（Ａ）ｎモチーフに相当する。）と非晶領域（典型的には、アミノ酸配列のＲＥＰに相当する。）を生じるアミノ酸配列であり、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるアミノ酸配列を意味する。ここで、式１中、（Ａ）ｎモチーフは、アラニン残基を主とするアミノ酸配列を示し、アミノ酸残基数は２～２７である。（Ａ）ｎモチーフのアミノ酸残基数は、２～２０、４～２７、４～２０、８～２０、１０～２０、４～１６、８～１６、又は１０～１６の整数であってよい。また、（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数の割合は４０％以上であればよく、６０％以上、７０％以上、８０％以上、８３％以上、８５％以上、８６％以上、９０％以上、９５％以上、又は１００％（アラニン残基のみで構成されることを意味する。）であってもよい。ドメイン配列中に複数存在する（Ａ）ｎモチーフは、少なくとも７つがアラニン残基のみで構成されてもよい。ＲＥＰは２～２００アミノ酸残基から構成されるアミノ酸配列を示す。ＲＥＰは、１０～２００アミノ酸残基から構成されるアミノ酸配列であってもよい。ｍは２～３００の整数を示し、１０～３００の整数であってもよい。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。 In the present specification, “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. Here, in Formula 1, 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. (A) 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.
　本実施形態に係る改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列に対して、例えば、１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行うことで得ることができる。アミノ酸残基の置換、欠失、挿入及び／又は付加は、部分特異的突然変異誘発法等の当業者に周知の方法により行うことができる。具体的には、Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓ．１０，６４８７（１９８２）、Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，１００，４４８（１９８３）等の文献に記載されている方法に準じて行うことができる。 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.
　天然由来のフィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質であり、具体的には、例えば、昆虫又はクモ類が産生するフィブロインが挙げられる。 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.
　昆虫が産生するフィブロインとしては、例えば、ボンビックス・モリ（Ｂｏｍｂｙｘ　ｍｏｒｉ）、クワコ（Ｂｏｍｂｙｘ　ｍａｎｄａｒｉｎａ）、天蚕（Ａｎｔｈｅｒａｅａ　ｙａｍａｍａｉ）、柞蚕（Ａｎｔｅｒａｅａ　ｐｅｒｎｙｉ）、楓蚕（Ｅｒｉｏｇｙｎａ　ｐｙｒｅｔｏｒｕｍ）、蓖蚕（Ｐｉｌｏｓａｍｉａ　Ｃｙｎｔｈｉａ　ｒｉｃｉｎｉ）、樗蚕（Ｓａｍｉａ　ｃｙｎｔｈｉａ）、栗虫（Ｃａｌｉｇｕｒａ　ｊａｐｏｎｉｃａ）、チュッサー蚕（Ａｎｔｈｅｒａｅａ　ｍｙｌｉｔｔａ）、ムガ蚕（Ａｎｔｈｅｒａｅａ　ａｓｓａｍａ）等のカイコが産生する絹タンパク質、及びスズメバチ（Ｖｅｓｐａ　ｓｉｍｉｌｌｉｍａ　ｘａｎｔｈｏｐｔｅｒａ）の幼虫が吐出するホーネットシルクタンパク質が挙げられる。 Examples of 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.
　昆虫が産生するフィブロインのより具体的な例としては、例えば、カイコ・フィブロインＬ鎖（ＧｅｎＢａｎｋアクセッション番号Ｍ７６４３０（塩基配列）、及びＡＡＡ２７８４０．１（アミノ酸配列））が挙げられる。 More specific examples of insect-produced fibroin include, for example, silkworm fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
　クモ類が産生するフィブロインとしては、例えば、オニグモ、ニワオニグモ、アカオニグモ、アオオニグモ及びマメオニグモ等のオニグモ属（Ａｒａｎｅｕｓ属）に属するクモ、ヤマシロオニグモ、イエオニグモ、ドヨウオニグモ及びサツマノミダマシ等のヒメオニグモ属（Ｎｅｏｓｃｏｎａ属）に属するクモ、コオニグモモドキ等のコオニグモモドキ属（Ｐｒｏｎｕｓ属）に属するクモ、トリノフンダマシ及びオオトリノフンダマシ等のトリノフンダマシ属（Ｃｙｒｔａｒａｃｈｎｅ属）に属するクモ、トゲグモ及びチブサトゲグモ等のトゲグモ属（Ｇａｓｔｅｒａｃａｎｔｈａ属）に属するクモ、マメイタイセキグモ及びムツトゲイセキグモ等のイセキグモ属（Ｏｒｄｇａｒｉｕｓ属）に属するクモ、コガネグモ、コガタコガネグモ及びナガコガネグモ等のコガネグモ属（Ａｒｇｉｏｐｅ属）に属するクモ、キジロオヒキグモ等のオヒキグモ属（Ａｒａｃｈｎｕｒａ属）に属するクモ、ハツリグモ等のハツリグモ属（Ａｃｕｓｉｌａｓ属）に属するクモ、スズミグモ、キヌアミグモ及びハラビロスズミグモ等のスズミグモ属（Ｃｙｔｏｐｈｏｒａ属）に属するクモ、ゲホウグモ等のゲホウグモ属（Ｐｏｌｔｙｓ属）に属するクモ、ゴミグモ、ヨツデゴミグモ、マルゴミグモ及びカラスゴミグモ等のゴミグモ属（Ｃｙｃｌｏｓａ属）に属するクモ、及びヤマトカナエグモ等のカナエグモ属（Ｃｈｏｒｉｚｏｐｅｓ属）に属するクモが産生するスパイダーシルクタンパク質、並びにアシナガグモ、ヤサガタアシナガグモ、ハラビロアシダカグモ及びウロコアシナガグモ等のアシナガグモ属（Ｔｅｔｒａｇｎａｔｈａ属）に属するクモ、オオシロカネグモ、チュウガタシロカネグモ及びコシロカネグモ等のシロカネグモ属（Ｌｅｕｃａｕｇｅ属）に属するクモ、ジョロウグモ及びオオジョロウグモ等のジョロウグモ属（Ｎｅｐｈｉｌａ属）に属するクモ、キンヨウグモ等のアズミグモ属（Ｍｅｎｏｓｉｒａ属）に属するクモ、ヒメアシナガグモ等のヒメアシナガグモ属（Ｄｙｓｃｈｉｒｉｏｇｎａｔｈａ属）に属するクモ、クロゴケグモ、セアカゴケグモ、ハイイロゴケグモ及びジュウサンボシゴケグモ等のゴケグモ属（Ｌａｔｒｏｄｅｃｔｕｓ属）に属するクモ、及びユープロステノプス属（Ｅｕｐｒｏｓｔｈｅｎｏｐｓ属）に属するクモ等のアシナガグモ科（Ｔｅｔｒａｇｎａｔｈｉｄａｅ科）に属するクモが産生するスパイダーシルクタンパク質が挙げられる。スパイダーシルクタンパク質としては、例えば、ＭａＳｐ（ＭａＳｐ１及びＭａＳｐ２）、ＡＤＦ（ＡＤＦ３及びＡＤＦ４）等の牽引糸タンパク質、ＭｉＳｐ（ＭｉＳｐ１及びＭｉＳｐ２）等が挙げられる。 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 belonging to the genus Arachnura such as a green-tailed spider, a spider belonging to the genus Acusilas such as a spider, a spider spider belonging to the genus Acusilas Spiders belonging to the genus Cytophora, spiders belonging to the genus Pythogmo (Poltys), spider spiders belonging to the genus Poltys, spider spiders such as the spider spider belonging to the genus Cyclosa such as the spider spider such as the spider spider, the spider spider, the spider spider and the crow spider spider, and the spider spider belonging to the genus Cyclosa Spider silk proteins produced by spiders belonging to the genus Chorizopes), and asinacea such as asinacea, assassinum, assassinum and assassinum Spiders belonging to the genus Gummo (Tetragnatha), spiders belonging to the genus Negrocarpus, such as the spiders belonging to the genus Necha Spiders belonging to the genus Azosoma (Menosira), spiders belonging to the genus Spermatoglyphus (Dyschiriognatha), such as the spider snail, spiders belonging to the genus Latrodectus such as the spider snail, the spider moth, the spider moth, and the spider moth Produced by spiders belonging to the family Agnagidae (Tetragnathidae) such as spiders belonging to the genus Prostenopus (Euprosthenops) The spider silk protein is mentioned. Examples of spider silk proteins include MaSp (MaSp1 and MaSp2), dragline proteins such as ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.
　クモ類が産生するスパイダーシルクタンパク質のより具体的な例としては、例えば、ｆｉｂｒｏｉｎ－３（ａｄｆ－３）［Ａｒａｎｅｕｓ　ｄｉａｄｅｍａｔｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ４７０１０（アミノ酸配列）、Ｕ４７８５５（塩基配列））、ｆｉｂｒｏｉｎ－４（ａｄｆ－４）［Ａｒａｎｅｕｓ　ｄｉａｄｅｍａｔｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ４７０１１（アミノ酸配列）、Ｕ４７８５６（塩基配列））、ｄｒａｇｌｉｎｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ｓｐｉｄｒｏｉｎ　１［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ０４５０４（アミノ酸配列）、Ｕ３７５２０（塩基配列））、ｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　１［Ｌａｔｒｏｄｅｃｔｕｓ　ｈｅｓｐｅｒｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＢＲ６８８５６（アミノ酸配列）、ＥＦ５９５２４６（塩基配列））、ｄｒａｇｌｉｎｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ｓｐｉｄｒｏｉｎ　２［Ｎｅｐｈｉｌａ　ｃｌａｖａｔａ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＬ３２４７２（アミノ酸配列）、ＡＦ４４１２４５（塩基配列））、ｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　１［Ｅｕｐｒｏｓｔｈｅｎｏｐｓ　ａｕｓｔｒａｌｉｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＣＡＪ００４２８（アミノ酸配列）、ＡＪ９７３１５５（塩基配列））、及びｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　２［Ｅｕｐｒｏｓｔｈｅｎｏｐｓ　ａｕｓｔｒａｌｉｓ］（ＧｅｎＢａｎｋアクセッション番号ＣＡＭ３２２４９．１（アミノ酸配列）、ＡＭ４９０１６９（塩基配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　１［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ１４５８９．１（アミノ酸配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　２［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ１４５９１．１（アミノ酸配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ－ｌｉｋｅ　ｐｒｏｔｅｉｎ［Ｎｅｐｈｉｌｅｎｇｙｓ　ｃｒｕｅｎｔａｔａ］（ＧｅｎＢａｎｋアクセッション番号ＡＢＲ３７２７８．１（アミノ酸配列）等が挙げられる。 More specific examples of 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. 1 (amino acid sequence), AM 490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession No. AAC 14589. 1 (amino acid sequence)), minor ampullate silk protein 2 [ Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullalate spidroin-like protein [Nephilengys cruentata] (GenBank Accession No. ABR37278.1 (amino acid sequence)), and the like.
　天然由来のフィブロインのより具体的な例としては、更に、ＮＣＢＩ　ＧｅｎＢａｎｋに配列情報が登録されているフィブロインを挙げることができる。例えば、ＮＣＢＩ　ＧｅｎＢａｎｋに登録されている配列情報のうちＤＩＶＩＳＩＯＮとしてＩＮＶを含む配列の中から、ＤＥＦＩＮＩＴＩＯＮにｓｐｉｄｒｏｉｎ、ａｍｐｕｌｌａｔｅ、ｆｉｂｒｏｉｎ、「ｓｉｌｋ及びｐｏｌｙｐｅｐｔｉｄｅ」、又は「ｓｉｌｋ及びｐｒｏｔｅｉｎ」がキーワードとして記載されている配列、ＣＤＳから特定のｐｒｏｄｕｃｔの文字列、ＳＯＵＲＣＥからＴＩＳＳＵＥ　ＴＹＰＥに特定の文字列の記載された配列を抽出することにより確認することができる。 More specific examples of naturally derived fibroin further include fibroin whose sequence information is registered in NCBI GenBank. For example, 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.
　本実施形態に係る改変フィブロインは、改変絹（シルク）フィブロイン（カイコが産生する絹タンパク質のアミノ酸配列を改変したもの）であってもよく、改変クモ糸フィブロイン（クモ類が産生するスパイダーシルクタンパク質のアミノ酸配列を改変したもの）であってもよい。改変フィブロインとしては、改変クモ糸フィブロインが好ましい。 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). As modified fibroin, modified spider silk fibroin is preferred.
　改変フィブロインの具体的な例として、クモの大瓶状腺で産生される大吐糸管しおり糸タンパク質に由来する改変フィブロイン（第１の改変フィブロイン）、グリシン残基の含有量が低減されたドメイン配列を有する改変フィブロイン（第２の改変フィブロイン）、（Ａ）ｎモチーフの含有量が低減されたドメイン配列を有する改変フィブロイン（第３の改変フィブロイン）、グリシン残基の含有量、及び（Ａ）ｎモチーフの含有量が低減された改変フィブロイン、局所的に疎水性指標の大きい領域を含むドメイン配列を有する改変フィブロイン（第５の改変フィブロイン）、並びにグルタミン残基の含有量が低減されたドメイン配列を有する改変フィブロイン（第６の改変フィブロイン）が挙げられる。 As a specific example of the modified fibroin, 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).
　第１の改変フィブロインとしては、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質が挙げられる。第１の改変フィブロインにおいて、（Ａ）ｎモチーフのアミノ酸残基数は、３～２０の整数が好ましく、４～２０の整数がより好ましく、８～２０の整数が更に好ましく、１０～２０の整数が更により好ましく、４～１６の整数が更によりまた好ましく、８～１６の整数が特に好ましく、１０～１６の整数が最も好ましくい。第１の改変フィブロインは、式１中、ＲＥＰを構成するアミノ酸残基の数は、１０～２００残基であることが好ましく、１０～１５０残基であることがより好ましく、２０～１００残基であることが更に好ましく、２０～７５残基であることが更により好ましい。第１の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるアミノ酸配列中に含まれるグリシン残基、セリン残基及びアラニン残基の合計残基数がアミノ酸残基数全体に対して、４０％以上であることが好ましく、６０％以上であることがより好ましく、７０％以上であることが更に好ましい。 The first modified fibroin includes a protein comprising a domain sequence represented by Formula 1: [(A) n Motif-REP] m . In the first modified fibroin, 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. In the first modified fibroin, in the formula 1, 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. In the first modified fibroin, 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.
