WO2020027153A1 - Modified fibroin fibers and production method therefor - Google Patents

Modified fibroin fibers and production method therefor Download PDF

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Publication number
WO2020027153A1
WO2020027153A1 PCT/JP2019/029892 JP2019029892W WO2020027153A1 WO 2020027153 A1 WO2020027153 A1 WO 2020027153A1 JP 2019029892 W JP2019029892 W JP 2019029892W WO 2020027153 A1 WO2020027153 A1 WO 2020027153A1
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amino acid
seq
modified fibroin
sequence
fibroin
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PCT/JP2019/029892
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French (fr)
Japanese (ja)
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久弘 工藤
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Ｓｐｉｂｅｒ株式会社
小島プレス工業株式会社
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Publication of WO2020027153A1 publication Critical patent/WO2020027153A1/en

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    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
    • D—TEXTILES; PAPER
    • D02—YARNS; MECHANICAL FINISHING OF YARNS OR ROPES; WARPING OR BEAMING
    • D02J—FINISHING OR DRESSING OF FILAMENTS, YARNS, THREADS, CORDS, ROPES OR THE LIKE
    • D02J1/00—Modifying the structure or properties resulting from a particular structure; Modifying, retaining, or restoring the physical form or cross-sectional shape, e.g. by use of dies or squeeze rollers
    • D02J1/22—Stretching or tensioning, shrinking or relaxing, e.g. by use of overfeed and underfeed apparatus, or preventing stretch

Definitions

  • the present invention relates to a modified fibroin fiber and a method for producing the same.
  • the wet spinning method and the dry-wet spinning method are also used when producing modified fibroin fibers containing modified fibroin as a main component (for example, see Patent Document 1).
  • modified fibroin fibers Various properties are required for the modified fibroin fiber depending on the application, and one of them may be required to have sufficient strength.
  • the modified fibroin fibers obtained by the wet method and the dry-wet method voids are generated inside the raw material fibers due to the removal of the solvent in the coagulation liquid, and the strength may be insufficient due to the voids.
  • an object of the present invention is to provide a modified fibroin fiber having high strength.
  • Another object of the present invention is to provide a method for producing a modified fibroin fiber having high strength.
  • the present invention relates to, for example, the following inventions.
  • a modified fibroin fiber comprising the modified fibroin and having a density greater than 1.34 g / cm 3 .
  • [3] (A) a first drawing step of drawing a raw fiber containing modified fibroin in a water bath, and (b) a heating step of heating and drying the raw fiber after the first drawing step, and heat-shrinking the fiber in the radial direction. And producing a modified fibroin fiber.
  • a modified fibroin fiber having high strength can be provided. Further, according to the present invention, a method for producing a modified fibroin fiber having high strength can be provided.
  • FIG. 7 (A) shows an undrawn yarn production device—first-stage drawing device
  • FIG. 7 (B) shows a second-stage drawing device.
  • the modified fibroin fiber according to the present embodiment contains modified fibroin, and has a density of more than 1.34 g / cm 3 .
  • 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. Including proteins.
  • the modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to one or both of the N-terminal side and the C-terminal side of the domain sequence.
  • the N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.
  • modified fibroin means artificially produced fibroin (artificial fibroin).
  • the modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be the same as the amino acid sequence of naturally occurring fibroin.
  • naturally-derived fibroin as used herein is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Is a protein containing the domain sequence to be determined.
  • Modified fibroin may be a directly used amino acid sequence of a naturally occurring fibroin, or a modified amino acid sequence based on the amino acid sequence of a naturally occurring fibroin (for example, cloned naturally occurring fibroin).
  • the amino acid sequence may be modified by modifying the gene sequence of fibroin), or may be artificially designed and synthesized without using naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence may be used). Which have a desired amino acid sequence by chemical synthesis).
  • domain sequence refers to a crystalline region unique 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).
  • the (A) n motif indicates an amino acid sequence mainly containing an alanine residue, and has 2 to 27 amino acid residues.
  • the number of amino acid residues in 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 n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues).
  • At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • REP may be an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300, and may be an integer of 10 to 300.
  • the plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • a plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin according to the present embodiment has, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to a cloned natural fibroin gene sequence.
  • an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to a cloned natural fibroin gene sequence can be obtained by performing the following modification.
  • Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982) and Methods ⁇ in ⁇ Enzymology, 100, 448 (1983).
  • Naturally occurring fibroin is a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Yes, specifically, for example, fibroin produced by insects or spiders.
  • fibroin produced by insects examples include Bombyx @ mori, Bombyx @ mandarina, natural silkworm (Antheraea @ yamamai), tussah (Anterea @ pernii), and maple silkworm (Erioganyerii). ), Silkworms produced by silkworms (Samia cynthia), chestnut worms (Caligura japonica), tussah silkworms (Antheraea mylitta), silkworms such as moga silkworms (Antheraea assama), and silkworms produced by larvae of the hornet beetle Hornet silk protein.
  • fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
  • Examples of the fibroin produced by the spiders include spiders belonging to the genus Araneus (genus Araneus), such as the spiders, the spiders, the red spider, the red spider, the green spider, etc.
  • Spiders belonging to the genus Argiope genus such as spiders belonging to the genus Procarpus spp.
  • Spiders belonging to the genus Pronus such as spiders belonging to the genus Procarpus spp.
  • Spiders belonging to the genus Pronus and spider spiders belonging to the genus Cynotarachne, such as the genus Cyrtarachne, such as Torinofundamashi and Otorinofundamashi.
  • Spiders belonging to the genus Ordgarius such as spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Orbalis, and spiders belonging to the genus Ordgarius, such as the spiders belonging to the genus Ordgarius.
  • Spiders belonging to the genus Argiope such as Argiope bruennichi, spiders belonging to the genus Argiope, such as Argiope spiders, spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas, such as spiders belonging to the genus Acusilas, and spider spiders belonging to the spider spiders, such as the spider spiders belonging to the genus Acusilas Spiders belonging to the genus Spider (Genus Poltys) such as spiders belonging to the genus (Cytophora) and spiders belonging to the genus (Potys), spiders belonging to the genus Spiders (genus Cyclosa) such as the spiders belonging to the genus Cyclosa and spiders belonging to the genus Cygnus spp.
  • Geneus Poltys such as spiders belonging to the genus (Cytophora) and spiders belonging to the genus (Potys
  • Spider silk proteins produced by spiders belonging to the genus Chorizopes), and asina such as red-headed spiders, red-backed spiders, blue-backed spiders and urocore-red spiders Spiders belonging to the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucaegium, such as the spiders Argiope bruennichi and spiders spiders belonging to the genus Leucauge, such as the spiders belonging to the genus Nephila sp.
  • Spiders belonging to the genus Dyschiriognatha such as spiders belonging to the genus Menosira and spiders belonging to the genus Dyschiriognatha, such as spiders belonging to the genus Dyschiriognatha, and spiders belonging to the genus Latus sp. Spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops (Euprosthenops) are produced. Spider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
  • spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from amino acid sequence of Nephila claviBAC04A4 and derived from amino acid sequence of ph ), U37520 (base sequence)), major ⁇ ampullate ⁇ spidro n 1 [Derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Derived from Nephila clavata (GenBank accession number
  • CAJ00428 amino acid sequence
  • AJ97155 base sequence
  • major ⁇ sample ⁇ spidroin ⁇ 2 [Euprosus] ] GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (base sequence)
  • minor ⁇ silk ⁇ protein ⁇ 1 [Nephila ⁇ clavipes] GenBank Accession No. AAC14589.1 (amino acid sequence)
  • minor ⁇ ampilatte [in] Nephila clavipes] GenBank Accession No. AAC14591.1 (amino acid sequence)
  • minor ampoulate spidroin-like protein [Nephilengys Cruenata] GenBank Accession No. ABR3727.1.
  • fibroin in which sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidroin, ampullate, fibroin, "silk and polypeptide", or “silk and protein” is described as a keyword in DEFINITION from among sequences including INV as DIVISION in the sequence information registered in NCBI @ GenBank. It can be confirmed by extracting a character string of a specific product from a sequence or CDS, and extracting a sequence in which a specific character string is described in TISSUE @ TYPE from SOURCE.
  • the modified fibroin according to this embodiment may be a modified silk (silk) fibroin (an amino acid sequence of a silk protein produced by a silkworm modified), and a modified spider silk fibroin (a spider silk protein produced by an arachnid). Modified amino acid sequence).
  • a modified spider silk fibroin is preferable.
  • the modified fibroin include a modified fibroin (first modified fibroin) derived from a large spinal cord marker protein produced in a spider's large ampullate gland, and a domain sequence having a reduced content of glycine residues.
  • first modified fibroin derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • domain sequence having a reduced content of glycine residues derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • a second modified fibroin derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • a second modified fibroin derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • second modified fibroin derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • a domain sequence having a reduced content of glycine residues derived from a large spinal cord marker protein produced in a spider's large ampul
  • the first modified fibroin includes a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the number of amino acid residues 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 still more preferable, an integer of 4 to 16 is still more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable.
  • the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and more preferably 20 to 100 residues.
  • the first modified fibroin has the total number of glycine, serine and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) n motif-REP] m
  • the total number is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more.
  • the first modified fibroin comprises a unit of the amino acid sequence represented by Formula 1: [(A) n motif-REP] m , and has a C-terminal sequence represented by any of SEQ ID NOS: 1 to 3 or
  • the polypeptide may be an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.
  • the amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is
  • the amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
  • the first modified fibroin (1-i) an amino acid sequence represented by SEQ ID NO: 4 (recombinant ⁇ spider ⁇ silk ⁇ protein ⁇ ADF3KaiLargeNRSH1) or (1-ii) an amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroin comprising an amino acid sequence having at least% sequence identity.
  • the sequence identity is preferably 95% or more.