　第１の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるアミノ酸配列の単位を含み、かつＣ末端配列が配列番号１～３のいずれかに示されるアミノ酸配列又は配列番号１～３のいずれかに示されるアミノ酸配列と９０％以上の相同性を有するアミノ酸配列であるポリペプチドであってもよい。 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.
　配列番号１に示されるアミノ酸配列は、ＡＤＦ３（ＧＩ：１２６３２８７、ＮＣＢＩ）のアミノ酸配列のＣ末端の５０残基のアミノ酸からなるアミノ酸配列と同一であり、配列番号２に示されるアミノ酸配列は、配列番号１に示されるアミノ酸配列のＣ末端から２０残基取り除いたアミノ酸配列と同一であり、配列番号３に示されるアミノ酸配列は、配列番号１に示されるアミノ酸配列のＣ末端から２９残基取り除いたアミノ酸配列と同一である。 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.
　第１の改変フィブロインのより具体的な例として、（１－ｉ）配列番号４（ｒｅｃｏｍｂｉｎａｎｔ　ｓｐｉｄｅｒ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ＡＤＦ３ＫａｉＬａｒｇｅＮＲＳＨ１）で示されるアミノ酸配列、又は（１－ｉｉ）配列番号４で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。配列同一性は、９５％以上であることが好ましい。 As a more specific example of the first modified fibroin, 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.
　配列番号４で示されるアミノ酸配列は、Ｎ末端に開始コドン、Ｈｉｓ１０タグ及びＨＲＶ３Ｃプロテアーゼ（Ｈｕｍａｎ　ｒｈｉｎｏｖｉｒｕｓ　３Ｃプロテアーゼ）認識サイトからなるアミノ酸配列（配列番号５）を付加したＡＤＦ３のアミノ酸配列において、第１～１３番目の反復領域をおよそ２倍になるように増やすとともに、翻訳が第１１５４番目アミノ酸残基で終止するように変異させたものである。配列番号４で示されるアミノ酸配列のＣ末端のアミノ酸配列は、配列番号３で示されるアミノ酸配列と同一である。 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.
　（１－ｉ）の改変フィブロインは、配列番号４で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (1-i) may consist of the amino acid sequence shown by SEQ ID NO: 4.
　第２の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、グリシン残基の含有量が低減されたアミノ酸配列を有する。第２の改変フィブロインは、天然由来のフィブロインと比較して、少なくともＲＥＰ中の１又は複数のグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものということができる。 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 .
　第２の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中のＧＧＸ及びＧＰＧＸＸ（但し、Ｇはグリシン残基、Ｐはプロリン残基、Ｘはグリシン以外のアミノ酸残基を示す。）から選ばれる少なくとも一つのモチーフ配列において、少なくとも１又は複数の当該モチーフ配列中の１つのグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものであってもよい。 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. May be
　第２の改変フィブロインは、上述のグリシン残基が別のアミノ酸残基に置換されたモチーフ配列の割合が、全モチーフ配列に対して、１０％以上であってもよい。 In the second modified fibroin, 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.
　第２の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、上記ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を除いた配列中の全ＲＥＰに含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を除いた配列中の総アミノ酸残基数をｗとしたときに、ｚ／ｗが３０％以上、４０％以上、５０％以上又は５０．９％以上であるアミノ酸配列を有するものであってもよい。（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数は８３％以上であってよいが、８６％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることが更に好ましく、１００％であること（アラニン残基のみで構成されることを意味する）が更により好ましい。 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 total number of amino acid residues of the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal end of From the (A) n motif located closest to the C-terminus to the C-terminal end of the above domain sequence, where w is the total number of amino acid residues in the sequence, z / w is 30% or more, It may have an amino acid sequence that is 40% or more, 50% or more, or 50.9% or more. (A) The 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.
　第２の改変フィブロインは、ＧＧＸモチーフの１つのグリシン残基を別のアミノ酸残基に置換することにより、ＸＧＸからなるアミノ酸配列の含有割合を高めたものであることが好ましい。第２の改変フィブロインは、ドメイン配列中のＧＧＸからなるアミノ酸配列の含有割合が、３０％以下であることが好ましく、２０％以下であることがより好ましく、１０％以下であることが更に好ましく、６％以下であることが更により好ましく、４％以下であることが更によりまた好ましく、２％以下であることが特に好ましい。ドメイン配列中のＧＧＸからなるアミノ酸配列の含有割合は、下記ＸＧＸからなるアミノ酸配列の含有割合（ｚ／ｗ）の算出方法と同様の方法で算出することができる。 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. In the second modified fibroin, 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.
　ｚ／ｗの算出方法を更に詳細に説明する。まず、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むフィブロイン（改変フィブロイン又は天然由来のフィブロイン）において、ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列に含まれる全てのＲＥＰから、ＸＧＸからなるアミノ酸配列を抽出する。ＸＧＸを構成するアミノ酸残基の総数がｚである。例えば、ＸＧＸからなるアミノ酸配列が５０個抽出された場合（重複はなし）、ｚは５０×３＝１５０である。また、例えば、ＸＧＸＧＸからなるアミノ酸配列の場合のように２つのＸＧＸに含まれるＸ（中央のＸ）が存在する場合は、重複分を控除して計算する（ＸＧＸＧＸの場合は５アミノ酸残基である）。ｗは、ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列に含まれる総アミノ酸残基数である。例えば、図１に示したドメイン配列の場合、ｗは４＋５０＋４＋１００＋４＋１０＋４＋２０＋４＋３０＝２３０である（最もＣ末端側に位置する（Ａ）ｎモチーフは除いている。）。次に、ｚをｗで除すことによって、ｚ／ｗ（％）を算出することができる。 The method of calculating z / w will be described in more detail. First, in fibroin (modified fibroin or naturally-derived 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. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (without duplication), z is 50 × 3 = 150. Also, for example, as in the case of the amino acid sequence consisting of XGXGX, when X (center X) contained in two XGX is present, calculation is made by subtracting the overlap (in the case of XGXGX, 5 amino acid residues is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located closest to the C-terminus to the C-terminus of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (the (A) n motif located at the most C-terminal side is excluded). Next, z / w (%) can be calculated by dividing z by w.
　ここで、天然由来のフィブロインにおけるｚ／ｗについて説明する。まず、上述のように、ＮＣＢＩ　ＧｅｎＢａｎｋにアミノ酸配列情報が登録されているフィブロインを例示した方法により確認したところ、６６３種類のフィブロイン（このうち、クモ類由来のフィブロインは４１５種類）が抽出された。抽出された全てのフィブロインのうち、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、フィブロイン中のＧＧＸからなるアミノ酸配列の含有割合が６％以下である天然由来のフィブロインのアミノ酸配列から、上述の算出方法により、ｚ／ｗを算出した。その結果を図２に示す。図２の横軸はｚ／ｗ（％）を示し、縦軸は頻度を示す。図２から明らかなとおり、天然由来のフィブロインにおけるｚ／ｗは、いずれも５０．９％未満である（最も高いもので、５０．８６％）。 Here, z / w in naturally derived fibroin will be described. First, as described above, when confirmed by the method exemplifying fibroin whose amino acid sequence information is registered in NCBI GenBank, 663 types of fibroin (of which 415 types of fibroin derived from spiders) were extracted. Among all the fibroins extracted, a naturally derived one containing a domain sequence represented by Formula 1: [(A) n Motif -REP] m and having a content of the amino acid sequence consisting of GGX in fibroin of 6% or less From the amino acid sequence of fibroin of the above, 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. As is clear from FIG. 2, z / w in all naturally occurring fibroin is less than 50.9% (highest, 50.86%).
　第２の改変フィブロインにおいて、ｚ／ｗは、５０．９％以上であることが好ましく、５６．１％以上であることがより好ましく、５８．７％以上であることが更に好ましく、７０％以上であることが更により好ましく、８０％以上であることが更によりまた好ましい。ｚ／ｗの上限に特に制限はないが、例えば、９５％以下であってもよい。 In the second modified fibroin, 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.
　第２の改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列から、グリシン残基をコードする塩基配列の少なくとも一部を置換して別のアミノ酸残基をコードするように改変することにより得ることができる。このとき、改変するグリシン残基として、ＧＧＸモチーフ及びＧＰＧＸＸモチーフにおける１つのグリシン残基を選択してもよいし、またｚ／ｗが５０．９％以上になるように置換してもよい。また、例えば、天然由来のフィブロインのアミノ酸配列から上記態様を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列からＲＥＰ中のグリシン残基を別のアミノ酸残基に置換したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 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. At this time, as 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. Alternatively, for example, 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. In any case, in addition to the modification corresponding to substitution of a glycine residue in REP from the amino acid sequence of naturally-derived fibroin with another amino acid residue, 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.
　上記の別のアミノ酸残基としては、グリシン残基以外のアミノ酸残基であれば特に制限はないが、バリン（Ｖ）残基、ロイシン（Ｌ）残基、イソロイシン（Ｉ）残基、メチオニン（Ｍ）残基、プロリン（Ｐ）残基、フェニルアラニン（Ｆ）残基及びトリプトファン（Ｗ）残基等の疎水性アミノ酸残基、グルタミン（Ｑ）残基、アスパラギン（Ｎ）残基、セリン（Ｓ）残基、リシン（Ｋ）残基及びグルタミン酸（Ｅ）残基等の親水性アミノ酸残基が好ましく、バリン（Ｖ）残基、ロイシン（Ｌ）残基、イソロイシン（Ｉ）残基、フェニルアラニン（Ｆ）残基及びグルタミン（Ｑ）残基がより好ましく、グルタミン（Ｑ）残基が更に好ましい。 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.
　第２の改変フィブロインのより具体的な例として、（２－ｉ）配列番号６（Ｍｅｔ－ＰＲＴ３８０）、配列番号７（Ｍｅｔ－ＰＲＴ４１０）、配列番号８（Ｍｅｔ－ＰＲＴ５２５）若しくは配列番号９（Ｍｅｔ－ＰＲＴ７９９）で示されるアミノ酸配列、又は（２－ｉｉ）配列番号６、配列番号７、配列番号８若しくは配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of the second modified fibroin, (2-i) 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 amino acid sequence represented by -PRT 799) or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9; Modified fibroin can be mentioned.
　（２－ｉ）の改変フィブロインについて説明する。配列番号６で示されるアミノ酸配列は、天然由来のフィブロインに相当する配列番号１０（Ｍｅｔ－ＰＲＴ３１３）で示されるアミノ酸配列のＲＥＰ中の全てのＧＧＸをＧＱＸに置換したものである。配列番号７で示されるアミノ酸配列は、配列番号６で示されるアミノ酸配列から、Ｎ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフを欠失させ、更にＣ末端配列の手前に［（Ａ）ｎモチーフ－ＲＥＰ］を１つ挿入したものである。配列番号８で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列の各（Ａ）ｎモチーフのＣ末端側に２つのアラニン残基を挿入し、更に一部のグルタミン（Ｑ）残基をセリン（Ｓ）残基に置換し、配列番号７の分子量とほぼ同じとなるようにＣ末端側の一部のアミノ酸を欠失させたものである。配列番号９で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中に存在する２０個のドメイン配列の領域（但し、当該領域のＣ末端側の数アミノ酸残基が置換されている。）を４回繰り返した配列のＣ末端に所定のヒンジ配列とＨｉｓタグ配列が付加されたものである。 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. It is substituted with a serine (S) residue and a partial amino acid at the C-terminal side is deleted so as to be approximately the same as the molecular weight of SEQ ID NO: 7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 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.
　配列番号１０で示されるアミノ酸配列（天然由来のフィブロインに相当）におけるｚ／ｗの値は、４６．８％である。配列番号６で示されるアミノ酸配列、配列番号７で示されるアミノ酸配列、配列番号８で示されるアミノ酸配列、及び配列番号９で示されるアミノ酸配列におけるｚ／ｗの値は、それぞれ５８．７％、７０．１％、６６．１％及び７０．０％である。また、配列番号１０、配列番号６、配列番号７、配列番号８及び配列番号９で示されるアミノ酸配列のギザ比率（後述する）１：１．８～１１．３におけるｘ／ｙの値は、それぞれ１５．０％、１５．０％、９３．４％、９２．７％、８９．３％及び８９．８％である。 The value of z / w in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The value of z / w in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8 and the amino acid sequence represented by SEQ ID NO: 70.1%, 66.1% and 70.0%. In addition, 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.
　（２－ｉ）の改変フィブロインは、配列番号６、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列からなるものであってもよい。 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.
　（２－ｉｉ）の改変フィブロインは、配列番号６、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（２－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 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.
　（２－ｉｉ）の改変フィブロインは、配列番号６、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＲＥＰ中に含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列中のＲＥＰの総アミノ酸残基数をｗとしたときに、ｚ／ｗが、好ましくは５０．９％以上である。 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.
　第２の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、改変フィブロインの単離、固定化、検出及び可視化等が可能となる。 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.
　タグ配列として、例えば、他の分子との特異的親和性（結合性、アフィニティ）を利用したアフィニティタグを挙げることができる。アフィニティタグの具体例として、ヒスチジンタグ（Ｈｉｓタグ）を挙げることができる。Ｈｉｓタグは、ヒスチジン残基が４から１０個程度並んだ短いペプチドで、ニッケル等の金属イオンと特異的に結合する性質があるため、金属キレートクロマトグラフィー（ｃｈｅｌａｔｉｎｇ　ｍｅｔａｌ　ｃｈｒｏｍａｔｏｇｒａｐｈｙ）による改変フィブロインの単離に利用することができる。タグ配列の具体例として、例えば、配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含むアミノ酸配列）が挙げられる。 As a tag sequence, for example, 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).
　また、グルタチオンに特異的に結合するグルタチオン－Ｓ－トランスフェラーゼ（ＧＳＴ）、マルトースに特異的に結合するマルトース結合タンパク質（ＭＢＰ）等のタグ配列を利用することもできる。 In addition, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can also be used.
　さらに、抗原抗体反応を利用した「エピトープタグ」を利用することもできる。抗原性を示すペプチド（エピトープ）をタグ配列として付加することにより、当該エピトープに対する抗体を結合させることができる。エピトープタグとして、ＨＡ（インフルエンザウイルスのヘマグルチニンのペプチド配列）タグ、ｍｙｃタグ、ＦＬＡＧタグ等を挙げることができる。エピトープタグを利用することにより、高い特異性で容易に改変フィブロインを精製することができる。 Furthermore, "epitope tags" utilizing antigen-antibody reactions can also be used. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like. By using an epitope tag, modified fibroin can be easily purified with high specificity.
　さらにタグ配列を特定のプロテアーゼで切り離せるようにしたものも使用することができる。当該タグ配列を介して吸着したタンパク質をプロテアーゼ処理することにより、タグ配列を切り離した改変フィブロインを回収することもできる。 Furthermore, those in which the tag sequence can be separated by a specific protease can also be used. The 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.
　タグ配列を含む改変フィブロインのより具体的な例として、（２－ｉｉｉ）配列番号１２（ＰＲＴ３８０）、配列番号１３（ＰＲＴ４１０）、配列番号１４（ＰＲＴ５２５）若しく配列番号１５（ＰＲＴ７９９）で示されるアミノ酸配列、又は（２－ｉｖ）配列番号１２、配列番号１３、配列番号１４若しくは配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 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.
　配列番号１６（ＰＲＴ３１３）、配列番号１２、配列番号１３、配列番号１４及び配列番号１５で示されるアミノ酸配列は、それぞれ配列番号１０、配列番号６、配列番号７、配列番号８及び配列番号９で示されるアミノ酸配列のＮ末端に配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。 The 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 The 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.
　（２－ｉｉｉ）の改変フィブロインは、配列番号１２、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列からなるものであってもよい。 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.
　（２－ｉｖ）の改変フィブロインは、配列番号１２、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（２－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 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.
　（２－ｉｖ）の改変フィブロインは、配列番号１２、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＲＥＰ中に含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列中のＲＥＰの総アミノ酸残基数をｗとしたときに、ｚ／ｗが５０．９％以上であることが好ましい。 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.
　第２の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 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.
　第３の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、（Ａ）ｎモチーフの含有量が低減されたアミノ酸配列を有する。第３の改変フィブロインのドメイン配列は、天然由来のフィブロインと比較して、少なくとも１又は複数の（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものということができる。 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.
　第３の改変フィブロインは、天然由来のフィブロインから（Ａ）ｎモチーフを１０～４０％欠失させたことに相当するアミノ酸配列を有するものであってもよい。 The third modified fibroin may have an amino acid sequence corresponding to 10-40% of the (A) n motif deleted from naturally occurring fibroin.
　第３の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、少なくともＮ末端側からＣ末端側に向かって１～３つの（Ａ）ｎモチーフ毎に１つの（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものであってもよい。 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
　第３の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、少なくともＮ末端側からＣ末端側に向かって２つ連続した（Ａ）ｎモチーフの欠失、及び１つの（Ａ）ｎモチーフの欠失がこの順に繰り返されたことに相当するアミノ酸配列を有するものであってもよい。 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.
　第３の改変フィブロインは、そのドメイン配列が、少なくともＮ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものであってもよい。 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 .
　第３の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、Ｎ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが２０％以上、３０％以上、４０％以上又は５０％以上であるアミノ酸配列を有するものであってもよい。（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数は８３％以上であってよいが、８６％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることが更に好ましく、１００％であること（アラニン残基のみで構成されることを意味する）が更により好ましい。 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 When the number of amino acid residues of the [REP] unit is sequentially compared, and the number of amino acid residues of the REP having a small number of amino acid residues is 1, 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 In addition, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The 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.
　ｘ／ｙの算出方法を図１を参照しながら更に詳細に説明する。図１には、改変フィブロインからＮ末端配列及びＣ末端配列を除いたドメイン配列を示す。当該ドメイン配列は、Ｎ末端側（左側）から（Ａ）ｎモチーフ－第１のＲＥＰ（５０アミノ酸残基）－（Ａ）ｎモチーフ－第２のＲＥＰ（１００アミノ酸残基）－（Ａ）ｎモチーフ－第３のＲＥＰ（１０アミノ酸残基）－（Ａ）ｎモチーフ－第４のＲＥＰ（２０アミノ酸残基）－（Ａ）ｎモチーフ－第５のＲＥＰ（３０アミノ酸残基）－（Ａ）ｎモチーフという配列を有する。 The method of calculating x / y will be described in more detail with reference to FIG. 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.
　隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットは、重複がないように、Ｎ末端側からＣ末端側に向かって、順次選択する。このとき、選択されない［（Ａ）ｎモチーフ－ＲＥＰ］ユニットが存在してもよい。図１には、パターン１（第１のＲＥＰと第２のＲＥＰの比較、及び第３のＲＥＰと第４のＲＥＰの比較）、パターン２（第１のＲＥＰと第２のＲＥＰの比較、及び第４のＲＥＰと第５のＲＥＰの比較）、パターン３（第２のＲＥＰと第３のＲＥＰの比較、及び第４のＲＥＰと第５のＲＥＰの比較）、パターン４（第１のＲＥＰと第２のＲＥＰの比較）を示した。なお、これ以外にも選択方法は存在する。 Two adjacent [(A) n motif-REP] units are sequentially selected from the N terminal side to the C terminal side so that there is no overlap. At this time, there may be [(A) n motif-REP] units not selected. In FIG. 1, pattern 1 (the comparison of the first REP and the second REP, and the comparison of the third REP and the fourth REP), the pattern 2 (the comparison of the first REP and the second REP, and Comparison of the fourth REP with the fifth REP), pattern 3 (comparison of the second REP with the third REP, and comparison of the fourth REP with the fifth REP), pattern 4 (with the first REP) Comparison of the second REP). There are other selection methods besides this.
　次に各パターンについて、選択した隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニット中の各ＲＥＰのアミノ酸残基数を比較する。比較は、よりアミノ酸残基数の少ない方を１としたときの、他方のアミノ酸残基数の比を求めることによって行う。例えば、第１のＲＥＰ（５０アミノ酸残基）と第２のＲＥＰ（１００アミノ酸残基）の比較の場合、よりアミノ酸残基数の少ない第１のＲＥＰを１としたとき、第２のＲＥＰのアミノ酸残基数の比は、１００／５０＝２である。同様に、第４のＲＥＰ（２０アミノ酸残基）と第５のＲＥＰ（３０アミノ酸残基）の比較の場合、よりアミノ酸残基数の少ない第４のＲＥＰを１としたとき、第５のＲＥＰのアミノ酸残基数の比は、３０／２０＝１．５である。 Next, for each pattern, 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. For example, in the case of comparison between the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is taken as 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparison between the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having a smaller number of amino acid residues is taken as 1, the fifth REP The ratio of the number of amino acid residues is 30/20 = 1.5.
　図１中、よりアミノ酸残基数の少ない方を１としたときに、他方のアミノ酸残基数の比が１．８～１１．３となる［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの組を実線で示した。本明細書中、この比をギザ比率と呼ぶ。よりアミノ酸残基数の少ない方を１としたときに、他方のアミノ酸残基数の比が１．８未満又は１１．３超となる［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの組は破線で示した。 In FIG. 1, assuming that the smaller number of amino acid residues is 1, the ratio of the number of other amino acid residues is 1.8 to 11.3. [(A) n Motif-REP] unit set It showed by a solid line. This ratio is referred to herein as the Giza ratio. When one having the smaller number of amino acid residues is 1, the ratio of the number of other amino acid residues is less than 1.8 or more than 11.3 [(A) n motif-REP] Indicated.
　各パターンにおいて、実線で示した隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの全てのアミノ酸残基数を足し合わせる（ＲＥＰのみではなく、（Ａ）ｎモチーフのアミノ酸残基数もである。）。そして、足し合わせた合計値を比較して、当該合計値が最大となるパターンの合計値（合計値の最大値）をｘとする。図１に示した例では、パターン１の合計値が最大である。 In 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.
　次に、ｘをドメイン配列の総アミノ酸残基数ｙで除すことによって、ｘ／ｙ（％）を算出することができる。 Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y of the domain sequence.
　第３の改変フィブロインにおいて、ｘ／ｙは、５０％以上であることが好ましく、６０％以上であることがより好ましく、６５％以上であることが更に好ましく、７０％以上であることが更により好ましく、７５％以上であることが更によりまた好ましく、８０％以上であることが特に好ましい。ｘ／ｙの上限に特に制限はなく、例えば、１００％以下であってよい。ギザ比率が１：１．９～１１．３の場合には、ｘ／ｙは８９．６％以上であることが好ましく、ギザ比率が１：１．８～３．４の場合には、ｘ／ｙは７７．１％以上であることが好ましく、ギザ比率が１：１．９～８．４の場合には、ｘ／ｙは７５．９％以上であることが好ましく、ギザ比率が１：１．９～４．１の場合には、ｘ／ｙは６４．２％以上であることが好ましい。 In the third modified fibroin, 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. There is no particular limitation on the upper limit of x / y, and it may be, for example, 100% or less. In the case of a Giza ratio of 1: 1.9 to 11.3, 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.
　第３の改変フィブロインが、ドメイン配列中に複数存在する（Ａ）ｎモチーフの少なくとも７つがアラニン残基のみで構成される改変フィブロインである場合、ｘ／ｙは、４６．４％以上であることが好ましく、５０％以上であることがより好ましく、５５％以上であることが更に好ましく、６０％以上であることが更により好ましく、７０％以上であることが更によりまた好ましく、８０％以上であることが特に好ましい。ｘ／ｙの上限に特に制限はなく、１００％以下であればよい。 When the third modified fibroin is a modified fibroin in which at least seven of the (A) n motifs in the domain sequence are composed of only alanine residues, 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.
　ここで、天然由来のフィブロインにおけるｘ／ｙについて説明する。まず、上述のように、ＮＣＢＩ　ＧｅｎＢａｎｋにアミノ酸配列情報が登録されているフィブロインを例示した方法により確認したところ、６６３種類のフィブロイン（このうち、クモ類由来のフィブロインは４１５種類）が抽出された。抽出された全てのフィブロインのうち、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列で構成される天然由来のフィブロインのアミノ酸配列から、上述の算出方法により、ｘ／ｙを算出した。ギザ比率が１：１．９～４．１の場合の結果を図３に示す。 Here, x / y in naturally occurring fibroin will be described. First, as described above, when confirmed by the method exemplifying fibroin whose amino acid sequence information is registered in NCBI GenBank, 663 types of fibroin (of which 415 types of fibroin derived from spiders) were extracted. From the amino acid sequence of naturally derived fibroin composed of the domain sequence represented by the formula 1: [(A) n motif-REP] m among all the fibroins extracted, x / y according to the above-mentioned calculation method Was calculated. The results for the Giza ratio of 1: 1.9 to 4.1 are shown in FIG.
　図３の横軸はｘ／ｙ（％）を示し、縦軸は頻度を示す。図３から明らかなとおり、天然由来のフィブロインにおけるｘ／ｙは、いずれも６４．２％未満である（最も高いもので、６４．１４％）。 The horizontal axis of FIG. 3 indicates x / y (%) and the vertical axis indicates frequency. As apparent from FIG. 3, x / y in naturally derived fibroin is less than 64.2% in all cases (highest, 64.14%).
　第３の改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列から、ｘ／ｙが６４．２％以上になるように（Ａ）ｎモチーフをコードする配列の１又は複数を欠失させることにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から、ｘ／ｙが６４．２％以上になるように１又は複数の（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列から（Ａ）ｎモチーフが欠失したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 The third modified fibroin, for example, 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. In any case, in addition to the modification corresponding to the deletion of the (A) n motif from the amino acid sequence of naturally derived fibroin, 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.
　第３の改変フィブロインのより具体的な例として、（３－ｉ）配列番号１７（Ｍｅｔ－ＰＲＴ３９９）、配列番号７（Ｍｅｔ－ＰＲＴ４１０）、配列番号８（Ｍｅｔ－ＰＲＴ５２５）若しくは配列番号９（Ｍｅｔ－ＰＲＴ７９９）で示されるアミノ酸配列、又は（３－ｉｉ）配列番号１７、配列番号７、配列番号８若しくは配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of 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 amino acid sequence represented by -PRT 799) or (3-ii) having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9; Modified fibroin can be mentioned.
　（３－ｉ）の改変フィブロインについて説明する。配列番号１７で示されるアミノ酸配列は、天然由来のフィブロインに相当する配列番号１０（Ｍｅｔ－ＰＲＴ３１３）で示されるアミノ酸配列から、Ｎ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフを欠失させ、更にＣ末端配列の手前に［（Ａ）ｎモチーフ－ＲＥＰ］を１つ挿入したものである。配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列は、第２の改変フィブロインで説明したとおりである。 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.
　配列番号１０で示されるアミノ酸配列（天然由来のフィブロインに相当）のギザ比率１：１．８～１１．３におけるｘ／ｙの値は１５．０％である。配列番号１７で示されるアミノ酸配列、及び配列番号７で示されるアミノ酸配列におけるｘ／ｙの値は、いずれも９３．４％である。配列番号８で示されるアミノ酸配列におけるｘ／ｙの値は、９２．７％である。配列番号９で示されるアミノ酸配列におけるｘ／ｙの値は、８９．８％である。配列番号１０、配列番号１７、配列番号７、配列番号８及び配列番号９で示されるアミノ酸配列におけるｚ／ｗの値は、それぞれ４６．８％、５６．２％、７０．１％、６６．１％及び７０．０％である。 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%.