  • the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human ⁇ rhinovirus @ 3C protease) recognition site at the N-terminus is added.
  • the 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue.
  • the amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.
  • the modified fibroin of (1-i) may have an amino acid sequence represented 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 at least one or more glycine residues in the REP replaced by another amino acid residue, as compared to a naturally occurring fibroin. .
  • the second modified fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine, as compared with a naturally-derived fibroin. At least one motif sequence selected from the group consisting of at least one glycine residue in one or more of the motif sequences has been replaced with another amino acid residue. You may.
  • the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence.
  • the second modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and from the (A) n motif located at the most C-terminal side to the domain sequence
  • the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus is represented by z, From, when the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is defined as w, z / w is 30% or more; It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
  • the second modified fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by substituting 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, further preferably 10% or less, and 6% or less. %, Still more preferably 4% or less, further preferably 2% or less.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGGX below.
  • z / w (%) can be calculated by dividing z by w.
  • 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. Is still more preferred, and even more preferably 80% or more.
  • the upper limit of z / w is not particularly limited, but may be, for example, 95% or less.
  • the second modified fibroin is modified, for example, by replacing at least a part of the base sequence encoding a glycine residue from the cloned natural fibroin gene sequence to encode another amino acid residue.
  • a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more.
  • the amino acid sequence can be obtained by designing an amino acid sequence satisfying the above aspect from the amino acid sequence of naturally occurring 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 corresponding to insertion, addition, and / or addition may be modified.
  • the other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but includes a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and a methionine ( M) residue, hydrophobic amino acid residue such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues, and valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.
  • the second modified fibroin examples include (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), 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.
  • the modified fibroin (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 6 is obtained by replacing all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin with GQX.
  • the amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In which one [(A) n motif-REP] was inserted.
  • the amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some amino acids on the C-terminal side have been deleted so that the molecular weight becomes almost the same as SEQ ID NO: 7.
  • the amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). Is repeated four times with a predetermined hinge sequence and His tag sequence added to the C-terminal.
  • the value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%.
  • the values 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: 9 are 58.7%, respectively. 70.1%, 66.1% and 70.0%.
  • the value of x / y at the jagged ratio (described later) of 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 as follows: They are 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.
  • the modified fibroin of (2-i) may have an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (2-ii) has 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, and contains XGX ( Where X represents an amino acid residue other than glycine.)
  • z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.
  • the second modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.
  • the tag sequence examples include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule.
  • affinity tag is a histidine tag (His tag).
  • His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography (chelating @ metal @ chromatography).
  • SEQ ID NO: 11 amino acid sequence including a His tag sequence and a hinge sequence.
  • tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose binding protein (MBP), which specifically binds to maltose, can be used.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • an “epitope tag” utilizing an antigen-antibody reaction can be used.
  • a peptide (epitope) showing antigenicity as a tag sequence an antibody against the epitope can be bound.
  • the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag.
  • a tag sequence that can be cleaved by a specific protease can be used.
  • protease treatment By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified fibroin from which the tag sequence has been separated can also be recovered.
  • modified fibroin containing a tag sequence (2-iii) the amino acid represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799)
  • Modified fibroin comprising a sequence or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .
  • amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.
  • An amino acid sequence represented by SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) is added to the N-terminal of the amino acid sequence shown.
  • the modified fibroin of (2-iii) may have an amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented 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 containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and contains XGX (where X represents an amino acid residue other than glycine.)
  • z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.
  • the second modified fibroin may include a secretion signal for releasing a 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 the host.
  • the third modified fibroin has an amino acid sequence whose domain sequence has a reduced content of the (A) n motif as compared to a naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) n motifs as compared to naturally occurring fibroin.
  • the third modified fibroin may have an amino acid sequence corresponding to 10 to 40% deletion of the (A) n motif 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 May have an amino acid sequence corresponding to the deletion of
  • the third modified fibroin has a domain sequence deletion of at least two (A) n motifs from the N-terminal side to the C-terminal side, and one (A) It may have an amino acid sequence corresponding to the deletion of the n motif repeated in this order.
  • the third modified fibroin may have a domain sequence having an amino acid sequence corresponding to the deletion of the (A) n motif at least every third sequence from the N-terminal side to the C-terminal side. .
  • the third modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) n motifs from the N-terminal side to the C-terminal side] -REP]
  • the number of amino acid residues of REP in the unit is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to When the maximum value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units that is 11.3 is x, and the total number of amino acid residues in 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.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
  • FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence 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 an n motif.
  • FIG. 1 shows pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP with the fourth REP), pattern 2 (comparison of the first REP and the second REP, and Pattern 4 (comparison of the second REP with the third REP, and comparison of the fourth REP with the fifth REP), pattern 4 (the comparison of the fourth REP with the fifth REP), and pattern 4 (the first REP with the fifth REP). Second REP comparison). Note that there are other selection methods.
  • the number of amino acid residues of each REP in two selected adjacent [(A) n motif-REP] units is compared.
  • each pattern the total number of amino acid residues of two adjacent [(A) n motif-REP] units shown by a solid line is added (not only the REP but also the number of amino acid residues of the (A) n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
  • x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.
  • x / y is preferably at least 50%, more preferably at least 60%, further preferably at least 65%, and even more preferably at least 70%. Preferably, it is still more preferably at least 75%, particularly preferably at least 80%.
  • the upper limit of x / y is not particularly limited, and may be, for example, 100% or less. When the indentation ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the indentation ratio is 1: 1.8 to 3.4, x / y is x / y.
  • / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9%, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably at least 64.2%.
  • x / y should be 46.4% or more. Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, and even more preferably 80% or more. It is particularly preferred that there is.
  • the upper limit of x / y is not particularly limited, and may be 100% or less.
  • x / y in naturally occurring fibroin will be described.
  • 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted.
  • x / y was calculated from the amino acid sequence of the naturally occurring fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above calculation method.
  • FIG. 3 shows the results when the indentation ratio is 1: 1.9 to 4.1.
  • the horizontal axis in FIG. 3 indicates x / y (%), and the vertical axis indicates frequency.
  • the x / y of the naturally derived fibroin is less than 64.2% (the highest is 64.14%).
  • the third modified fibroin deletes one or more of the sequence encoding the (A) n motif from the cloned natural fibroin gene sequence such that x / y is 64.2% or more. Can be obtained. Further, for example, an amino acid sequence corresponding to deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more based on the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence.
  • amino acid residues are further substituted, deleted, inserted and / or added.
  • Amino acid sequence modification corresponding to the above may be performed.
  • the third modified fibroin (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met-PRT525) -PRT799), or (3-ii) an amino acid sequence 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.
  • the modified fibroin (3-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 17 differs from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin in that every two amino acids from the N-terminal side to the C-terminal side (A) n The motif was deleted, and one [(A) n motif-REP] was inserted before the C-terminal sequence.
  • the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for the second modified fibroin.
  • the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) has an x / y value of 15.0% at a giza ratio of 1: 1.8 to 11.3.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 17 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%.
  • the values of z / w in the amino acid sequences represented 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 be composed of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented 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 containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (3-ii) has 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, and is N-terminal to C-terminal.
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other Amino acid residues of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (a giza ratio of 1: 1.8 to 11.3).
  • x / y be 64.2% or more, where x is the maximum value of the sum of the base numbers and y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may include the above-described tag sequence at one or both of the N-terminus and the C-terminus.
  • modified fibroin containing a tag sequence (3-iii) the amino acid represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799)
  • Modified fibroin comprising a sequence or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .
  • amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding SEQ ID NO: 11 to the N-terminal of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. (Including a His tag sequence and a hinge sequence).
  • the modified fibroin of (3-iii) may have an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified fibroin of (3-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented 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 containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 and is N-terminal to C-terminal.
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other
  • the maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 is defined as 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 include a secretion signal for releasing a 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 the host.
  • the fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of a glycine residue in addition to the content of the (A) n motif reduced as compared to a naturally-derived fibroin.
  • the domain sequence of the fourth modified fibroin is at least one or more (A) n motifs deleted, and further at least one or more glycine residues in the REP, as compared to naturally occurring fibroin. It can be said that it has an amino acid sequence equivalent to being replaced with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having both the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects and the like are as described for the second modified fibroin and the third modified fibroin.
  • 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) ), The amino acid sequence represented by SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), 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
  • a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by.
  • Specific embodiments of the modified fibroin comprising the amino acid sequence represented by 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 are as described above.
  • the fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP have been replaced by amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin, and / or It may have an amino acid sequence locally including a region having a large hydrophobicity index, corresponding to insertion of one or more amino acid residues having a large hydrophobicity index therein.
  • a region having a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
  • the amino acid residue having a large hydrophobicity index is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A). More preferably, it is a residue.
  • the fifth modified fibroin may have one or more amino acid residues in the REP replaced with amino acid residues having a higher hydrophobicity index, and / or one or more amino acids in the REP as compared to a naturally occurring fibroin.
  • one or more amino acid residues may be substituted, deleted, inserted and / or added as compared with naturally occurring fibroin.
  • the fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned natural fibroin gene sequence, It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP.
  • a group for example, an amino acid residue having a positive hydrophobicity index
  • one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP.
  • one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in 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 Formula 1: [(A) n motif-REP] m and extends from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
  • the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p,
  • q the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q
  • p / q is 6 .2% or more.
  • hydrophobicity index of amino acid residues
  • a publicly known index Kyte J, & Doolittle R (1982) "A simple method for display, the hydropathic charactor of aa protein, J.Pol.Mol. 105-132).
  • HI hydropathic index
  • sequence A [(A) n motif-REP] m (Hereinafter, referred to as “sequence A”).
  • sequence A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues).
  • the average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.
  • p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more.
  • the upper limit of p / q is not particularly limited, but may be, for example, 45% or less.
  • the fifth modified fibroin is, for example, one or a plurality of hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP so that the amino acid sequence of the cloned natural fibroin is satisfied so as to satisfy the above-mentioned p / q conditions.
  • a hydrophobic amino acid residue eg, an amino acid residue having a positive hydrophobicity index
  • inserting one or more hydrophobic amino acid residues into the REP By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index.
  • an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues in the REP have been replaced by amino acid residues with a higher hydrophobicity index and / or one or more amino acid residues in the REP as compared to naturally occurring fibroin.
  • a 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, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A Is preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
  • the fifth modified fibroin (5-i) the amino acid sequence represented by SEQ ID NO: 19 (Met-PRT665), 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 90% or more sequence identity with the amino acid sequence represented 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 represented by SEQ ID NO: 19 consists of three amino acid residues every other REP, except for the domain sequence of the terminal at the C-terminal side, with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410).
  • two amino acid sequences (VLI) were inserted, some glutamine (Q) residues were further substituted with serine (S) residues, and some C-terminal amino acids were deleted.
  • the amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525). is there.
  • the amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8.
  • the modified fibroin of (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and is located at the most C-terminal side (A) n
  • amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When 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 at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.
  • the fifth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus.
  • modified fibroin containing the tag sequence (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) ) Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 correspond to the amino acid sequence represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).
  • the modified fibroin of (5-iii) may have an amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located at the most C-terminal side (A) n
  • all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more
  • P / q is preferably at least 6.2%.
  • the fifth modified fibroin may include a secretion signal for releasing a 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 the host.
  • the sixth modified fibroin has an amino acid sequence in which the content of glutamine residues is reduced as compared with naturally occurring fibroin.
  • the sixth modified fibroin preferably contains at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence of REP.
  • the content of the GPGXX motif is usually 1% or more, and 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.
  • the “GPGXX motif content” is a value calculated by the following method.
  • Formula 1 Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) 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 at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence
  • the number obtained by multiplying the total number by 3 ie, the total number of G and P in the GPGXX motif
  • the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is represented by (A)
  • the content of the GPGXX motif content is represented by the content of the G
  • “the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” to “the most C-terminal side” (A) Sequence from n motif to C-terminus of domain sequence (sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of fibroin, and m may be small. In this case (that is, when the domain sequence is short), the calculation result of the GPGXX motif content is affected, so that this effect is eliminated.
  • “GPGXX motif” is located at the C-terminus of the REP, even when “XX” is, for example, “AA”, it is treated as a “GPGXX motif”.
  • FIG. 5 is a schematic diagram showing the domain sequence of a modified fibroin.
  • the method of calculating the content rate of the GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin shown in FIG. 5 (“[(A) n motif-REP] m- (A) n motif” type), all REPs are located at the most C-terminal side.
  • (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 as“ region A ”in FIG. 5).
  • 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%. .
  • the “glutamine residue content” is a value calculated by the following method.
  • Formula 1 Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence.
  • the total number of glutamine residues contained in the region is defined as u, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence, and further (A) n
  • the glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif.
  • the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.
  • the sixth modified fibroin corresponds to the fact that its domain sequence has one or more glutamine residues in the REP deleted or replaced with other amino acid residues, as compared to the naturally occurring fibroin. It may have an amino acid sequence.
  • the “other amino acid residue” may be an amino acid residue other than a glutamine residue, but 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 include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M )
  • Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can.
  • an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable.
  • the sixth modified fibroin preferably has a hydrophobicity of REP of -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more, and 0.3 or more. Is still more preferable, and it is particularly preferable that it is 0.4 or more.
  • the upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or may be 0.7 or less.
  • “REP hydrophobicity” is a value calculated by the following method.
  • Formula 1 Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence.
  • the sum of the hydrophobicity indices of each amino acid residue in the region is defined as v, and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal 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 REPs excluding n motifs.
  • the degree of hydrophobicity of the REP the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as follows. The same is true.
  • the sixth modified fibroin may have a domain sequence that is missing one or more glutamine residues in the REP and / or one or more glutamine residues in the REP, as compared to the naturally occurring fibroin.
  • 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 remove one or more glutamine residues in the REP. By substituting the amino acid residue with, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the above and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • modified fibroin (6-i) SEQ ID NO: 25 (Met-PRT988), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT889) -PRT916), modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 29 (Met-PRT699), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698) or SEQ ID NO: 32 (Met-PRT966), or (6-ii) having 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: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32 Modified fibroin containing the amino acid sequence having the same.
  • the modified fibroin of (6-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 25 is obtained by substituting VL for all QQ in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410).
  • the amino acid sequence represented by SEQ ID NO: 26 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with TS, and substituting the remaining Q with A.
  • the amino acid sequence represented by SEQ ID NO: 27 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VL, and substituting the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO: 28 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with VI and substituting the remaining Q with L.
  • the amino acid sequence represented by SEQ ID NO: 29 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VF and substituting the remaining Q with I.
  • amino acid sequence represented by SEQ ID NO: 30 is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with VL.
  • the amino acid sequence represented by SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 8 with VL and substituting the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO: 32 replaces all QQs in the sequence obtained by repeating twice the domain of the 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) with VF, In addition, the remaining Q is replaced with I.
  • amino acid sequences represented 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 a glutamine residue content of 9% or less. (Table 2).
  • the modified fibroin of (6-i) consists of 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: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32 There may be.
  • the modified fibroin of (6-ii) has 90% or more of 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: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32 And amino acid sequences having the same sequence identity.
  • the modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif A protein containing a sequence.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.
  • modified fibroin containing the tag sequence (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698) or SEQ ID NO: 40 (PRT966), or (6-iv) 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 a modified fibroin having an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 40.
  • amino acid sequences 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, 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 were added with the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) at the N-terminus. Things.
  • amino acid sequence represented by SEQ ID NO: 40 has a glutamine residue content of 9% or less (Table 3).
  • the modified fibroin of (6-iii) comprises 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 There may be.
  • the modified fibroin of (6-iv) has an 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 by 90% or more. And amino acid sequences having the same sequence identity.
  • the modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif A protein containing a sequence.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less.
  • the modified fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a secretion signal for releasing a 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 the host.
  • the modified fibroin is at least two or more of the characteristics of 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. Modified fibroin having both of the following characteristics may be used.
  • the modified fibroin according to any of the above embodiments may be, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.
  • the method for producing the nucleic acid encoding the modified fibroin is not particularly limited.
  • the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding a natural fibroin and modifying it by a genetic engineering technique, or a chemical synthesis method. can do.
  • the method for chemically synthesizing nucleic acids is not particularly limited.
  • AKTA oligopilot plus10010 / 100 Genes can be chemically synthesized by a method of linking oligonucleotides synthesized automatically by PCR or the like.
  • a nucleic acid encoding a modified fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence comprising an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence is synthesized. May be.
  • the regulatory sequence is a sequence that controls the expression of the modified fibroin in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host.
  • An inducible promoter that functions in a host cell and is capable of inducing the expression of a modified fibroin may be used as the promoter.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor such as an increase or decrease in temperature, osmotic pressure, or pH value.
  • the type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like.
  • a plasmid vector a virus vector
  • a cosmid vector a fosmid vector
  • an artificial chromosome vector an artificial chromosome vector
  • those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed are suitably used.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
  • microorganisms belonging to the genus Escherichia include, for example, Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri.
  • Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis and the like.
  • Microorganisms belonging to the genus Microbacterium include, for example, Microbacterium ammonia phyllum.
  • Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum.
  • Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (such as mold).
  • yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like.
  • 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 modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051).
  • any method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. ⁇ Natl. ⁇ Acad. ⁇ Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, competent, and the like.
  • a method for expressing a nucleic acid by a host transformed with an expression vector in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .
  • the modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium.
  • the method of culturing the host in the culture medium can be performed according to a method usually used for culturing the host.
  • the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.
  • a prokaryote such as Escherichia coli or a eukaryote such as yeast
  • a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.
  • the carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolysate, acetic acid and propionic acid, and the like. Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • the inorganic salts for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.
  • ⁇ Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culturing time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium.
  • an inducer may be added to the medium as necessary.
  • lac promoter isopropyl- ⁇ -D-thiogalactopyranoside or the like is used.
  • indole acryl is used.
  • An acid or the like may be added to the medium.
  • the expressed and modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton. The host cells are crushed with a Gaulin homogenizer, Dynomill or the like to obtain a cell-free extract.
  • a method commonly used for isolating and purifying proteins that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia).
  • a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei)
  • cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia).
  • Electrophoretic methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Purification using methods such as alone or in combination It is possible to obtain the goods.
  • the modified fibroin When the modified fibroin is expressed by forming an insoluble form in the cells, the host cells are similarly recovered, crushed, and centrifuged to collect the insoluble form of the modified fibroin as a precipitate fraction.
  • the recovered insoluble form of the modified fibroin can be solubilized with a protein denaturant.
  • a purified sample 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 secreted extracellularly, 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 sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the modified fibroin fiber according to the present embodiment is obtained by spinning the above-described modified fibroin, and contains the above-described modified fibroin as a main component.
  • the density of the modified fibroin fiber according to this embodiment is more than 1.34 g / cm 3 .
  • the density of the modified fibroin fiber is 1.35 g / cm 3 or more, 1.36 g / cm 3 or more, 1.37 g / cm 3 or more, 1.38 g / cm 3 or more, 1.39 g / cm 3 or more, or 1.40 g. / may be cm 3 or more, 1.45 g / cm 3 or less, 1.44 g / cm 3 or less, 1.43 g / cm 3 or less, there at 1.42 g / cm 3 or less, or 1.41 g / cm 3 or less May be.
  • the density of the modified fibroin fiber means a value measured according to JISL 1013.