　（３－ｉ）の改変フィブロインは、配列番号１７、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列からなるものであってもよい。 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.
　（３－ｉｉ）の改変フィブロインは、配列番号１７、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（３－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 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.
　（３－ｉｉ）の改変フィブロインは、配列番号１７、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＮ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３（ギザ比率が１：１．８～１１．３）となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが６４．２％以上であることが好ましい。 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.
　第３の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方に上述したタグ配列を含んでいてもよい。 The third modified fibroin may contain the above-described tag sequence at either or both of the N-terminus and the C-terminus.
　タグ配列を含む改変フィブロインのより具体的な例として、（３－ｉｉｉ）配列番号１８（ＰＲＴ３９９）、配列番号１３（ＰＲＴ４１０）、配列番号１４（ＰＲＴ５２５）若しくは配列番号１５（ＰＲＴ７９９）で示されるアミノ酸配列、又は（３－ｉｖ）配列番号１８、配列番号１３、配列番号１４若しくは配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As a more specific example of the 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 .
　配列番号１８、配列番号１３、配列番号１４及び配列番号１５で示されるアミノ酸配列は、それぞれ配列番号１７、配列番号７、配列番号８及び配列番号９で示されるアミノ酸配列のＮ末端に配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。 The 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.
　（３－ｉｉｉ）の改変フィブロインは、配列番号１８、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列からなるものであってもよい。 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.
　（３－ｉｖ）の改変フィブロインは、配列番号１８、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（３－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 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.
　（３－ｉｖ）の改変フィブロインは、配列番号１８、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＮ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが６４．２％以上であることが好ましい。 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. Preferably, x / y is 64.2% or more, where y is the total number of amino acid residues in the domain sequence.
　第３の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 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.
　第４の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、（Ａ）ｎモチーフの含有量が低減されたことに加え、グリシン残基の含有量が低減されたアミノ酸配列を有するものである。第４の改変フィブロインのドメイン配列は、天然由来のフィブロインと比較して、少なくとも１又は複数の（Ａ）ｎモチーフが欠失したことに加え、更に少なくともＲＥＰ中の１又は複数のグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものということができる。すなわち、第４の改変フィブロインは、上述した第２の改変フィブロインと、第３の改変フィブロインと、の両方の特徴を併せ持つ改変フィブロインである。具体的な態様等は、第２の改変フィブロイン、、及び第３の改変フィブロインで説明したとおりである。 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.
　第４の改変フィブロインのより具体的な例として、（４－ｉ）配列番号７（Ｍｅｔ－ＰＲＴ４１０）、配列番号８（Ｍｅｔ－ＰＲＴ５２５）、配列番号９（Ｍｅｔ－ＰＲＴ７９９）、配列番号１３（ＰＲＴ４１０）、配列番号１４（ＰＲＴ５２５）若しくは配列番号１５（ＰＲＴ７９９）で示されるアミノ酸配列、又は（４－ｉｉ）配列番号７、配列番号８、配列番号９、配列番号１３、配列番号１４若しくは配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。配列番号７、配列番号８、配列番号９、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列を含む改変フィブロインの具体的な態様は上述のとおりである。 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 There may be mentioned 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.
　第５の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する、局所的に疎水性指標の大きい領域を含むアミノ酸配列を有するものであってよい。 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.
　局所的に疎水性指標の大きい領域は、連続する２～４アミノ酸残基で構成されていることが好ましい。 The region locally having a large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
　上述の疎水性指標の大きいアミノ酸残基は、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）から選ばれるアミノ酸残基であることがより好ましい。 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.
　第５の改変フィブロインは、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に、天然由来のフィブロインと比較して、１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変があってもよい。 In the fifth modified fibroin, 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. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, 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.
　第５の改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列からＲＥＰ中の１又は複数の親水性アミノ酸残基（例えば、疎水性指標がマイナスであるアミノ酸残基）を疎水性アミノ酸残基（例えば、疎水性指標がプラスであるアミノ酸残基）に置換すること、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数の親水性アミノ酸残基を疎水性アミノ酸残基に置換したこと、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数の親水性アミノ酸残基を疎水性アミノ酸残基に置換したこと、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 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. It 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. In any case, 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 In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.
　第５の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を上記ドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を上記ドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であるアミノ酸配列を有してもよい。 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, When the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located most C-terminal to the C-terminus of the domain sequence from the above domain sequence is q, p / q is 6 And may have an amino acid sequence that is 2% or more.
　アミノ酸残基の疎水性指標については、公知の指標（Ｈｙｄｒｏｐａｔｈｙ　ｉｎｄｅｘ：Ｋｙｔｅ　Ｊ，＆Ｄｏｏｌｉｔｔｌｅ　Ｒ（１９８２）“Ａ　ｓｉｍｐｌｅ　ｍｅｔｈｏｄ　ｆｏｒ　ｄｉｓｐｌａｙｉｎｇ　ｔｈｅ　ｈｙｄｒｏｐａｔｈｉｃ　ｃｈａｒａｃｔｅｒ　ｏｆ　ａ　ｐｒｏｔｅｉｎ”，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１５７，ｐｐ．１０５－１３２）を使用する。具体的には、各アミノ酸の疎水性指標（ハイドロパシー・インデックス、以下「ＨＩ」とも記す。）は、下記表１に示すとおりである。 With regard to the 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). Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as "HI") of each amino acid is as shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　ｐ／ｑの算出方法を更に詳細に説明する。算出には、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列（以下、「配列Ａ」とする）を用いる。まず、配列Ａに含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値を算出する。疎水性指標の平均値は、連続する４アミノ酸残基に含まれる各アミノ酸残基のＨＩの総和を４（アミノ酸残基数）で除して求める。疎水性指標の平均値は、全ての連続する４アミノ酸残基について求める（各アミノ酸残基は、１～４回平均値の算出に用いられる。）。次いで、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域を特定する。あるアミノ酸残基が、複数の「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」に該当する場合であっても、領域中には１アミノ酸残基として含まれることになる。そして、当該領域に含まれるアミノ酸残基の総数がｐである。また、配列Ａに含まれるアミノ酸残基の総数がｑである。 The method of calculating p / q will be described in more detail. In the calculation, the sequence from the domain sequence represented by the formula 1: [(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. First, in all REPs included in 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.
　例えば、「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が２０カ所抽出された場合（重複はなし）、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域には、連続する４アミノ酸残基（重複はなし）が２０含まれることになり、ｐは２０×４＝８０である。また、例えば、２つの「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が１アミノ酸残基だけ重複して存在する場合、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域には、７アミノ酸残基含まれることになる（ｐ＝２×４－１＝７。「－１」は重複分の控除である。）。例えば、図４に示したドメイン配列の場合、「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が重複せずに７つ存在するため、ｐは７×４＝２８となる。また、例えば、図４に示したドメイン配列の場合、ｑは４＋５０＋４＋４０＋４＋１０＋４＋２０＋４＋３０＝１７０である（Ｃ末端側の最後に存在する（Ａ）ｎモチーフは含めない）。次に、ｐをｑで除すことによって、ｐ／ｑ（％）を算出することができる。図４の場合２８／１７０＝１６．４７％となる。 For example, when 20 consecutive 4 amino acid residues in which the average value of the hydrophobicity index is 2.6 or more are extracted (without duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2 The region of .6 or more contains 20 consecutive 4 amino acid residues (without duplication), and p is 20 × 4 = 80. Also, for example, when two “four consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” overlap by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues is determined In the region where the average value of is 2.6 or more, 7 amino acid residues are included (p = 2 × 4-1 = 7. “−1” is a subtraction of the overlap). For example, in the case of the domain sequence shown in FIG. 4, p is 7 × 4 = because 7 consecutive 4 amino acid residues for which the average value of the hydrophobicity index is 2.6 or more do not overlap. It will be 28. Also, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (does not include the (A) n motif present at the C-terminal end). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.
　第５の改変フィブロインにおいて、ｐ／ｑは、６．２％以上であることが好ましく、７％以上であることがより好ましく、１０％以上であることが更に好ましく、２０％以上であることが更により好ましく、３０％以上であることが更によりまた好ましい。ｐ／ｑの上限は、特に制限されないが、例えば、４５％以下であってもよい。 In the fifth modified fibroin, 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.
　第５の改変フィブロインは、例えば、クローニングした天然由来のフィブロインのアミノ酸配列を、上記のｐ／ｑの条件を満たすように、ＲＥＰ中の１又は複数の親水性アミノ酸残基（例えば、疎水性指標がマイナスであるアミノ酸残基）を疎水性アミノ酸残基（例えば、疎水性指標がプラスであるアミノ酸残基）に置換すること、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入することにより、局所的に疎水性指標の大きい領域を含むアミノ酸配列に改変することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から上記のｐ／ｑの条件を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当する改変を行ってもよい。 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. Substitution of a negative amino acid residue with a hydrophobic amino acid residue (eg, an amino acid residue with a positive hydrophobicity index) and / or 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. Alternatively, for example, it can be obtained by designing an amino acid sequence satisfying the above p / q condition from the amino acid sequence of naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, 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 In addition to the modification corresponding to insertion of an amino acid residue having a large hydrophobicity index, modification corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. .
　疎水性指標の大きいアミノ酸残基としては、特に制限はないが、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）が好ましく、バリン（Ｖ）、ロイシン（Ｌ）及びイソロイシン（Ｉ）がより好ましい。 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.
　第５の改変フィブロインのより具体的な例として、（５－ｉ）配列番号１９（Ｍｅｔ－ＰＲＴ７２０）、配列番号２０（Ｍｅｔ－ＰＲＴ６６５）若しくは配列番号２１（Ｍｅｔ－ＰＲＴ６６６）で示されるアミノ酸配列、又は（５－ｉｉ）配列番号１９、配列番号２０若しくは配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of 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.
　（５－ｉ）の改変フィブロインについて説明する。配列番号１９で示されるアミノ酸配列は、配列番号７（Ｍｅｔ－ＰＲＴ４１０）で示されるアミノ酸配列に対し、Ｃ末端側の端末のドメイン配列を除いて、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を２カ所挿入し、更に一部のグルタミン（Ｑ）残基をセリン（Ｓ）残基に置換し、かつＣ末端側の一部のアミノ酸を欠失させたものである。配列番号２０で示されるアミノ酸配列は、配列番号８（Ｍｅｔ－ＰＲＴ５２５）で示されるアミノ酸配列に対し、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を１カ所挿入したものである。配列番号２１で示されるアミノ酸配列は、配列番号８で示されるアミノ酸配列に対し、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を２カ所挿入したものである。 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.
　（５－ｉ）の改変フィブロインは、配列番号１９、配列番号２０又は配列番号２１で示されるアミノ酸配列からなるものであってもよい。 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.
　（５－ｉｉ）の改変フィブロインは、配列番号１９、配列番号２０又は配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（５－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 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.
　（５－ｉｉ）の改変フィブロインは、配列番号１９、配列番号２０又は配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつ最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であることが好ましい。 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.
　第５の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。 The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.
　タグ配列を含む改変フィブロインのより具体的な例として、（５－ｉｉｉ）配列番号２２（ＰＲＴ７２０）、配列番号２３（ＰＲＴ６６５）若しくは配列番号２４（ＰＲＴ６６６）で示されるアミノ酸配列、又は（５－ｉｖ）配列番号２２、配列番号２３若しくは配列番号２４で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 More specific examples of the modified fibroin containing the tag sequence 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).
　配列番号２２、配列番号２３及び配列番号２４で示されるアミノ酸配列は、それぞれ配列番号１９、配列番号２０及び配列番号２１で示されるアミノ酸配列のＮ末端に配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。 The 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).
　（５－ｉｉｉ）の改変フィブロインは、配列番号２２、配列番号２３又は配列番号２４で示されるアミノ酸配列からなるものであってもよい。 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.
　（５－ｉｖ）の改変フィブロインは、配列番号２２、配列番号２３又は配列番号２４で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（５－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 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.
　（５－ｉｖ）の改変フィブロインは、配列番号２２、配列番号２３又は配列番号２４で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつ最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であることが好ましい。 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.
　第５の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 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.
　第６の改変フィブロインは、天然由来のフィブロインと比較して、グルタミン残基の含有量が低減されたアミノ酸配列を有する。 The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues as compared to naturally occurring fibroin.
　第６の改変フィブロインは、ＲＥＰのアミノ酸配列中に、ＧＧＸモチーフ及びＧＰＧＸＸモチーフから選ばれる少なくとも一つのモチーフが含まれていることが好ましい。 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.
　第６の改変フィブロインが、ＲＥＰ中にＧＰＧＸＸモチーフを含む場合、ＧＰＧＸＸモチーフ含有率は、通常１％以上であり、５％以上であってもよく、１０％以上であるのが好ましい。ＧＰＧＸＸモチーフ含有率の上限に特に制限はなく、５０％以下であってよく、３０％以下であってもよい。 When the sixth modified fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.
　本明細書において、「ＧＰＧＸＸモチーフ含有率」は、以下の方法により算出される値である。
　式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロイン（改変フィブロイン又は天然由来のフィブロイン）において、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、その領域に含まれるＧＰＧＸＸモチーフの個数の総数を３倍した数（即ち、ＧＰＧＸＸモチーフ中のＧ及びＰの総数に相当）をｓとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｔとしたときに、ＧＰＧＸＸモチーフ含有率はｓ／ｔとして算出される。 In the present specification, “GPGXX motif content” is a value calculated by the following method.