  • the strength of the modified fibroin fiber according to the present embodiment may be more than 200 MPa, 220 MPa or more, 240 MPa or more, 260 MPa or more, 280 MPa or more, 300 MPa or more, 320 MPa or more, 340 MPa or more, or 350 MPa or more, 600 MPa or less, 500 MPa or less, 400 MPa. Or 380 MPa or less.
  • the strength of the modified fibroin fiber can be measured by the method described in Examples described later.
  • the method for producing a modified fibroin fiber comprises: (a) a first drawing step of drawing a raw fiber containing a modified fibroin in water; and (b) heating the raw fiber that has passed through the first drawing step. And a densification step of drying and thermally shrinking in the radial direction.
  • modified fibroin fibers having high strength for example, having a density of more than 1.34 g / cm 3 .
  • the method for producing a modified fibroin fiber according to the present embodiment may further include (c) a second drawing step of drawing the raw material fiber after the densification step. In this case, the strength of the modified fibroin fiber obtained by the production method is further increased.
  • the raw fiber containing the modified fibroin can be produced through a spinning process.
  • a dope solution containing a solvent for dissolution and modified fibroin dissolved in the solvent for dissolution is extruded into a coagulation liquid to obtain raw fibers containing modified fibroin as undrawn yarn. That is, the raw fiber containing the modified fibroin can be obtained by removing the solvent for dissolution from the dope solution (also referred to as desolvation or coagulation) and forming the fiber by a spinning step.
  • the dissolving solvent examples include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP).
  • DMSO dimethyl sulfoxide
  • DMF N, N-dimethylformamide
  • HFIP hexafluoroisopropanol
  • the dope solution may further contain a dissolution accelerator such as an inorganic salt, if necessary.
  • the concentration of the modified fibroin in the dope solution is preferably 3% by mass or more, more preferably 10% by mass or more, even more preferably 20% by mass or more based on the total amount of the dope solution. From the viewpoint of maintaining good solubility of the modified fibroin, the concentration of the modified fibroin in the dope solution is preferably 60% by mass or less, more preferably 50% by mass or less, and more preferably 40% by mass or less. Is more preferable, and it is still more preferable that it is 30 mass% or less.
  • the coagulation liquid is not particularly limited as long as it can remove the solvent.
  • lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone can be used.
  • Water may be appropriately added to the coagulation liquid.
  • the temperature of the coagulating liquid is, for example, 5 to 30 ° C. If the temperature of the coagulation liquid is within this range, spinning can be performed more stably.
  • an undrawn yarn is obtained by extruding a dope solution from a spinneret (spinner) into a coagulating solution in a desolvation tank.
  • the extrusion speed may be, for example, 0.2 to 6.0 ml / hour per hole, and may be 1.4 to 4 ml. 0.0 ml / hour. If the extrusion speed is in this range, spinning can be performed more stably.
  • First stretching step In the first drawing step, the raw fiber containing the modified fibroin is drawn (first drawing) in a water bath.
  • the first stretching may be performed in one-stage stretching, or may be performed in two or more stages.
  • the modified fibroin is a protein in which molecules such as the modified spider silk fibroin are difficult to orient
  • the molecules can be oriented in multiple stages by performing multi-stage stretching, and the total stretching ratio can be increased. Fiber with a high fiber density can be obtained.
  • the first stretching is performed in a water bath. That is, the first stretching is a wet heat stretching.
  • the water bath may contain a solvent other than water.
  • the temperature of the water bath in the first stretching step may be 5-60 ° C, 10-55 ° C, 15-52 ° C, 20-50 ° C, 30-48 ° C or 35-45 ° C.
  • the temperature of the water bath in the first stretching step is preferably 60 ° C or less.
  • the temperature of the water bath is preferably 5 ° C. or higher.
  • the wet heat draw ratio in the first drawing may be, for example, 1 to 3 times the undrawn yarn.
  • the raw fiber (hereinafter also referred to as “first drawn yarn”) that has passed through the first drawing step is heated and dried, and is thermally shrunk in the radial direction (the direction perpendicular to the fiber axis).
  • first drawn yarn the raw fiber that has passed through the first drawing step is heated and dried, and is thermally shrunk in the radial direction (the direction perpendicular to the fiber axis).
  • the dope solution containing the modified fibroin particularly the modified spider silk fibroin
  • voids are formed inside the raw material fibers.
  • a solvent such as water may be present in the void inside the raw fiber.
  • a solvent eg, water
  • water such as water present in the voids included in the first drawn yarn
  • the densification step may be a step of removing water present in the voids contained in the raw fiber (first drawn yarn) that has passed through the first drawing step and flattening the voids.
  • the densification step may be performed such that the moisture content of the raw fiber (first drawn yarn) that has passed through the first drawing step is 15% or less, 10% or less, or 5% or less.
  • the water content of the raw fiber (first drawn yarn) that has passed through the first drawing step means the ratio of the mass of water to the total mass of the first drawn yarn.
  • the moisture content of the first drawn yarn can be measured by a Karl Fischer method or the like.
  • the densification step may be performed in a drying device.
  • the drying device may be a contact type drying device such as a hot roller or a dry heat plate, or may be a non-contact type drying device such as a hot air drying furnace.
  • the drying device is preferably a non-contact type drying device. In this case, the yarn breakage is further suppressed.
  • the temperature (heating temperature) and the time (heating time) in the densification step are not particularly limited as long as moisture in the voids present in the first drawn yarn can be removed and the fiber can shrink in the radial direction.
  • the temperature (heating temperature) in the densification step is 70 to 150 ° C, 75 to 140 ° C, 78 to 135 ° C, 80 to 120 ° C, 82 to 110 ° C, or 85 to 100 ° C. It may be.
  • the time (heating time) in the densification step may be 5 to 40 seconds, 7 to 35 seconds, or 8 to 30 seconds.
  • the densification step is preferably performed in a state where tension is applied to the first drawn yarn to prevent shrinkage in the length direction.
  • the amount of shrinkage (diameter reduction amount) of the first drawn yarn in the radial direction is further increased, and the voids are further flattened. Furthermore, by flattening the voids in the densification step, the occurrence of yarn breakage in the second stretching step described later is further suppressed.
  • the densification step may be performed, for example, with the end of the first drawn yarn fixed.
  • the fixing of the first drawn yarn may be capable of reducing the shrinkage in the length direction.
  • the first drawn yarn may be fixed by, for example, stretching a drawn yarn of a predetermined length without looseness in a state where no external force is applied (natural state), and fixing both ends of the fiber in this state.
  • the end portion of the first drawn yarn may be fixed at least at the end portion of the first drawn yarn, and the whole raw material fiber may be fixed by sandwiching the whole raw material fiber between two metal plates. Good.
  • the means for fixing the raw fibers is not particularly limited, and any fixing means may be used.
  • the fixing means is preferably a detachable one, and examples thereof include an adhesive tape, an adhesive, and a clamp.
  • the densification step may be performed between the first stretching step and the second stretching step.
  • the densification step may be performed once, or may be performed in multiple stages by providing a drying device in multiple stages, but since the effect of the present invention is more remarkably exhibited, it is preferable to perform the process once. Is preferred.
  • ⁇ Second stretching step> the raw fibers that have undergone the densification step are drawn (second drawing).
  • the voids flattened in the densification step can be further flattened.
  • the resulting modified fibroin fiber has a higher density, and as a result, a modified fibroin having higher strength is obtained.
  • the second stretching step is performed after flattening the voids in the densification step, the occurrence of yarn breakage in the second stretching step is further suppressed.
  • the second stretching may be performed in one-stage stretching or in two or more stages.
  • the second stretching may be wet heat stretching or dry heat stretching.
  • the wet heat stretching in the second stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating. Note that the wet heat stretching performed during the steam heating is referred to as steam stretching.
  • the second stretching is preferably a steam stretching or a dry heat stretching, particularly preferably a steam stretching.
  • the second stretching step preferably includes performing steam stretching. It is expected that high stretching can be achieved more reliably by steam stretching because of high thermal conductivity or by the moisture permeated into the fiber functioning as a plasticizer or the like.
  • the temperature for performing the wet heat stretching other than the steam stretching may be, for example, 50 to 90 ° C, and preferably 75 to 85 ° C.
  • the temperature for performing the steam stretching may be, for example, 100 to 200 ° C., 100 to 150 ° C., or 100 to 120 ° C.
  • the wet heat stretching ratio in the second stretching may be 1 to 10 times or 2 to 8 times with respect to the raw material fiber that has passed through the densification step.
  • Dry heat drawing can be performed using an electric tube furnace, a dry heat plate or the like.
  • the second stretching step preferably includes a stretching operation on a dry heat plate.
  • the temperature at which the dry heat stretching is performed (dry heat drawing temperature) may be, for example, 140 ° C. to 270 ° C., or may be 150 ° C. to 260 ° C.
  • the dry heat drawing ratio in the second drawing may be, for example, 0.5 to 8 times or 1 to 6 times with respect to the raw material fiber that has passed through the densification step. If the second stretching step is performed by dry heat stretching, it is expected that re-entry of water into the void from which the solvent such as water has been removed in the densification step is prevented.
  • the wet heat stretching and the dry heat stretching may each be performed alone, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and further the third stage stretching is performed by dry heat stretching.
  • wet heat stretching and dry heat stretching can be performed in an appropriate combination.
  • the lower limit of the total draw ratio is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more of the undrawn yarn. , 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less, It is 12 times or less, 11 times or less, and 10 times or less.
  • the first stretching step, the densification step, and the second stretching step may or may not be performed continuously.
  • the raw material fiber after the densification step is used as a wound body, and while the wound body is unwound, the second fiber is unwound.
  • a stretching step may be performed.
  • FIGS. 6 and 7 are explanatory diagrams showing an example of an apparatus for producing a modified fibroin fiber.
  • the manufacturing apparatus shown in FIG. 6 is a spin stretching apparatus that continuously performs a spinning step and a first stretching step.