Formula 1: [(A) n Motif -REP] m , or Formula 2: [(A) n Motif -REP] m- (A) fibroin containing a domain sequence represented by n motif (modified fibroin or naturally derived In fibroin), the number of GPGXX motifs contained in the region 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. Be
　ＧＰＧＸＸモチーフ含有率の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としているのは、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列」（ＲＥＰに相当する配列）には、フィブロインに特徴的な配列と相関性の低い配列が含まれることがあり、ｍが小さい場合（つまり、ドメイン配列が短い場合）、ＧＰＧＸＸモチーフ含有率の算出結果に影響するので、この影響を排除するためである。なお、ＲＥＰのＣ末端に「ＧＰＧＸＸモチーフ」が位置する場合、「ＸＸ」が例えば「ＡＡ」の場合であっても、「ＧＰＧＸＸモチーフ」として扱う。 In the calculation of 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. When “GPGXX motif” is located at the C-terminal of REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.
　図５は、改変フィブロインのドメイン配列を示す模式図である。図５を参照しながらＧＰＧＸＸモチーフ含有率の算出方法を具体的に説明する。まず、図５に示した改変フィブロインのドメイン配列（「［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフ」タイプである。）では、全てのＲＥＰが「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」（図５中、「領域Ａ」で示した配列。）に含まれているため、ｓを算出するためのＧＰＧＸＸモチーフの個数は７であり、ｓは７×３＝２１となる。同様に、全てのＲＥＰが「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」（図５中、「領域Ａ」で示した配列。）に含まれているため、当該配列から更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数ｔは５０＋４０＋１０＋２０＋３０＝１５０である。次に、ｓをｔで除すことによって、ｓ／ｔ（％）を算出することができ、図５の改変フィブロインの場合２１／１５０＝１４．０％となる。 FIG. 5 is a schematic view showing the domain sequence of modified fibroin. The method of calculating the GPGXX motif content rate will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin ("[(A) n Motif -REP] m- (A) n Motif" type) shown in FIG. 5, all the REPs are located "most C-terminally (A) A sequence obtained by removing the sequence from the n motif to the C terminus of the domain sequence from the domain sequence "(the sequence shown in" region A "in FIG. 5), so that s can be calculated The number of GPGXX motifs is 7, and s is 7 × 3 = 21. Similarly, 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 total number t of the amino acid residues of all the REP from which the (A) n motif has been further removed from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t, and it becomes 21/150 = 14.0% in the case of the modified fibroin of FIG.
　第６の改変フィブロインは、グルタミン残基含有率が９％以下であることが好ましく、７％以下であることがより好ましく、４％以下であることが更に好ましく、０％であることが特に好ましい。 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%. .
　本明細書において、「グルタミン残基含有率」は、以下の方法により算出される値である。
　式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロイン（改変フィブロイン又は天然由来のフィブロイン）において、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列（図５の「領域Ａ」に相当する配列。）に含まれる全てのＲＥＰにおいて、その領域に含まれるグルタミン残基の総数をｕとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｔとしたときに、グルタミン残基含有率はｕ／ｔとして算出される。グルタミン残基含有率の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, “glutamine residue content” is a value calculated by the following method.
Formula 1: [(A) n Motif -REP] m , or Formula 2: [(A) n Motif -REP] m- (A) fibroin containing a domain sequence represented by n motif (modified fibroin or naturally derived 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). in REP, then 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. In the calculation of glutamine residue content, 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.
　第６の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のグルタミン残基を欠失したこと、又は他のアミノ酸残基に置換したことに相当するアミノ酸配列を有するものであってよい。 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.
　「他のアミノ酸残基」は、グルタミン残基以外のアミノ酸残基であればよいが、グルタミン残基よりも疎水性指標の大きいアミノ酸残基であることが好ましい。アミノ酸残基の疎水性指標は表１に示すとおりである。 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.
　表１に示すとおり、グルタミン残基よりも疎水性指標の大きいアミノ酸残基としては、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）アラニン（Ａ）、グリシン（Ｇ）、スレオニン（Ｔ）、セリン（Ｓ）、トリプトファン（Ｗ）、チロシン（Ｙ）、プロリン（Ｐ）及びヒスチジン（Ｈ）から選ばれるアミノ酸残基を挙げることができる。これらの中でも、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）から選ばれるアミノ酸残基であることがより好ましく、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）及びフェニルアラニン（Ｆ）から選ばれるアミノ酸残基であることが更に好ましい。 As shown in Table 1, as amino acid residues having a larger hydrophobicity index than glutamine residues, isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) ) To mention amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H) it can. Among these, it is more preferable that 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).
　第６の改変フィブロインは、ＲＥＰの疎水性度が、－０．８以上であることが好ましく、－０．７以上であることがより好ましく、０以上であることが更に好ましく、０．３以上であることが更により好ましく、０．４以上であることが特に好ましい。ＲＥＰの疎水性度の上限に特に制限はなく、１．０以下であってよく、０．７以下であってもよい。 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.
　本明細書において、「ＲＥＰの疎水性度」は、以下の方法により算出される値である。
　式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロイン（改変フィブロイン又は天然由来のフィブロイン）において、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列（図５の「領域Ａ」に相当する配列。）に含まれる全てのＲＥＰにおいて、その領域の各アミノ酸残基の疎水性指標の総和をｖとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｔとしたときに、ＲＥＰの疎水性度はｖ／ｔとして算出される。ＲＥＰの疎水性度の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, “the hydrophobicity of REP” is a value calculated by the following method.
Formula 1: [(A) n Motif -REP] m , or Formula 2: [(A) n Motif -REP] m- (A) fibroin containing a domain sequence represented by n motif (modified fibroin or naturally derived 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). In the REP, the total of the hydrophobicity index of each amino acid residue in the region is v, and 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 A) 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.
　第６の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のグルタミン残基を欠失したこと、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変があってもよい。 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 In addition to 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. .
　第６の改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列からＲＥＰ中の１又は複数のグルタミン残基を欠失させること、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数のグルタミン残基を欠失したこと、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。 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.
　第６の改変フィブロインのより具体的な例として、（６－ｉ）配列番号２５（Ｍｅｔ－ＰＲＴ８８８）、配列番号２６（Ｍｅｔ－ＰＲＴ９６５）、配列番号２７（Ｍｅｔ－ＰＲＴ８８９）、配列番号２８（Ｍｅｔ－ＰＲＴ９１６）、配列番号２９（Ｍｅｔ－ＰＲＴ９１８）、配列番号３０（Ｍｅｔ－ＰＲＴ６９９）、配列番号３１（Ｍｅｔ－ＰＲＴ６９８）若しくは配列番号３２（Ｍｅｔ－ＰＲＴ９６６）で示されるアミノ酸配列を含む改変フィブロイン、又は（６－ｉｉ）配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１若しくは配列番号３２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む改変フィブロインを挙げることができる。 As a more specific example of the sixth modified fibroin, (6-i) 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 or SEQ ID NO: 32 Mention may be made of modified fibroin which comprises the amino acid sequence it has.
　（６－ｉ）の改変フィブロインについて説明する。配列番号２５で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）中のＱＱを全てＶＬに置換したものである。配列番号２６で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中のＱＱを全てＴＳに置換し、かつ残りのＱをＡに置換したものである。配列番号２７で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中のＱＱを全てＶＬに置換し、かつ残りのＱをＩに置換したものである。配列番号２８で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中のＱＱを全てＶＩに置換し、かつ残りのＱをＬに置換したものである。配列番号２９で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中のＱＱを全てＶＦに置換し、かつ残りのＱをＩに置換したものである。 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.
　配列番号３０で示されるアミノ酸配列は、配列番号８で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ５２５）中のＱＱを全てＶＬに置換したものである。配列番号３１で示されるアミノ酸配列は、配列番号８で示されるアミノ酸配列中のＱＱを全てＶＬに置換し、かつ残りのＱをＩに置換したものである。 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.
　配列番号３２で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）中に存在する２０個のドメイン配列の領域を２回繰り返した配列中のＱＱを全てＶＦに置換し、かつ残りのＱをＩに置換したものである。 The 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.
　配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１及び配列番号３２で示されるアミノ酸配列は、いずれもグルタミン残基含有率は９％以下である（表２）。 The 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).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　（６－ｉ）の改変フィブロインは、配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１又は配列番号３２で示されるアミノ酸配列からなるものであってもよい。 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.
　（６－ｉｉ）の改変フィブロインは、配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１又は配列番号３２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（６－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 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.
　（６－ｉｉ）の改変フィブロインは、グルタミン残基含有率が９％以下であることが好ましい。また、（６－ｉｉ）の改変フィブロインは、ＧＰＧＸＸモチーフ含有率が１０％以上であることが好ましい。 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.
　第６の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、改変フィブロインの単離、固定化、検出及び可視化等が可能となる。 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.
　タグ配列を含む改変フィブロインのより具体的な例として、（６－ｉｉｉ）配列番号３３（ＰＲＴ８８８）、配列番号３４（ＰＲＴ９６５）、配列番号３５（ＰＲＴ８８９）、配列番号３６（ＰＲＴ９１６）、配列番号３７（ＰＲＴ９１８）、配列番号３８（ＰＲＴ６９９）、配列番号３９（ＰＲＴ６９８）若しくは配列番号４０（ＰＲＴ９６６）で示されるアミノ酸配列を含む改変フィブロイン、又は（６－ｉｖ）配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９若しくは配列番号４０で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む改変フィブロインを挙げることができる。 More specific examples of the 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.
　配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９及び配列番号４０で示されるアミノ酸配列は、それぞれ配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１及び配列番号３２で示されるアミノ酸配列のＮ末端に配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。Ｎ末端にタグ配列を付加しただけであるため、グルタミン残基含有率に変化はなく、配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９及び配列番号４０で示されるアミノ酸配列は、いずれもグルタミン残基含有率が９％以下である（表３）。 The 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 (including His tag sequence and hinge sequence) was added to the N terminus of the amino acid sequence shown in It is a thing. There is no change in the glutamine residue content, since only the tag sequence is added to the N terminus, and 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 And the amino acid sequence shown by SEQ ID NO: 40 has a glutamine residue content of 9% or less (Table 3).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　（６－ｉｉｉ）の改変フィブロインは、配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９又は配列番号４０で示されるアミノ酸配列からなるものであってもよい。 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.
　（６－ｉｖ）の改変フィブロインは、配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９又は配列番号４０で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（６－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 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.
　（６－ｉｖ）の改変フィブロインは、グルタミン残基含有率が９％以下であることが好ましい。また、（６－ｉｖ）の改変フィブロインは、ＧＰＧＸＸモチーフ含有率が１０％以上であることが好ましい。 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.
　第６の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 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.
　改変フィブロインは、第１の改変フィブロイン、第２の改変フィブロイン、第３の改変フィブロイン、第４の改変フィブロイン、第５の改変フィブロイン、及び第６の改変フィブロインが有する特徴のうち、少なくとも２つ以上の特徴を併せ持つ改変フィブロインであってもよい。 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
＜改変フィブロインの製造方法＞
　改変フィブロインは、例えば、目的とする改変フィブロインをコードする核酸配列と、当該核酸配列に作動可能に連結された１又は複数の調節配列とを有する発現ベクターで形質転換された宿主により、当該核酸を発現させることにより生産したものを用いることができる。 <Method of producing modified fibroin>
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.
　目的とする改変フィブロインをコードする核酸の製造方法は特に制限されない。例えば、天然のフィブロインをコードする遺伝子を利用して、ポリメラーゼ連鎖反応（ＰＣＲ）などで増幅しクローニングし、遺伝子工学的手法により改変する方法、又は、化学的に合成する方法によって、当該核酸を製造することができる。核酸の化学的な合成方法も特に制限されず、例えば、ＮＣＢＩのウェブデータベースなどより入手したフィブロインのアミノ酸配列情報をもとに、ＡＫＴＡ　ｏｌｉｇｏｐｉｌｏｔ　ｐｌｕｓ　１０／１００（ＧＥヘルスケア・ジャパン株式会社製）などで自動合成したオリゴヌクレオチドをＰＣＲなどで連結する方法によって核酸を化学的に合成することができる。この際に、改変フィブロインの精製や確認を容易にするため、上記のアミノ酸配列のＮ末端に開始コドン及びＨｉｓ１０タグからなるアミノ酸配列を付加したアミノ酸配列からなるタンパク質、をコードする核酸を合成してもよい。 There are no particular limitations on the method of producing the nucleic acid encoding the target modified fibroin. For example, 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.
　調節配列は、宿主における組換えタンパク質の発現を制御する配列（例えば、プロモーター、エンハンサー、リボソーム結合配列、転写終結配列等）であり、宿主の種類に応じて適宜選択することができる。プロモーターとして、宿主細胞中で機能し、目的とする改変フィブロインを発現誘導可能な誘導性プロモーターを用いてもよい。誘導性プロモーターは、誘導物質（発現誘導剤）の存在、リプレッサー分子の非存在、又は温度、浸透圧若しくはｐＨ値の上昇若しくは低下等の物理的要因により、転写を制御できるプロモーターである。 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. As 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.
　発現ベクターの種類は、プラスミドベクター、ウイルスベクター、コスミドベクター、フォスミドベクター、人工染色体ベクター等であってよく、宿主の種類に応じて適宜選択することができる。発現ベクターとしては、宿主細胞において自立複製が可能、又は宿主の染色体中への組込みが可能で、目的とするタンパク質をコードする核酸を転写できる位置にプロモーターを含有しているものが好適に用いられる。 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. As the expression vector, 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. .
　宿主として、原核生物、並びに酵母、糸状真菌、昆虫細胞、動物細胞及び植物細胞等の真核生物のいずれも好適に用いることができる。 As a host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.