  • the spinning and drawing apparatus 100 includes an extruder 1, an undrawn yarn manufacturing apparatus 2, a wet heat drawing apparatus 3, a drying apparatus 10, and a dry heat drawing apparatus 4.
  • the dope solution 6 is stored in a storage tank 7 and pushed out of a base 9 by a gear pump 8.
  • a dope solution may be filled in a cylinder and extruded from a nozzle using a syringe pump.
  • the extruded dope liquid 6 is supplied into the coagulation liquid 11 of the coagulation liquid tank 20 via the air gap 19 or directly without passing through the air gap 19.
  • the solvent is removed from the dope solution 6 in the coagulation solution 11, and an undrawn yarn (raw material fiber) is formed.
  • the undrawn yarn is supplied into the hot water 12 in the drawing bath 21 and the first-stage drawing is performed.
  • the stretching ratio is determined by the speed ratio between the supply nip roller 13 and the take-off nip roller 14.
  • the drawn yarn (first drawn yarn) that has passed through the first-stage drawing is then supplied to the drying device 10 and heated in the yarn path 22a.
  • the heated first drawn yarn is supplied to the dry heat drawing device 17, and the second stage drawing is performed in the yarn path 22b.
  • the drawn yarn (second drawn yarn) that has passed through the second-stage drawing is wound up to form a wound body 5.
  • the stretching ratio in the second stage stretching is determined by the speed ratio between the supply nip roller 15 and the take-off nip roller 16.
  • 18a to 18g are yarn guides.
  • FIG. 7 The production apparatus shown in FIG. 7 is a spinning and stretching apparatus that performs a spinning step and a stretching step (two-stage stretching).
  • FIG. 7A shows an undrawn yarn production device 30, a first-stage drawing device 40, and a drying device 60
  • FIG. 7B shows a second-stage drawing device 50.
  • the yarn may be wound in each device or stored in a container without being wound.
  • the dope liquid 32 is put in an extruder (micro syringe) 31, and is moved in the direction of arrow P using a syringe pump, and the dope liquid 32 is extruded from the nozzle 33 to form a coagulating liquid tank.
  • the dope liquid 32 is supplied to the coagulating liquid 35 in 34 to make an undrawn yarn 36 (raw fiber).
  • the undrawn yarn 36 is supplied into the hot water 38 in the drawing bath 37 to perform the first-stage drawing.
  • the first-stage drawn yarn (first drawn yarn) is supplied to the drying device 60 and heated in the yarn path 61.
  • the first-stage drawn yarn after heating is wound into a wound body 39.
  • the stretching ratio is determined by the speed ratio between the supply nip roller 41 and the take-off nip roller 42.
  • the first-stage drawn yarn is drawn from the wound body 39, supplied to the dry heat drawing device 43, and subjected to the second-stage drawing in the yarn path 47.
  • the stretching ratio is determined by the speed ratio between the supply nip roller 45 and the take-off nip roller 46.
  • the second-stage drawn yarn (second drawn yarn) is wound around the wound body 44.
  • a modified fibroin (PRT799) having the amino acid sequence represented by SEQ ID NO: 15 was designed.
  • a nucleic acid encoding the designed modified fibroin was synthesized.
  • An NdeI site was added to the 5 'end of the nucleic acid, and an EcoRI site was added downstream of the stop codon.
  • This nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was digested with NdeI and EcoRI and cut out, followed by recombination into a protein expression vector pET-22b (+) to obtain an expression vector.
  • Escherichia coli BLR (DE3) was transformed with the obtained pET-22b (+) expression vector.
  • the transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 became 0.005.
  • the temperature of the culture was maintained at 30 ° C., and the flask was cultured until the OD 600 reached 5 (about 15 hours) to obtain a seed culture.
  • the seed culture solution was added to a jar fermenter to which 500 mL of a production medium (Table 5) had been added so that the OD 600 was 0.05.
  • the temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the concentration of dissolved oxygen in the culture was maintained at 20% of the saturated concentration of dissolved oxygen.
  • a feed solution (455 g / 1 L of glucose, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Culture was performed 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 the expression of the desired modified fibroin. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of a band corresponding to the size of the target modified fibroin depending on the addition of IPTG revealed the presence of the target modified fibroin. Expression was confirmed.
  • the precipitate after washing 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) so as to have a concentration of 100 mg / mL, and then suspended at 60 ° C. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation. Water was removed from the collected aggregated protein using a freeze dryer to obtain a freeze-dried powder of the desired modified fibroin.
  • 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • modified fibroin fibers of Examples 1 to 4 and Comparative Example 1 were produced.
  • the modified fibroin fibers of Examples 1 to 3 were obtained by spinning and drawing using the spinning and drawing apparatus 100 shown in FIG.
  • the modified fibroin fiber of Example 4 was spun and drawn by using a spin drawing apparatus provided with a wet heat drawing apparatus (steam drawing (steam drawing) apparatus) instead of the dry heat drawing apparatus in the spin drawing apparatus 100 shown in FIG. It is what went.
  • the modified fibroin fiber of Comparative Example 1 was obtained by spinning and drawing using a spinning and drawing apparatus without the drying apparatus 10 in the spinning and drawing apparatus 100 of FIG.
  • the manufacturing conditions are as follows.

Abstract

The present invention pertains to modified fibroin fibers comprising a modified fibroin and having a density greater than 1.34 g/cm3.

Description

改変フィブロイン繊維及びその製造方法Modified fibroin fiber and method for producing the same
　本発明は、改変フィブロイン繊維及びその製造方法に関する。 (4) The present invention relates to a modified fibroin fiber and a method for producing the same.
　従来から、人造繊維の製造方法として、繊維形成成分を所定の溶媒中に溶解してなる紡糸原液をノズルから凝固浴液中に吐出させ、凝固浴液中で凝固させて繊維を形成する湿式紡糸法及び乾湿式紡糸法が知られている。 2. Description of the Related Art Conventionally, as a method for producing artificial fibers, a wet spinning method in which a spinning solution obtained by dissolving a fiber-forming component in a predetermined solvent is discharged from a nozzle into a coagulation bath solution and coagulated in the coagulation bath solution to form fibers. Methods and dry-wet spinning methods are known.
　湿式紡糸法及び乾湿式紡糸法は、改変フィブロインを主成分として含む改変フィブロイン繊維を製造する際にも利用されている（例えば、特許文献１参照）。 The wet spinning method and the dry-wet spinning method are also used when producing modified fibroin fibers containing modified fibroin as a main component (for example, see Patent Document 1).
特許第５５４０１５４号公報Japanese Patent No. 5540154
　改変フィブロイン繊維は、用途に応じて様々な特性が要求され、その一つとして十分な強度を有していることが求められる場合がある。ところが、湿式法及び乾湿式法で得られる改変フィブロイン繊維においては、凝固液中での溶媒の離脱により原料繊維内部にボイドが生じ、それが原因で強度不足となることがあった。 Various properties are required for the modified fibroin fiber depending on the application, and one of them may be required to have sufficient strength. However, in the modified fibroin fibers obtained by the wet method and the dry-wet method, voids are generated inside the raw material fibers due to the removal of the solvent in the coagulation liquid, and the strength may be insufficient due to the voids.
　そこで、本発明は、高い強度を有する改変フィブロイン繊維を提供することを目的とする。本発明はまた、高い強度を有する改変フィブロイン繊維の製造方法を提供することも目的とする。 Therefore, an object of the present invention is to provide a modified fibroin fiber having high strength. Another object of the present invention is to provide a method for producing a modified fibroin fiber having high strength.
　本発明は、例えば、以下の各発明に関する。
［１］
　改変フィブロインを含み、密度が１．３４ｇ／ｃｍ３超である、改変フィブロイン繊維。
［２］
　改変フィブロインが改変クモ糸フィブロインである、［１］に記載の改変フィブロイン繊維。
［３］
　（ａ）改変フィブロインを含む原料繊維を水浴中で延伸する第一の延伸工程と、（ｂ）第一の延伸工程を経た原料繊維を加熱して、乾燥させると共に、径方向に熱収縮させる緻密化工程と、を備える、改変フィブロイン繊維の製造方法。
［４］
　緻密化工程が、第一の延伸工程を経た原料繊維に含まれるボイドに存在する水分を除去すると共に、該ボイドを扁平化する工程である、［３］に記載の改変フィブロイン繊維の製造方法。
［５］
　水浴の温度が５～６０℃である、［３］又は［４］に記載の改変フィブロイン繊維の製造方法。
［６］
　（ｃ）緻密化工程を経た原料繊維を延伸する第二の延伸工程を更に備える、［３］～［５］のいずれかに改変フィブロイン繊維の製造方法。
［７］
　第二の延伸工程が、スチーム延伸を行うことを含む、［６］に記載の改変フィブロイン繊維の製造方法。
［８］
　第二の延伸工程が、乾熱延伸を行うことを含む、［６］又は［７］に記載の改変フィブロイン繊維の製造方法。
［９］
　改変フィブロインが改変クモ糸フィブロインである、［３］～［８］のいずれかに記載の改変フィブロイン繊維の製造方法。 The present invention relates to, for example, the following inventions.
[1]
A modified fibroin fiber comprising the modified fibroin and having a density greater than 1.34 g / cm 3 .
[2]
The modified fibroin fiber according to [1], wherein the modified fibroin is a modified spider silk fibroin.
[3]
(A) a first drawing step of drawing a raw fiber containing modified fibroin in a water bath, and (b) a heating step of heating and drying the raw fiber after the first drawing step, and heat-shrinking the fiber in the radial direction. And producing a modified fibroin fiber.
[4]
The method for producing a modified fibroin fiber according to [3], wherein the densification step is a step of removing water present in the voids contained in the raw fiber after the first drawing step and flattening the void.
[5]
The method for producing a modified fibroin fiber according to [3] or [4], wherein the temperature of the water bath is 5 to 60 ° C.