　原核生物の好ましい例として、エシェリヒア属、ブレビバチルス属、セラチア属、バチルス属、ミクロバクテリウム属、ブレビバクテリウム属、コリネバクテリウム属及びシュードモナス属等に属する細菌を挙げることができる。エシェリヒア属に属する微生物として、例えば、エシェリヒア・コリ等を挙げることができる。ブレビバチルス属に属する微生物として、例えば、ブレビバチルス・アグリ等を挙げることができる。セラチア属に属する微生物として、例えば、セラチア・リクエファシエンス等を挙げることができる。バチルス属に属する微生物として、例えば、バチルス・サチラス等を挙げることができる。ミクロバクテリウム属に属する微生物として、例えば、ミクロバクテリウム・アンモニアフィラム等を挙げることができる。ブレビバクテリウム属に属する微生物として、例えば、ブレビバクテリウム・ディバリカタム等を挙げることができる。コリネバクテリウム属に属する微生物として、例えば、コリネバクテリウム・アンモニアゲネス等を挙げることができる。シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属に属する微生物として、例えば、シュードモナス・プチダ等を挙げることができる。 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. Examples of microorganisms belonging to the genus Serratia include Serratia liquofaciens and the like. As a microorganism belonging to the genus Bacillus, for example, Bacillus subtilis and the like can be mentioned. Examples of the microorganism belonging to the genus Microbacterium include, for example, Microbacterium ammoniafilum and the like. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes and the like. As a microorganism belonging to the genus Pseudomonas, for example, Pseudomonas putida etc. can be mentioned.
　原核生物を宿主とする場合、目的とする改変フィブロインをコードする核酸を導入するベクターとしては、例えば、ｐＢＴｒｐ２（ベーリンガーマンハイム社製）、ｐＧＥＸ（Ｐｈａｒｍａｃｉａ社製）、ｐＵＣ１８、ｐＢｌｕｅｓｃｒｉｐｔＩＩ、ｐＳｕｐｅｘ、ｐＥＴ２２ｂ、ｐＣｏｌｄ、ｐＵＢ１１０、ｐＮＣＯ２（特開２００２－２３８５６９号公報）等を挙げることができる。 When a prokaryote is used as a host, 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.
　真核生物の宿主としては、例えば、酵母及び糸状真菌（カビ等）を挙げることができる。酵母としては、例えば、サッカロマイセス属、ピキア属、シゾサッカロマイセス属等に属する酵母を挙げることができる。糸状真菌としては、例えば、アスペルギルス属、ペニシリウム属、トリコデルマ（Ｔｒｉｃｈｏｄｅｒｍａ）属等に属する糸状真菌を挙げることができる。 Eukaryotic hosts can include, for example, yeast and filamentous fungi (molds and the like). As a yeast, the yeast which belongs to Saccharomyces genus, Pichia genus, Schizosaccharomyces genus etc. can be mentioned, for example. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
　真核生物を宿主とする場合、目的とする改変フィブロインをコードする核酸を導入するベクターとしては、例えば、ＹＥｐ１３（ＡＴＣＣ３７１１５）、ＹＥｐ２４（ＡＴＣＣ３７０５１）等を挙げることができる。 When a eukaryote is used as a host, 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.
　上記宿主細胞への発現ベクターの導入方法としては、上記宿主細胞へＤＮＡを導入する方法であればいずれも用いることができる。例えば、カルシウムイオンを用いる方法〔Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ，６９，２１１０　（１９７２）〕、エレクトロポレーション法、スフェロプラスト法、プロトプラスト法、酢酸リチウム法、コンピテント法等を挙げることができる。 As a method of introducing the expression vector into the host cell, any method of introducing DNA into the host cell can be used. For example, a method using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like.
　発現ベクターで形質転換された宿主による核酸の発現方法としては、直接発現のほか、モレキュラー・クローニング第２版に記載されている方法等に準じて、分泌生産、融合タンパク質発現等を行うことができる。 As 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. .
　目的とする改変フィブロインは、例えば、発現ベクターで形質転換された宿主を培養培地中で培養し、培養培地中に当該改変フィブロインを生成及び蓄積させ、該培養培地から採取することにより製造することができる。宿主を培養培地中で培養する方法は、宿主の培養に通常用いられる方法に従って行うことができる。 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.
　宿主が大腸菌等の原核生物又は酵母等の真核生物である場合、宿主の培養培地として、該宿主が資化し得る炭素源、窒素源及び無機塩類等を含有し、該宿主の培養を効率的に行える培地であれば天然培地、及び合成培地のいずれを用いてもよい。 When the host is a prokaryote such as E. coli or a eukaryote such as yeast, 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. Either a natural medium or a synthetic medium may be used as long as the medium can be used.
　炭素源としては、上記形質転換された宿主が資化し得るものであればよく、例えば、グルコース、フラクトース、スクロース、及びこれらを含有する糖蜜、デンプン及びデンプン加水分解物等の炭水化物、酢酸及びプロピオン酸等の有機酸、並びにエタノール及びプロパノール等のアルコール類などを用いることができる。 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.
　窒素源としては、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム及びリン酸アンモニウム等の無機酸又は有機酸のアンモニウム塩、その他の含窒素化合物、並びにペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼイン加水分解物、大豆粕及び大豆粕加水分解物、各種発酵菌体及びその消化物などを用いることができる。 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.
　無機塩としては、例えば、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅及び炭酸カルシウムなどを用いることができる。 As the inorganic salt, for example, potassium monophosphate, potassium monobasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
　大腸菌等の原核生物又は酵母等の真核生物の培養は、例えば、振盪培養又は深部通気攪拌培養等の好気的条件下で行うことができる。培養温度は、例えば、１５～４０℃である。培養時間は、通常１６時間～７日間である。培養中の培養培地のｐＨは３．０～９．０に保持することが好ましい。培養培地のｐＨの調整は、無機酸、有機酸、アルカリ溶液、尿素、炭酸カルシウム及びアンモニア等を用いて行うことができる。 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.
　培養中必要に応じて、アンピシリン及びテトラサイクリン等の抗生物質を培養培地に添加してもよい。プロモーターとして誘導性のプロモーターを用いた発現ベクターで形質転換した微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例えば、ｌａｃプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはイソプロピル－β－Ｄ－チオガラクトピラノシド等を、ｔｒｐプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培地に添加してもよい。 Antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during culture. When a microorganism transformed with an expression vector using an inducible promoter as a promoter is cultured, an inducer may be added to the medium as needed. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside etc., and culturing a microorganism transformed with an expression vector using a trp promoter, indole acrylic An acid or the like may be added to the medium.
　宿主が生成蓄積した目的とする改変フィブロインの単離、精製は通常用いられている方法で行うことができる。例えば、当該改変フィブロインが、細胞内に溶解状態で発現した場合には、培養終了後、宿主細胞を遠心分離により回収し、水系緩衝液に懸濁した後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー及びダイノミル等により宿主細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清から、タンパク質の単離精製に通常用いられている方法によって精製標品を得ることができる。また、当該改変フィブロインが細胞内に不溶体を形成して発現した場合は、同様に宿主細胞を回収後、破砕し、遠心分離を行うことにより、沈殿画分として改変フィブロインの不溶体を回収する。回収した改変フィブロインの不溶体は、タンパク質変性剤で可溶化することができる。該操作の後、上記と同様の単離精製法により改変フィブロインの精製標品を得ることができる。 Isolation and purification of the desired modified fibroin produced and accumulated by the host can be performed by a commonly used method. For example, when the modified fibroin is expressed in a dissolved state in cells, after completion of culture, 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. Also, 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. After the operation, a purified preparation of the modified fibroin can be obtained by the same isolation and purification method as described above.
　当該改変フィブロインが細胞外に分泌された場合には、培養上清から当該改変フィブロインを回収することができる。すなわち、培養物を遠心分離等の手法により処理することにより培養上清を取得し、該培養上清から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。 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.
　タンパク質の単離精製に通常用いられている方法としては、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル（ＤＥＡＥ）－セファロース、ＤＩＡＩＯＮ　ＨＰＡ－７５（三菱化成社製）等のレジンを用いた陰イオン交換クロマトグラフィー法、Ｓ－Ｓｅｐｈａｒｏｓｅ　ＦＦ（Ｐｈａｒｍａｃｉａ社製）等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の方法を挙げることができる。これらの方法は、単独又は組み合わせて使用してもよい。 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.
　加撚工程において使用する繊維は、改変フィブロインを含む。当該繊維は、好ましくは改変フィブロインからなる繊維（以下、改変フィブロイン繊維ともいう）である。改変フィブロイン繊維としては、好ましくは改変クモ糸フィブロインからなる繊維（改変クモ糸フィブロイン繊維）である。 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). The modified fibroin fibers are preferably fibers consisting of modified spider silk fibroin (modified spider silk fibroin fibers).
　改変フィブロインを含む繊維は、改変フィブロインを含む組成物を公知の紡糸方法により紡糸することによって製造することができる。以下では、改変フィブロインからなる繊維を例に、改変フィブロイン繊維の調製方法について説明する。すなわち、改変フィブロイン繊維を製造する際には、まず、上述した方法に準じて製造した改変フィブロインを、ジメチルスルホキシド（ＤＭＳＯ）、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）、又はヘキサフルオロイソプロノール（ＨＦＩＰ）、ギ酸等の溶媒に、必要に応じて、溶解促進剤としての無機塩と共に添加し、溶解させてドープ液を作製する。次いで、このドープ液（紡糸原液）を用いて、湿式紡糸、乾式紡糸又は乾湿式紡糸等の公知の紡糸方法により紡糸して、目的とする改変フィブロイン繊維を得ることができる。 The fiber containing the modified fibroin can be produced by spinning a composition containing the modified fibroin by a known spinning method. In the following, 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.
　図６は、改変フィブロイン繊維を製造するための紡糸装置の一例を示す概略図である。図６に示す紡糸装置１０は、乾湿式紡糸用の紡糸装置の一例であり、押出し装置１と、凝固浴槽２０と、洗浄浴槽２１と、乾燥装置４とを、上流側から順に有している。 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. .
　押出し装置１は貯槽７を有しており、ここにドープ液（紡糸原液）６が貯留される。凝固浴槽２０に凝固液１１（例えば、メタノール）が貯留される。ドープ液６は、貯槽７の下端部に取り付けられたギヤポンプ８により、凝固液１１との間にエアギャップ１９を開けて設けられたノズル９から押し出される。押し出されたドープ液６は、エアギャップ１９を経て凝固液１１内に供給される。凝固液１１内でドープ液６から溶媒が除去されてタンパク質が凝固する。凝固した改変フィブロインは、洗浄浴槽２１に導かれ、洗浄浴槽２１内の洗浄液１２により洗浄された後、洗浄浴槽２１内に設置された第一ニップローラ１３と第二ニップローラ１４により、乾燥装置４へと送られる。このとき、例えば、第二ニップローラ１４の回転速度を第一ニップローラ１３の回転速度よりも速く設定すると、回転速度比に応じた倍率で延伸された改変フィブロイン繊維３６が得られる。洗浄液１２中で延伸された改変フィブロイン繊維３６は、洗浄浴槽２１内を離脱してから、乾燥装置４内を通過する際に乾燥され、その後、ワインダーにて巻き取られる。このようにして、改変フィブロイン繊維３６が、紡糸装置１０により、最終的にワインダーに巻き取られた巻回物５として得られる。なお、１８ａ～１８ｇは糸ガイドである。 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) is stored in coagulation bath 20. 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.
　凝固液１１としては、ノズル９から押し出されたドープ液６から、溶媒を抽出（脱溶媒）できる有機溶剤であればよい。このような有機溶剤としては、例えば、メタノール、エタノール及び２－プロパノール等の炭素数１～５の低級アルコール、並びにアセトン等を挙げることができる。凝固液１１は、適宜水を含んでいてもよい。凝固液１１の温度は、０～３０℃であることが好ましい。凝固した改変フィブロインが凝固液１１中を通過する距離（実質的には、糸ガイド１８ａから糸ガイド１８ｂまでの距離）は、脱溶媒が効率的に行える、凝固液１１中での改変フィブロイン繊維の滞在時間を確保可能な長さがあればよい。凝固液１１中での滞留時間は、例えば、０．０１～３分であってよく、０．５～１分であってもよい。また、凝固液１１中で延伸（前延伸）をしてもよい。 The coagulating solution 11 may be an organic solvent capable of extracting (desolving) the solvent from the dope 6 extruded from the nozzle 9. Examples of such 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.
　洗浄液１２としては、主として水を用いることができる。洗浄液１２は、凝固液１１に使用できるよう剤として列挙したものを含んでいてもよい。なお、改変フィブロイン繊維を得る際に洗浄浴槽２１内で実施される延伸は、温水中、温水に有機溶剤等を加えた溶液中等で行う、いわゆる湿熱延伸であってもよい。この湿熱延伸の温度としては、例えば、０～９０℃であってよく、２０～７０℃が好ましく、３０～６０℃がより好ましい。湿熱延伸での未延伸糸（又は前延伸糸）の延伸倍率は、例えば、１～１０倍であってもよく、２～８倍であってもよい。 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.
　本実施形態における乾燥装置４内を通過する際に、改変フィブロイン繊維を更に延伸（いわゆる乾熱延伸）してもよい。 The modified fibroin fibers may be further drawn (so-called dry heat drawing) when passing through the drying device 4 in the present embodiment.
　最終的な改変フィブロイン繊維の延伸倍率は、その下限値が、未延伸糸（又は前延伸糸）に対して、好ましくは、１倍超、２倍以上、３倍以上、４倍以上、５倍以上、６倍以上、７倍以上、８倍以上、９倍以上のうちのいずれかであり、上限値が、好ましくは１００倍以下、８０倍以下、６０倍以下、４０倍以下、３０倍以下、２０倍以下、１５倍以下、１４倍以下、１３倍以下、１２倍以下、１１倍以下、１０倍以下である。 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.
　加熱工程では、加撚工程にて撚りを付与した糸を、撚りを維持した状態で加熱する。 In the heating step, the yarn provided with twist in the twisting step is heated while maintaining the twist.
　加熱工程において、糸の撚りを維持する方法は、特に制限されるものでなく、例えば、紙管等に糸を巻きつけた状態で、全体をネットなどでたるみなく拘束してもよい。または、撚糸機で撚りを付与している状態で（インラインで）、加熱工程を実施してもよい。 In the heating step, the method for maintaining the twist of the yarn is not particularly limited. For example, in a state where the yarn is wound around a paper tube or the like, the whole may be restrained with a net or the like without slack. Alternatively, the heating step may be performed in a state in which twisting is applied by a twisting machine (in-line).