[6]
(C) The method for producing a modified fibroin fiber according to any one of [3] to [5], further comprising a second drawing step of drawing the raw material fiber after the densification step.
[7]
The method for producing a modified fibroin fiber according to [6], wherein the second drawing step includes performing steam drawing.
[8]
The method for producing a modified fibroin fiber according to [6] or [7], wherein the second drawing step includes performing dry heat drawing.
[9]
The method for producing a modified fibroin fiber according to any one of [3] to [8], wherein the modified fibroin is a modified spider silk fibroin.
　本発明によれば、高い強度を有する改変フィブロイン繊維を提供することができる。また、本発明によれば、高い強度を有する改変フィブロイン繊維の製造方法を提供することができる。 According to the present invention, a modified fibroin fiber having high strength can be provided. Further, according to the present invention, a method for producing a modified fibroin fiber having high strength can be provided.
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。It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. 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。It is a figure which shows the distribution of the value of z / w (%) of the fibroin derived from nature. 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。It is a figure which shows the distribution of the value of x / y (%) of fibroin derived from nature. 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。It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. 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。It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. 改変フィブロイン繊維の製造装置の一例を示す説明図である。It is explanatory drawing which shows an example of the manufacturing apparatus of a modified fibroin fiber. 改変フィブロイン繊維の製造装置の一例を示す説明図である。図７（Ａ）は、未延伸糸製造装置－１段目延伸装置を示し、図７（Ｂ）は２段目延伸装置を示す。It is explanatory drawing which shows an example of the manufacturing apparatus of a modified fibroin fiber. FIG. 7 (A) shows an undrawn yarn production device—first-stage drawing device, and FIG. 7 (B) shows a second-stage drawing device.
　以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
〔改変フィブロイン繊維〕
　本実施形態に係る改変フィブロイン繊維は、改変フィブロインを含み、密度が１．３４ｇ／ｃｍ３超である。 (Modified fibroin fiber)
The modified fibroin fiber according to the present embodiment contains modified fibroin, and has a density of more than 1.34 g / cm 3 .
＜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. Including proteins. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed 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。本明細書でいう「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, the term “modified fibroin” means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be the same as the amino acid sequence of naturally occurring fibroin. The term “naturally-derived fibroin” as used herein is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Is a protein containing the domain sequence to be determined.
　「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。 "Modified fibroin" may be a directly used amino acid sequence of a naturally occurring fibroin, or a modified amino acid sequence based on the amino acid sequence of a naturally occurring fibroin (for example, cloned naturally occurring fibroin). The amino acid sequence may be modified by modifying the gene sequence of fibroin), or may be artificially designed and synthesized without using naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence may be used). Which have 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、（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。 As used herein, the term “domain sequence” refers to a crystalline region unique 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). The amino acid represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Means an array. Here, the (A) n motif indicates an amino acid sequence mainly containing an alanine residue, and has 2 to 27 amino acid residues. (A) The number of amino acid residues in 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 . (A) The ratio of the number of alanine residues to the total number of amino acid residues in the n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. The plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.
　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、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 has, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to a cloned natural fibroin gene sequence. Can be obtained by performing the following modification. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982) and Methods {in} Enzymology, 100, 448 (1983).
　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 containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Yes, specifically, for example, fibroin produced by insects or spiders.
　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 the fibroin produced by insects include Bombyx @ mori, Bombyx @ mandarina, natural silkworm (Antheraea @ yamamai), tussah (Anterea @ pernii), and maple silkworm (Erioganyerii). ), Silkworms produced by silkworms (Samia cynthia), chestnut worms (Caligura japonica), tussah silkworms (Antheraea mylitta), silkworms such as moga silkworms (Antheraea assama), and silkworms produced by larvae of the hornet beetle Hornet silk protein.
　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。 よ り More specific examples of fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank Accession No. 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。 Examples of the fibroin produced by the spiders include spiders belonging to the genus Araneus (genus Araneus), such as the spiders, the spiders, the red spider, the red spider, the green spider, etc. Spiders belonging to the genus Argiope genus (Genus Pronus), such as spiders belonging to the genus Procarpus spp., Spiders belonging to the genus Pronus, and spider spiders belonging to the genus Cynotarachne, such as the genus Cyrtarachne, such as Torinofundamashi and Otorinofundamashi. Spiders belonging to the genus Ordgarius, such as spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Orbalis, and spiders belonging to the genus Ordgarius, such as the spiders belonging to the genus Ordgarius. Spiders belonging to the genus Argiope, such as Argiope bruennichi, spiders belonging to the genus Argiope, such as Argiope spiders, spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas, such as spiders belonging to the genus Acusilas, and spider spiders belonging to the spider spiders, such as the spider spiders belonging to the genus Acusilas Spiders belonging to the genus Spider (Genus Poltys) such as spiders belonging to the genus (Cytophora) and spiders belonging to the genus (Potys), spiders belonging to the genus Spiders (genus Cyclosa) such as the spiders belonging to the genus Cyclosa and spiders belonging to the genus Cygnus spp. Spider silk proteins produced by spiders belonging to the genus Chorizopes), and asina such as red-headed spiders, red-backed spiders, blue-backed spiders and urocore-red spiders Spiders belonging to the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucaegium, such as the spiders Argiope bruennichi and spiders spiders belonging to the genus Leucauge, such as the spiders belonging to the genus Nephila sp. Spiders belonging to the genus Dyschiriognatha such as spiders belonging to the genus Menosira and spiders belonging to the genus Dyschiriognatha, such as spiders belonging to the genus Dyschiriognatha, and spiders belonging to the genus Latus sp. Spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops (Euprosthenops) are produced. Spider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
　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 number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from amino acid sequence of Nephila claviBAC04A4 and derived from amino acid sequence of ｐｈ ), U37520 (base sequence)), major \ ampullate \ spidro n 1 [Derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Derived from Nephila clavata (GenBank accession number ABA45, amino acid sequence of A23) )), Major \ sample \ spidroin \ 1 [from Euprosthenops \ australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ97155 (base sequence)), and major \ sample \ spidroin \ 2 [Euprosus] ] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor \ silk \ protein \ 1 [Nephila \ clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor \ ampilatte [in] Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampoulate spidroin-like protein [Nephilengys Cruenata] (GenBank Accession No. ABR3727.1.
　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。 よ り More specific examples of naturally occurring fibroin include fibroin in which sequence information is registered in NCBI GenBank. For example, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" is described as a keyword in DEFINITION from among sequences including INV as DIVISION in the sequence information registered in NCBI @ GenBank. It can be confirmed by extracting a character string of a specific product from a sequence or CDS, and extracting a sequence in which a specific character string is described in TISSUE @ TYPE from SOURCE.
　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 (an amino acid sequence of a silk protein produced by a silkworm modified), and a modified spider silk fibroin (a spider silk protein produced by an arachnid). Modified amino acid sequence). As the modified fibroin, a modified spider silk fibroin is preferable.
　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（第４の改変フィブロイン）、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。 Specific examples of the modified fibroin include a modified fibroin (first modified fibroin) derived from a large spinal cord marker protein produced in a spider's large ampullate gland, and a domain sequence having a reduced content of glycine residues. (A second modified fibroin), (A) a modified fibroin having a domain sequence with a reduced content of the n motif (a third modified fibroin), a glycine residue content, and (A) n Modified fibroin having a reduced motif content (fourth modified fibroin), modified fibroin having a domain sequence containing a region having a large hydrophobicity index (fifth modified fibroin), and glutamine residue content Modified fibroin having a reduced domain sequence (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 containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . In the first modified fibroin, the number of amino acid residues 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 still more preferable, an integer of 4 to 16 is still more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable. In the first modified fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and more preferably 20 to 100 residues. Is even more preferable, and even more preferably 20 to 75 residues. The first modified fibroin has the total number of glycine, serine and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) n motif-REP] m The total number is preferably 40% or more, more preferably 60% or more, and even 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 Formula 1: [(A) n motif-REP] m , and has a C-terminal sequence represented by any of SEQ ID NOS: 1 to 3 or The polypeptide may be an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one 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 the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is The amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. 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 more specific examples of the first modified fibroin, (1-i) an amino acid sequence represented by SEQ ID NO: 4 (recombinant \ spider \ silk \ protein \ ADF3KaiLargeNRSH1) or (1-ii) an amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroin comprising an amino acid sequence having at least% 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 represented by SEQ ID NO: 4 is the same as the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human \ rhinovirus @ 3C protease) recognition site at the N-terminus is added. The 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue. The amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by 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 have an amino acid sequence represented 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 at least one or more glycine residues in the REP replaced by another amino acid residue, as compared to a 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。 The second modified fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine, as compared with a naturally-derived fibroin. At least one motif sequence selected from the group consisting of at least one glycine residue in one or more of the motif sequences has been replaced with another amino acid residue. You may.
　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。 は In the second modified fibroin, the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of 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 comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and from the (A) n motif located at the most C-terminal side to the domain sequence The total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus is represented by z, From, when the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is defined as w, z / w is 30% or more; It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only 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。 ２ The second modified fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by substituting 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, further preferably 10% or less, and 6% or less. %, Still more preferably 4% or less, further preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGGX 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 a fibroin containing a domain sequence represented by Formula 1: [(A) n motif-REP] m (modified fibroin or naturally occurring fibroin), (A) n located closest to the C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (there is no duplication), z is 50 × 3 = 150. Further, for example, when there is an X (center X) included in two XGXs as in the case of an amino acid sequence consisting of XGXGX, calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues are used. is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located closest to the C-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 the naturally derived fibroin will be described. First, as described above, when fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the exemplified method, 663 kinds of fibroins (among them, 415 kinds of spider-derived fibroins) were extracted. Of all the extracted fibroins, a naturally occurring one containing a domain sequence represented by Formula 1: [(A) n motif-REP] m and containing 6% or less of a GGX amino acid sequence in the fibroin Z / w was calculated from the amino acid sequence of the fibroin by the above calculation method. The result is shown in FIG. The horizontal axis in FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is evident from FIG. 2, the z / w of the naturally derived fibroin is less than 50.9% (the highest one is 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. Is still more preferred, and even more preferably 80% or more. The upper limit of z / w is not particularly limited, but 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 is modified, for example, by replacing at least a part of the base sequence encoding a glycine residue from the cloned natural fibroin gene sequence to encode another amino acid residue. Obtainable. At this time, as the glycine residue to be modified, a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more. Alternatively, for example, the amino acid sequence can be obtained by designing an amino acid sequence satisfying the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, in addition to the modification corresponding to the substitution of another amino acid residue for the glycine residue in the REP from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. The amino acid sequence corresponding to insertion, addition, and / or addition may be modified.