　加熱の手段は、特に限定されるものでなく、ヒーター、オーブン、恒温槽等を用いることができる。 The means for heating is not particularly limited, and a heater, an oven, a thermostat, or the like can be used.
　加熱温度は、好ましくは５０℃超であり、より好ましくは１００℃以上であり、更に好ましくは１１５℃以上であり、更により好ましくは１３０℃以上である。加熱温度が、５０℃以上であると、糸に加えた撚りの固定をより充分なものとすることができる。加熱温度は、改変フィブロイン繊維の分解等をより抑制する観点から、好ましくは２４０℃以下であり、より好ましくは１８０℃以下であり、さらに好ましくは１５０℃以下である。例えば、上記加熱工程は、５０℃超の雰囲気下で上記糸を加熱する工程であってよく、また上記加熱工程は、２４０℃以下の雰囲気下で上記糸を加熱する工程であってよい。加熱温度は、一定であってもよく、又は段階的に昇温するように調整してもよい。加熱温度は、好ましくは一定となるように調整される。本明細書において、加熱温度とは、糸が曝される環境温度を意味し、通常は、対象とする糸の加熱手段（例えば、ヒーター）における設定温度を意味する。 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. For example, the heating step may be a step of heating the yarn in an atmosphere of more than 50 ° C., and 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. In the present specification, 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).
　加熱時間は、糸の状態や加熱温度等によって適宜に変更され、例えば、撚糸機で撚りを付与している状態（インライン）では、０．１秒以上であってもよく、1秒以上であってもよく、３秒以上であってもよい。また、紙管等に糸を巻きつけた状態では、加熱時間は、０．５時間以上であってもよく、１時間以上であってもよく、３時間以上であってもよく、６時間以上であってもよく、１２時間以上であってもよい。加熱時間が０．５時間以上であることにより、糸に撚りをより確実に付与することができる。加熱時間はまた、例えば、改変フィブロイン繊維の劣化を充分に低減する観点から、２４時間以下であってよく、又は１８時間以下であってよい。 The heating time is appropriately changed depending on the state of the yarn, the heating temperature, etc. For example, in 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. In the state where the yarn is wound around a paper tube or the like, 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. When the heating time is 0.5 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.
　第一実施形態に係る撚糸の製造方法により得られる撚糸は、加撚工程において付与された撚りが充分に維持、固定されたものとなる。得られる撚糸の撚数は、例えば、２００Ｔ／ｍ以上、６００Ｔ／ｍ以上、又は８００Ｔ／ｍ以上とすることができる。また、付与された撚りが充分に固定されていることは、撚糸の残留トルクを測定することにより確認することができる。残留トルクの測定は、後述する実施例に記載の方法による。 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. In addition, it can be confirmed by measuring the residual torque of the twisted yarn that the applied twist is sufficiently fixed. The measurement of the residual torque is according to the method described in the examples described later.
＜仮撚り糸の製造方法＞
　第二実施形態に係る仮撚り糸の製造方法は、改変フィブロイン繊維を含む糸を撚る加撚工程と、前記糸の撚りを維持した状態で、前記糸を加熱する加熱工程と、加熱された前記糸を解撚する解撚工程と、を備える。加撚工程と、加熱工程と、は互いに独立した工程であってもよく、同時に行われる工程であってもよく、好ましくは同時に行われる。 <Method of manufacturing false twist yarn>
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.
　本実施形態に係る仮撚り糸の製造方法と、第一実施形態に係る撚糸の製造方法と共通する部分については、撚糸の製造方法において説明した条件等を適用することができる。ここでは、説明を省略し、第一実施形態と異なる部分について説明する。 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. Here, the description is omitted, and parts different from the first embodiment will be described.
　加撚工程は、改変フィブロイン繊維に撚りを加える。撚りを加える手段は、公知の手段を用いることができ、例えば、仮撚機などを使用できる。仮撚機としては、例えば、ピン仮撚機、フリクション仮撚機、ベルト仮撚機等が挙げられる。撚り方向は、Ｓ撚り（右撚）であってよく、Ｚ撚り（左撚）であってもよい。 The twisting process adds twist to the modified fibroin fiber. As means for applying twist, known means can be used, and for example, a false twister can be used. As 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).
　第二施形態に係る仮撚り糸の製造方法において、撚数は、好ましくは８００Ｔ／ｍ以上であり、より好ましくは１０００Ｔ／ｍ以上であり、また１２００Ｔ／ｍ以上であってもよい。加撚工程における撚数を８００Ｔ／ｍ以上とすることにより、糸により効率的に撚りを付与することができる。また本実施形態に係る仮撚り糸の製造方法において、撚数は、例えば、１８００Ｔ／ｍ以下であってよく、または１６００Ｔ／ｍ以下であってもよい。 In the method for producing a false twist yarn according to the second embodiment, 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. By setting the number of twists in the twisting step to 800 T / m or more, twist can be efficiently applied to the yarn. Further, in the method of manufacturing the false twist yarn according to the present embodiment, the number of twists may be, for example, 1800 T / m or less, or 1600 T / m or less.
　解撚工程では、加撚工程、加熱工程を経た糸を、加撚工程とは逆の方向へ撚る。 In the untwisting step, the yarn subjected to the twisting step and the heating step is twisted in the opposite direction to the twisting step.
　解撚工程における撚数は、加撚工程における撚数と同等であってよく、又は加撚工程における撚数以上であってよい。解撚工程における撚数は、仮撚機の設定値を変更することにより、調整することができる。 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.
　第二実施形態に係る仮撚り糸の製造方法は、例えば、図７に示すような仮撚機１００を用いてもよい。図７は、仮撚り糸を製造するための仮撚装置の一例を示す概略図である。図７に示す仮撚機１００は、送り出しローラー４０と、第一フィードローラー４２と、第一ヒーター５０と、加撚部６０と、第二フィードローラー４４と、第二ヒーター５２と、第三フィードローラー４６と、巻取りローラー４８とを、上流側から順に有している。 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.
　巻回物５から送り出しローラー４０を介して改変フィブロイン繊維３６が仮撚機内部へと送り出される。送り出された改変フィブロイン繊維３６は、第一ヒーター５０において加熱され、温度を保ったままの状態で、加撚部６０の上部において撚りが加えられる。加熱された状態で糸に撚りが加えられることにより、糸に付与した撚りが固定される。次いで、加撚部６０の下部において、初めに加えた撚り方向とは逆の方向に、糸に撚りが加えられる。これにより、解撚される。解撚された糸は、第二フィードローラー４４により第二ヒーター５２に送り出され、さらに、第三フィードローラー４６及び巻取りローラーを介して、紙管に巻き取られ、最終的に、目的とする仮撚り糸が巻回物７０として得られる。この間、複数のフィードローラーの駆動速度を調整することで、ヒーター内における糸の滞在時間などを変更することができる。 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. Next, in the lower part of the twisting section 60, 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. During this time, it is possible to change the staying time of the yarn in the heater and the like by adjusting the driving speeds of the plurality of feed rollers.
＜糸の撚り加工方法＞
　第三実施形態に係る糸の撚り加工方法は、改変フィブロイン繊維を含む糸を撚ることと、前記糸の撚りを維持した状態で、前記糸を加熱することと、を含む。 <Method of twisting yarn>
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.
　本実施形態に係る糸の撚り加工方法における、改変フィブロイン、改変フィブロイン繊維を含む糸に撚りを加える条件、加熱条件等は、第一実施形態に係る撚糸の製造方法において説明した条件等を適用することができる。 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.
　第一実施形態に係る撚糸の製造方法により得られる撚糸、及び第二実施形態に係る仮撚り糸の製造方法により得られる仮撚り糸は、いずれも充分な撚りが糸に付与され、且つ充分に固定されたものであることから、例えば、複合繊維、テキスタイル、アパレル製品、各種編織物等に好適に用いることができる。 In each of 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.
　第一、第二、及び第三実施形態に係る方法で、上記のような効果が得られる理由は明らかではないが、改変フィブロイン繊維を含む糸が撚りを付与された状態で当該糸が加熱されることにより、改変フィブロイン繊維を含む糸の熱収縮が生じることで、撚りがかかった状態で塑性変形し、その状態の撚りが固定されるものと、本発明者らは推察している。 Although the reason why the above effects can be obtained by the methods according to the first, second and third embodiments is not clear, 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.
　以下、実施例により本発明をより具体的に説明するが、本発明は実施例に限定されるものではない。 Hereinafter, the present invention will be more specifically described by way of examples, but the present invention is not limited to the examples.
＜クモ糸フィブロインの製造＞
（１）プラスミド発現株の作製
　ネフィラ・クラビペス（Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ）由来のフィブロイン（ＧｅｎＢａｎｋアクセッション番号：Ｐ４６８０４．１、ＧＩ：１１７４４１５）の塩基配列及びアミノ酸配列に基づき、配列番号１５で示されるアミノ酸配列を有する改変フィブロイン（以下、「ＰＲＴ７９９」ともいう。）を設計した。なお、配列番号１５で示されるアミノ酸配列は、ネフィラ・クラビペス由来のフィブロインのアミノ酸配列に対して、生産性の向上を目的としてアミノ酸残基の置換、挿入及び欠失を施したアミノ酸配列を有し、さらにＮ末端に配列番号１１で示されるアミノ酸配列（タグ配列及びヒンジ配列）が付加されている。 <Manufacture of spider silk fibroin>
(1) Preparation of plasmid expression strain Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P46804.1, GI: 1174415) derived from Nephila clavipes, the amino acid sequence shown in SEQ ID NO: 15 is A modified fibroin (hereinafter also referred to as "PRT 799") was designed. In addition, the 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.
　次に、ＰＲＴ７９９をコードする核酸を合成した。当該核酸には、５’末端にＮｄｅＩサイト及び終止コドン下流にＥｃｏＲＩサイトを付加した。当該核酸をクローニングベクター（ｐＵＣ１１８）にクローニングした。その後、同核酸をＮｄｅＩ及びＥｃｏＲＩで制限酵素処理して切り出した後、タンパク質発現ベクターｐＥＴ－２２ｂ（＋）に組換えて発現ベクターを得た。 Next, the 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.
（２）タンパク質の発現
　配列番号１０で示されるアミノ酸配列を有するタンパク質をコードする核酸を含むｐＥＴ２２ｂ（＋）発現ベクターで、大腸菌ＢＬＲ（ＤＥ３）を形質転換した。当該形質転換大腸菌を、アンピシリンを含む２ｍＬのＬＢ培地で１５時間培養した。当該培養液を、アンピシリンを含む１００ｍＬのシード培養用培地（表４）にＯＤ６００が０．００５となるように添加した。培養液温度を３０℃に保ち、ＯＤ６００が５になるまでフラスコ培養を行い（約１５時間）、シード培養液を得た。 (2) 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.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　当該シード培養液を５００ｍＬの生産培地（表５）を添加したジャーファーメンターにＯＤ６００が０．０５となるように添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持するようにした。 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.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　生産培地中のグルコースが完全に消費された直後に、フィード液（グルコース４５５ｇ／１Ｌ、Ｙｅａｓｔ　Ｅｘｔｒａｃｔ　１２０ｇ／１Ｌ）を１ｍＬ／分の速度で添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持するようにし、２０時間培養を行った。その後、１Ｍのイソプロピル－β－チオガラクトピラノシド（ＩＰＴＧ）を培養液に対して終濃度１ｍＭになるよう添加し、目的のタンパク質を発現誘導させた。ＩＰＴＧ添加後２０時間経過した時点で、培養液を遠心分離し、菌体を回収した。ＩＰＴＧ添加前とＩＰＴＧ添加後の培養液から調製した菌体を用いてＳＤＳ－ＰＡＧＥを行い、ＩＰＴＧ添加に依存した目的とするクモ糸フィブロインサイズのバンドの出現により、目的とするクモ糸フィブロインの発現を確認した。 Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture solution was maintained at 37 ° C., and the culture was 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-β-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.
（３）タンパク質の精製
　ＩＰＴＧを添加してから２時間後に回収した菌体を２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｂｕｆｆｅｒ（ｐＨ７．４）で洗浄した。洗浄後の菌体を約１ｍＭのＰＭＳＦを含む２０ｍＭＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）に懸濁させ、高圧ホモジナイザー（ＧＥＡ　Ｎｉｒｏ　Ｓｏａｖｉ社製）で細胞を破砕した。破砕した細胞を遠心分離し、沈殿物を得た。得られた沈殿物を、高純度になるまで２０ｍＭＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）で洗浄した。洗浄後の沈殿物を１００ｍｇ／ｍＬの濃度になるように８Ｍ　グアニジン緩衝液（８Ｍグアニジン塩酸塩、１０ｍＭリン酸二水素ナトリウム、２０ｍＭＮａＣｌ、１ｍＭＴｒｉｓ－ＨＣｌ、ｐＨ７．０）で懸濁し、６０℃で３０分間、スターラーで撹拌し、溶解させた。溶解後、透析チューブ（三光純薬株式会社製のセルロースチューブ３６／３２）を用いて水で透析を行った。透析後に得られた白色の凝集タンパク質を遠心分離により回収し、凍結乾燥機で水分を除き、凍結乾燥粉末を回収することにより、クモ糸フィブロイン「ＰＲＴ７９９」を得た。 (3) Purification of Protein Two hours after addition of IPTG, the cells collected were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) to high purity. 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".
＜クモ糸フィブロイン繊維の調製＞
（１）ドープ液の調製
　ジメチルスルホキシド（ＤＭＳＯ）に、上述のクモ糸フィブロイン（ＰＲＴ７９９）を濃度２４質量％となるよう添加した後、溶解促進剤としてＬｉＣｌを濃度４．０質量％となるように添加した。その後、シェーカーを使用して、クモ糸フィブロインを３時間かけて溶解させ、ＤＭＳＯ溶液を得た。得られたＤＭＳＯ溶液中のゴミと泡を取り除き、ドープ液とした。ドープ液の溶液粘度は９０℃において５０００ｃＰ（センチポアズ）であった。 <Preparation of spider silk fibroin fiber>
(1) Preparation of Dope Solution After the spider silk fibroin (PRT 799) described above is added to dimethylsulfoxide (DMSO) to a concentration of 24 mass%, LiCl as a dissolution accelerator to a concentration of 4.0 mass% Added. Thereafter, using a shaker, spider silk fibroin was dissolved for 3 hours to obtain a DMSO solution. The dust and bubbles in the obtained DMSO solution were removed to make a dope. The solution viscosity of the dope solution was 5000 cP (centipoise) at 90.degree.
（２）紡糸
　上記のようにして得られたドープ液と図６に示される紡糸装置１０を用いて公知の乾湿式紡糸を行って、クモ糸フィブロインからなるモノフィラメントを得た。なお、ここでは、乾湿式紡糸を下記の条件で行った。
　凝固液（メタノール）の温度：５～１０℃
　延伸倍率：４．５２倍
　乾燥温度：８０℃ (2) Spinning A known dry-wet spinning was carried out using the dope solution obtained as described above and the spinning apparatus 10 shown in FIG. 6 to obtain monofilaments composed of spider silk fibroin. Here, dry-wet spinning was performed under the following conditions.
Coagulation liquid (methanol) temperature: 5 to 10 ° C
Stretching ratio: 4.52 times Drying temperature: 80 ° C
（実施例１）
　上記で得られたモノフィラメントを複数本束ねて、イタリー式基撚糸機を用いて、撚数６０６Ｔ／ｍの設定にて、Ｓ撚りをかけながら撚りを掛けた糸をボビンに巻取り巻回体を得た。得られた撚糸は約１６０デニールであった。当該巻回体を、糸がほどけないように巻回体ごとネットで固定し、６０℃、相対湿度０％ＲＨの恒温恒湿器内に収容し、６時間かけて糸を加熱した。こうして、目的とする撚糸１を得た。 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.
＜繊度の測定＞
　上記撚糸１を、２０℃、相対湿度６５％ＲＨの恒温恒湿器内に１時間保管した。その後、紙管から撚糸１から９０ｃｍの撚糸を５本切り出し、それぞれ質量を測定した。当該５本の撚糸それぞれの質量の平均し、得られた平均値の１００００倍した値を繊度とした。結果が表６に示される。 <Measurement of fineness>
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.
＜撚数の測定（検撚）＞
　上記撚糸１から撚糸を所定長さで巻き出して、その５０ｃｍの長さ分を、測定寸法５０ｃｍに調整された検撚機にセットした。その後、検撚機により、撚糸の撚りがなくなるまで撚り返し（加撚工程とは逆方向の撚りをかけ）、糸の撚りがなくなるまでの撚り返しの回数を測定した。この操作を１０回繰り返して、それぞれの撚り返し回数を平均し、得られた平均値を２倍して撚糸１の撚数とした。結果が表６に示される。 <Measurement of the number of twists (twisting)
The twisted yarn was unwound from the twisted yarn 1 by a predetermined length, and the 50 cm length was set in a twisting machine adjusted to a measurement dimension of 50 cm. After that, twisting was performed by using a twisting machine until twisting of the twisted yarn was lost (twisting in the direction opposite to the twisting step was applied), and the number of twisting until the twisting of the yarn was canceled was measured. This operation is repeated 10 times, each twisting number is averaged, and the obtained average value is doubled to obtain the number of twists of the twisted yarn 1. The results are shown in Table 6.
＜残留トルク測定＞
　上記撚糸１から１．５ｍの撚糸を１０本切り出した。切り出した撚糸１本の半分の位置（撚糸の一端から７５ｃｍの位置）に、重りを付けると共に、切り出した撚糸の両端部を合わせた。撚糸が撚り戻ろうとする力（トルク）を利用して、撚り合わせることでサンプルを得た。得られたサンプルを、測定寸法５０ｃｍに調整された検撚機に取り付けた。検撚機により、撚糸の撚りがなくなるまで撚り返し（加撚工程とは逆方向の撚りをかけ）、糸の撚りがなくなるまでの撚り返しの回数を測定した。１０本の撚糸それぞれの撚り返し回数を平均し、得られた平均値を２倍して撚糸１の残留トルクとした。残留トルクが大きいと糸に付与した撚りの固定が不十分であることを意味し、残留トルクが小さいと糸に付与した撚りの固定が十分であることを意味する。結果が表６に示される。 <Measurement of residual torque>
Ten twisted yarns of 1.5 m were cut out from the twisted yarn 1. A weight was attached to the position of one half of the cut-out twisted yarn (position 75 cm from one end of the twisted yarn), and both ends of the cut-out twisted yarn were aligned. The sample was obtained by twisting using the force (torque) with which the twisted yarn tries to untwist. The obtained sample was mounted on a twisting machine adjusted to a measuring dimension of 50 cm. By means of a twisting machine, twisting was performed until twisting of the twisted yarn was lost (twisting in the direction opposite to the twisting step was applied), and the number of twisting until the twisting of the yarn was lost was measured. The number of times of twisting of each of the ten twisted yarns was averaged, and the obtained average value was doubled to obtain the residual torque of the twisted yarn 1. A large residual torque means that the fixing of the twist applied to the yarn is insufficient, and a small residual torque means that the fixing of the twist applied to the yarn is sufficient. The results are shown in Table 6.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（実施例２～６）
　表６に記載の加熱温度、加熱時間に代えた以外は、実施例１と同様にして、目的とする撚糸を得た。得られた撚糸それぞれについて、実施例１と同様に繊度、撚数、及び残留トルクを測定した。結果が表６に示される。 (Examples 2 to 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.
（比較例１）
　実施例１で得られた加熱する前の撚りを付与した糸を、比較例におけるサンプルとした。当該サンプルについて、実施例１と同様に、繊度、撚数、及び残留トルクを測定した。結果が表６に示される。 (Comparative 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.
（実施例７）
　上記クモ糸フィブロイン繊維の調製において得られたクモ糸フィブロインからなるモノフィラメントを用いて、図７に示される仮撚機１００を用いて、表５に示す条件で目的とする仮撚り糸１を得た。得られた仮撚り糸１は全て捲縮が維持されていることが目視にて確認された。また、得られた仮撚り糸１について、繊度と伸度を、下記のとおり測定した。伸度については、仮撚り加工する前のクモ糸フィブロイン繊維の伸度に対する変化率を下記式１に従って算出した。結果が表７に示される。なお、使用したクモ糸フィブロインは、繊度２３０ｄｔｅｘ（２０７デニール）であった。
式１：伸度の変化率＝［（仮撚り糸の伸度）－（仮撚り加工前のクモ糸フィブロイン繊維の伸度）］／（仮撚り加工前のクモ糸フィブロイン繊維の伸度）×１００ (Example 7)
Using the monofilament made of spider silk fibroin obtained in the preparation of the above-mentioned spider silk fibroin fiber, the target false twisted yarn 1 was obtained under the conditions shown in Table 5 using the false twister 100 shown in FIG. It was visually confirmed that all false twist yarns 1 obtained were maintained crimped. In addition, the fineness and the elongation of the obtained false-twisted yarn 1 were measured as follows. About elongation, the change rate with respect to the elongation of the spider silk fibroin fiber before carrying out a false twist process was computed according to following formula 1. The results are shown in Table 7. The spider silk fibroin used had a fineness of 230 dtex (207 denier).
Formula 1: Change in elongation = [(Elongation of false twisted yarn)-(Elongation of spider silk fibroin fiber before false twist processing)] / (Elongation of spider yarn fibroin fiber before false twist processing) × 100
＜伸度測定＞
　上記仮撚り糸１に対して、（株）エー・アンド・デイ製　テンシロン万能材料試験機を用いて伸度を測定した。 <Elongation measurement>
The elongation of the false twisted yarn 1 was measured using a Tensilon universal material tester manufactured by A & D.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
（実施例８～１１）
　表７に記載の条件に変更した以外は、実施例７と同様にして、目的とする仮撚り糸を得た。得られた仮撚り糸それぞれについて、実施例７と同様に、繊度と伸度を測定した。結果が表７に示される。表７の結果から、本発明手法に従って仮撚りを行うことにより、伸度が向上することが認められた。また、仮撚り糸１を仮撚り後に再加熱しない方が、より高い伸度の向上効果が得られる傾向が確認された。 (Examples 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.
　１…押出し装置、４…乾燥装置、６…ドープ液、１０…紡糸装置、２０…凝固浴槽、２１…洗浄浴槽、３６…改変フィブロイン繊維、１００…仮撚機。 DESCRIPTION OF SYMBOLS 1 ... Extrusion apparatus, 4 ... Drying apparatus, 6 ... Doping liquid, 10 ... Spinning apparatus, 20 ... Coagulation tub, 21 ... Washing tub, 36 ... Modified fibroin fiber, 100 ... False twisting machine.

Claims (11)

  1. 　改変フィブロイン繊維を含む糸を撚る加撚工程と、
    　前記糸の撚りを維持した状態で、前記糸を加熱する加熱工程と、を備える、撚糸の製造方法。     A twisting step of twisting a yarn containing the modified fibroin fiber,
    And a heating step of heating the yarn while maintaining the yarn twist.
  2. 　前記改変フィブロイン繊維が、改変クモ糸フィブロイン繊維である、請求項１に記載の撚糸の製造方法。 The method for producing a twisted yarn according to claim 1, wherein the modified fibroin fiber is a modified spider yarn fibroin fiber.
  3. 　前記加熱工程が、５０℃超の雰囲気下で前記糸を加熱する工程である、請求項１又は２に記載の撚糸の製造方法。 The method for producing a twisted yarn according to claim 1, wherein the heating step is a step of heating the yarn under an atmosphere of more than 50 ° C. 4.
  4. 　前記加熱工程が、２４０℃以下の雰囲気下で前記糸を加熱する工程である、請求項１～３のいずれか一項に記載の撚糸の製造方法。 The method for producing a twisted yarn according to any one of claims 1 to 3, wherein the heating step is a step of heating the yarn under an atmosphere of 240 ° C or lower.
  5. 　前記加撚工程が、前記糸の撚数が２００Ｔ／ｍ以上であるように前記糸を撚る工程である、請求項１～４のいずれか一項に記載の撚糸の製造方法。 The method for producing a twisted yarn according to any one of claims 1 to 4, wherein the twisting step is a step of twisting the yarn such that the number of twists of the yarn is 200 T / m or more.
  6. 　改変フィブロイン繊維を含む糸を撚る加撚工程と、
    　前記糸の撚りを維持した状態で、前記糸を加熱する加熱工程と、
    　加熱された前記糸を解撚する解撚工程と、を備える、仮撚り糸の製造方法。     A twisting step of twisting a yarn containing the modified fibroin fiber,
    A heating step of heating the yarn while maintaining the twist of the yarn;
    And a twisting step of untwisting the heated yarn.
  7. 　前記改変フィブロイン繊維が、改変クモ糸フィブロイン繊維である、請求項６に記載の仮撚り糸の製造方法。 The method for producing a false twist yarn according to claim 6, wherein the modified fibroin fiber is a modified spider yarn fibroin fiber.
  8. 　前記加熱工程が、５０℃超の雰囲気下で前記糸を加熱する工程である、請求項６又は７に記載の仮撚り糸の製造方法。 The method for producing a false twist yarn according to claim 6 or 7, wherein the heating step is a step of heating the yarn under an atmosphere of more than 50 ° C.
  9. 　前記加熱工程が、２４０℃以下の雰囲気下で前記糸を加熱する工程である、請求項６～８のいずれか一項に記載の仮撚り糸の製造方法。 The method for producing a false twist yarn according to any one of claims 6 to 8, wherein the heating step is a step of heating the yarn under an atmosphere of 240 ° C or lower.
  10. 　前記加撚工程が、前記糸の撚数が８００Ｔ／ｍ以上であるように前記糸を撚る工程である、請求項６～９のいずれか一項に記載の仮撚り糸の製造方法。 The method for producing a false twist yarn according to any one of claims 6 to 9, wherein the twisting step is a step of twisting the yarn such that the number of twists of the yarn is 800 T / m or more.
  11. 　改変フィブロイン繊維を含む糸を撚ることと、
    　前記糸の撚りを維持した状態で、前記糸を加熱することと、を含む、糸の撚り加工方法。     Twisting a yarn containing the modified fibroin fiber,
    Heating the yarn in a state in which the yarn is kept twisted.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019182040A1 (en) * 2018-03-22 2019-09-26 株式会社島精機製作所 Protein fiber crimping method, protein fiber production method, protein fibers, spun yarn, and textile product

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JPS6170074A (en) * 1984-09-12 1986-04-10 水島　繁三郎 Shape memory silk yarn and its production
JPH01183534A (en) * 1988-01-18 1989-07-21 Shigesaburo Mizushima Production of shape-memorizing yarn of natural fiber
JPH09119033A (en) * 1995-10-27 1997-05-06 Matsuoka Kigyo Kk Latently crimping raw silk and its production, and crimping silk yarn obtained from the latently crimping silk
JP2014029054A (en) * 2012-06-28 2014-02-13 Spiber Inc Spun-dyed protein fiber, and method for producing the same

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Publication number Priority date Publication date Assignee Title
JPS6170074A (en) * 1984-09-12 1986-04-10 水島　繁三郎 Shape memory silk yarn and its production
JPH01183534A (en) * 1988-01-18 1989-07-21 Shigesaburo Mizushima Production of shape-memorizing yarn of natural fiber
JPH09119033A (en) * 1995-10-27 1997-05-06 Matsuoka Kigyo Kk Latently crimping raw silk and its production, and crimping silk yarn obtained from the latently crimping silk
JP2014029054A (en) * 2012-06-28 2014-02-13 Spiber Inc Spun-dyed protein fiber, and method for producing the same

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019182040A1 (en) * 2018-03-22 2019-09-26 株式会社島精機製作所 Protein fiber crimping method, protein fiber production method, protein fibers, spun yarn, and textile product

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