　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 other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but includes a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and a methionine ( M) residue, hydrophobic amino acid residue such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues, and valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are 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。 More specific examples of the second modified fibroin include (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), 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 (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 6 is obtained by replacing all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin with GQX. The amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In which one [(A) n motif-REP] was inserted. The amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some amino acids on the C-terminal side have been deleted so that the molecular weight becomes almost the same as SEQ ID NO: 7. The amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). Is repeated four times with a predetermined hinge sequence and His tag sequence added to the C-terminal.
　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％及び８９．８％である。 Ｚ The value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The values 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: 9 are 58.7%, respectively. 70.1%, 66.1% and 70.0%. In addition, the value of x / y at the jagged ratio (described later) of 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 as follows: They are 15.0%, 15.0%, 93.4%, 92.7% 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 have an amino acid sequence represented 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) contains 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. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（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５０．９％以上であることが好ましい。 The modified fibroin of (2-ii) has 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, and contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of 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。 ２The second modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.
　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。 Examples of the tag sequence include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography (chelating @ metal @ chromatography). Can be used for Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 11 (amino acid sequence including a His tag sequence and a 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。 タ グ Alternatively, tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose binding protein (MBP), which specifically binds to maltose, can 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, an “epitope tag” utilizing an antigen-antibody reaction can 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 an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag. By using the epitope tag, the 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。 Further, a tag sequence that can be cleaved by a specific protease can be used. By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified fibroin from which the tag sequence has been separated can also be recovered.
　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）若しくは配列番号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。 As more specific examples of the modified fibroin containing a tag sequence, (2-iii) the amino acid represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) Modified fibroin comprising a sequence or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .
　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 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. An amino acid sequence represented by SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) is added to the N-terminal 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 have an amino acid sequence represented 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) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented 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 containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The 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 represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of 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。 {Circle around (2)} The second modified fibroin may include a secretion signal for releasing a 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 the 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 whose domain sequence has a reduced content of the (A) n motif as compared to a naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (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 to 40% deletion of the (A) n motif 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 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 domain sequence deletion of at least two (A) n motifs from the N-terminal side to the C-terminal side, and one (A) It may have an amino acid sequence corresponding to the deletion of the n motif 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 a domain sequence having an amino acid sequence corresponding to the deletion of the (A) n motif at least every third sequence from 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 comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) n motifs from the N-terminal side to the C-terminal side] -REP] The number of amino acid residues of REP in the unit is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to When the maximum value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units that is 11.3 is x, and the total number of amino acid residues in 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 number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only 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 a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence 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 an 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 an unselected [(A) n motif-REP] unit. FIG. 1 shows pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP with the fourth REP), pattern 2 (comparison of the first REP and the second REP, and Pattern 4 (comparison of the second REP with the third REP, and comparison of the fourth REP with the fifth REP), pattern 4 (the comparison of the fourth REP with the fifth REP), and pattern 4 (the first REP with the fifth REP). Second REP comparison). Note that there are other selection methods.
　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 selected adjacent [(A) n motif-REP] units is compared. The comparison is performed by determining the ratio of the number of the other amino acid residues when the smaller number of the amino acid residues is set to 1. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is set to 1, the second REP is The ratio of the number of amino acid residues is 100/50 = 2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having a smaller number of amino acid residues is set to 1, the fifth REP 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, when the smaller number of amino acid residues is set to 1, the [(A) n motif-REP] unit pair in which the ratio of the other amino acid residues is 1.8 to 11.3 is set. Shown by solid line. In the present specification, this ratio is called a jagged ratio. Assuming that the smaller number of amino acid residues is 1, the pair of [(A) n motif-REP] units in which the ratio of the other amino acid residues is less than 1.8 or more than 11.3 is indicated by a broken line. 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 total number of amino acid residues of two adjacent [(A) n motif-REP] units shown by a solid line is added (not only the REP but also the number of amino acid residues of the (A) n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
　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 in 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 at least 50%, more preferably at least 60%, further preferably at least 65%, and even more preferably at least 70%. Preferably, it is still more preferably at least 75%, particularly preferably at least 80%. The upper limit of x / y is not particularly limited, and may be, for example, 100% or less. When the indentation ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the indentation ratio is 1: 1.8 to 3.4, x / y is x / y. / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9%, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably at least 64.2%.
　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 present in a plurality in the domain sequence are composed of only alanine residues, x / y should be 46.4% or more. Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, and even more preferably 80% or more. It is particularly preferred that there is. 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 the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the exemplified method, 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted. Among all the extracted fibroins, x / y was calculated from the amino acid sequence of the naturally occurring fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above calculation method. Was calculated. FIG. 3 shows the results when the indentation ratio is 1: 1.9 to 4.1.
　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％）。 横 The horizontal axis in FIG. 3 indicates x / y (%), and the vertical axis indicates frequency. As is evident from FIG. 3, the x / y of the naturally derived fibroin is less than 64.2% (the highest is 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 sequence encoding the (A) n motif from the cloned natural fibroin gene sequence such that x / y is 64.2% or more. Can be obtained. Further, for example, an amino acid sequence corresponding to deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more based on the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence. In any case, in addition to the modification corresponding to the deletion of the (A) n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. Amino acid sequence modification corresponding to the above may be performed.
　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-PRT525), or SEQ ID NO: 9 (Met-PRT525) -PRT799), or (3-ii) an amino acid sequence 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 (3-i) will be described. The amino acid sequence represented by SEQ ID NO: 17 differs from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin in that every two amino acids from the N-terminal side to the C-terminal side (A) n The motif was deleted, and one [(A) n motif-REP] was inserted before the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for 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。 Ｘ The amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) has an x / y value of 15.0% at a giza ratio of 1: 1.8 to 11.3. The value of x / y in the amino acid sequence represented by SEQ ID NO: 17 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences represented 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 be composed of the amino acid sequence represented 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) contains an amino acid sequence 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. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（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 (3-ii) has 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, and is N-terminal to C-terminal. , The number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other Amino acid residues of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (a giza ratio of 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 of the base numbers and 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、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 include the above-described tag sequence at one 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 more specific examples of the modified fibroin containing a tag sequence, (3-iii) the amino acid represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) Modified fibroin comprising a sequence or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .
　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 represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding SEQ ID NO: 11 to the N-terminal of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. (Including a His tag sequence and a hinge sequence).
　（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 have an amino acid sequence represented 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) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented 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 containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The 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 represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 and is N-terminal to C-terminal. , The number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other The maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 is defined as 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 include a secretion signal for releasing a 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 the 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 whose domain sequence has a reduced content of a glycine residue in addition to the content of the (A) n motif reduced as compared to a naturally-derived fibroin. Have The domain sequence of the fourth modified fibroin is at least one or more (A) n motifs deleted, and further at least one or more glycine residues in the REP, as compared to naturally occurring fibroin. It can be said that it has an amino acid sequence equivalent to being replaced with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having both the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects 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) ), The amino acid sequence represented by SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), 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 And a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by. Specific embodiments of the modified fibroin comprising the amino acid sequence represented by 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 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 has a domain sequence in which one or more amino acid residues in REP have been replaced by amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin, and / or It may have an amino acid sequence locally including a region having a large hydrophobicity index, corresponding to insertion of one or more amino acid residues having a large hydrophobicity index 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。 領域 A region having a locally 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 is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A). More preferably, 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。 The fifth modified fibroin may have one or more amino acid residues in the REP replaced with amino acid residues having a higher hydrophobicity index, and / or one or more amino acids in the REP as compared to a naturally occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, one or more amino acid residues may be substituted, deleted, inserted and / or added as compared with naturally occurring fibroin. There may be an amino acid sequence modification corresponding to the 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, one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned natural fibroin gene sequence, It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP. In addition, for example, one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP. Can be obtained by designing an amino acid sequence corresponding to the insertion of the amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in 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 Formula 1: [(A) n motif-REP] m and extends from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence. In all REPs included in the sequence excluding the sequence from the domain sequence, the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p, When the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q, p / q is 6 .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。 Regarding the hydrophobicity index of amino acid residues, a publicly known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for display, the hydropathic charactor of aa protein, J.Pol.Mol. 105-132). Specifically, the hydropathic index (hydropathic 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. For the calculation, the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m (Hereinafter, referred to as “sequence A”). First, in all REPs included in sequence A, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.
　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 “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” are extracted at 20 locations (without duplication), the average value of the hydrophobicity index of the 4 consecutive amino acid residues is 2 The region of .6 or more would contain 20 consecutive 4 amino acid residues (no duplication), and p would be 20 × 4 = 80. For example, when two “consecutive four 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 four consecutive amino acid residues A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “−1” is a deduction for the overlap). For example, in the case of the domain sequence shown in FIG. 4, there are seven consecutive “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without overlapping. 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 (the last (A) n motif on the C-terminal side is not included). 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, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more. The upper limit of p / q is not particularly limited, but 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 a plurality of hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP so that the amino acid sequence of the cloned natural fibroin is satisfied so as to satisfy the above-mentioned p / q conditions. Substituting a negative amino acid residue with a hydrophobic amino acid residue (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into the REP By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Further, for example, it can also be obtained by designing an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP have been replaced by amino acid residues with a higher hydrophobicity index and / or one or more amino acid residues in the REP as compared to naturally occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, a 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, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A Is 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 a more specific example of the fifth modified fibroin, (5-i) the amino acid sequence represented by SEQ ID NO: 19 (Met-PRT665), 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 90% or more sequence identity with the amino acid sequence represented 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 represented by SEQ ID NO: 19 consists of three amino acid residues every other REP, except for the domain sequence of the terminal at the C-terminal side, with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410). In this amino acid sequence, two amino acid sequences (VLI) were inserted, some glutamine (Q) residues were further substituted with serine (S) residues, and some C-terminal amino acids were deleted. The amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525). is there. The amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented 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 be composed of the amino acid sequence represented 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) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（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 represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and is located at the most C-terminal side (A) n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When 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 at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.
　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 include a tag sequence at one 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。 As more specific examples of the modified fibroin containing the tag sequence, (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) ) Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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 represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 correspond to the amino acid sequence represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).
　（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 have an amino acid sequence represented 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) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（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 represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located at the most C-terminal side (A) n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When 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 at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.
　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。 (5) The fifth modified fibroin may include a secretion signal for releasing a 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 the 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 in which the content of glutamine residues is reduced as compared with 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。 ６ The sixth modified fibroin preferably contains at least one motif selected from GGX motif and 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。 場合 When the sixth modified fibroin contains a GPGXX motif in the REP, the content of the GPGXX motif is usually 1% or more, and 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, the “GPGXX motif content” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) 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 at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total number by 3 (ie, the total number of G and P in the GPGXX motif) is represented by s, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is represented by (A) When the total number of amino acid residues of all REPs excluding the (A) n motif is represented by t, the content of the GPGXX motif is calculated as s / t. It is.
　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 the content ratio of the GPGXX motif, “the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” to “the most C-terminal side” (A) Sequence from n motif to C-terminus of domain sequence (sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of fibroin, and m may be small. In this case (that is, when the domain sequence is short), the calculation result of the GPGXX motif content is affected, so that this effect is eliminated. When the “GPGXX motif” is located at the C-terminus of the REP, even when “XX” is, for example, “AA”, it is treated as a “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 diagram showing the domain sequence of a modified fibroin. The method of calculating the content rate of the GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin shown in FIG. 5 (“[(A) n motif-REP] m- (A) n motif” type), all REPs are located at the most C-terminal side. (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 as“ region A ”in FIG. 5). The number of GPGXX motifs is 7, and s is 7 × 3 = 21. Similarly, all REPs are “sequences obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” (the sequence shown as “region A” in FIG. 5). ), The total number t of amino acid residues of all REPs excluding the (A) n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t, and in the case of the modified fibroin of FIG. 5, 21/150 = 14.0%.
　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, the “glutamine residue content” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the total number of glutamine residues contained in the region is defined as u, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence, and further (A) n The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif. In the calculation of the glutamine residue content, the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.
　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 the fact that its domain sequence has one or more glutamine residues in the REP deleted or replaced with other amino acid residues, as compared to the 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, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can. Among them, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. , 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 REP of -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more, and 0.3 or more. Is still more preferable, and it is particularly preferable that it is 0.4 or more. The upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or may be 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, “REP hydrophobicity” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is defined as v, and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal 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 REPs excluding n motifs. In the calculation of the degree of hydrophobicity of the REP, the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as follows. The same is true.
　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 may have a domain sequence that is missing one or more glutamine residues in the REP and / or one or more glutamine residues in the REP, as compared to the naturally occurring fibroin. In addition to the modification corresponding to the substitution of with another amino acid residue, there may be an amino acid sequence modification equivalent to the 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 remove one or more glutamine residues in the REP. By substituting the amino acid residue with In addition, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the above 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 more specific examples of the sixth modified fibroin, (6-i) SEQ ID NO: 25 (Met-PRT988), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT889) -PRT916), modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 29 (Met-PRT699), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698) or SEQ ID NO: 32 (Met-PRT966), or (6-ii) having 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: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32 Modified fibroin containing the amino acid sequence having the same.
　（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 represented by SEQ ID NO: 25 is obtained by substituting VL for all QQ in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410). The amino acid sequence represented by SEQ ID NO: 26 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with TS, and substituting the remaining Q with A. The amino acid sequence represented by SEQ ID NO: 27 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VL, and substituting the remaining Q with I. The amino acid sequence represented by SEQ ID NO: 28 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with VI and substituting the remaining Q with L. The amino acid sequence represented by SEQ ID NO: 29 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VF and substituting the remaining Q 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。 ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 30 is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence represented by SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 8 with VL and substituting the remaining Q 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 represented by SEQ ID NO: 32 replaces all QQs in the sequence obtained by repeating twice the domain of the 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) with VF, In addition, the remaining Q is replaced with 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 represented 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 a glutamine residue content of 9% or less. (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 represented 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 There 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 of 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: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32 And amino acid sequences having the same sequence identity. The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif A protein containing a sequence. The sequence identity is preferably 95% or more.
　（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. Further, the modified fibroin of (6-ii) preferably 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。 ６The sixth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.
　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。 As more specific examples of the modified fibroin containing the tag sequence, (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698) or SEQ ID NO: 40 (PRT966), or (6-iv) 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 a modified fibroin having an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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: 35, 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 were added with the amino acid sequence represented by SEQ ID NO: 11 (including His tag sequence and hinge sequence) at the N-terminus. Things. Since only a tag sequence was added to the N-terminus, there was no change in the glutamine residue content, 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 represented 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) comprises 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 There 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 an 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 by 90% or more. And amino acid sequences having the same sequence identity. The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif A protein containing a sequence. The sequence identity is preferably 95% or more.
　（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. In addition, the modified fibroin of (6-iv) preferably 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 a 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 the 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 of 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. Modified fibroin having both of the following characteristics may be used.
＜改変フィブロインの製造方法＞
　上記いずれの実施形態に係る改変フィブロインも、例えば、当該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することができる。 <Method for producing modified fibroin>
The modified fibroin according to any of the above embodiments may be, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.
　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）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遺伝子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及び／又は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改変フィブロインをコードする核酸を合成してもよい。 方法 The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding a natural fibroin and modifying it by a genetic engineering technique, or a chemical synthesis method. can do. The method for chemically synthesizing nucleic acids is not particularly limited. For example, based on amino acid sequence information of fibroin obtained from the NCBI web database or the like, AKTA oligopilot plus１００10 / 100 (GE Healthcare Japan Co., Ltd.) Genes can be chemically synthesized by a method of linking oligonucleotides synthesized automatically by PCR or the like. At this time, to facilitate purification and / or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence comprising an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence is synthesized. May be.
　調節配列は、宿主における改変フィブロイン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、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 the modified fibroin in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host. An inducible promoter that functions in a host cell and is capable of inducing the expression of a modified fibroin may be used as the promoter. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor such as an increase or decrease in temperature, osmotic pressure, or 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、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、改変フィブロイン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。 種類 The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. As the expression vector, those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed are 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 the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
　原核生物の宿主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。 好 ま し い Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis and the like. Microorganisms belonging to the genus Microbacterium include, for example, Microbacterium ammonia phyllum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
　原核生物を宿主とする場合、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 nucleic acid encoding a modified fibroin is introduced include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-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。 Examples of eukaryotic hosts include yeast and filamentous fungi (such as mold). Examples of the yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like. Examples of the 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、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。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。 場合 When a eukaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. {Natl. {Acad. {Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, competent, 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, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .
　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 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 the modified fibroin from the culture medium. The method of culturing the host in the culture medium can be performed 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、培養培地として、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、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 Escherichia coli or a eukaryote such as yeast, a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.
　炭素源としては、上記形質転換微生物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を用いることができる。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を用いることができる。無機塩類としては、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を用いることができる。 The carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolysate, acetic acid and propionic acid, and the like. Organic acids and alcohols such as ethanol and propanol can be used. As the nitrogen source, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used. As the inorganic salts, for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate 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。 培養 Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 ° C. The culturing time is usually 16 hours to 7 days. The pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
　また、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。 抗 生 During the culture, if necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used. When culturing a microorganism transformed with an expression vector using the trp promoter, indole acryl is used. An acid or the like may be added to the medium.
　発現させた改変フィブロインの単離及び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、すなわち、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単独又は組み合わせて使用し、精製標品を得ることができる。 単 離 The expressed and modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton. The host cells are crushed with a Gaulin homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method commonly used for isolating and purifying proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia). Electrophoretic methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Purification using methods such as alone or in combination It is possible to obtain the goods.
　また、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。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、その培養上清から、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 expressed by forming an insoluble form in the cells, the host cells are similarly recovered, crushed, and centrifuged to collect the insoluble form of the modified fibroin as a precipitate fraction. The recovered insoluble form of the modified fibroin can be solubilized with a protein denaturant. After this operation, a purified sample of the modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, 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 sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
＜改変フィブロイン繊維＞
　本実施形態に係る改変フィブロイン繊維は、上述した改変フィブロインを紡糸したものであり、上述した改変フィブロインを主成分として含む。 <Modified fibroin fiber>
The modified fibroin fiber according to the present embodiment is obtained by spinning the above-described modified fibroin, and contains the above-described modified fibroin as a main component.
　本実施形態に係る改変フィブロイン繊維の密度は、１．３４ｇ／ｃｍ３超である。改変フィブロイン繊維の密度は、１．３５ｇ／ｃｍ３以上、１．３６ｇ／ｃｍ３以上、１．３７ｇ／ｃｍ３以上、１．３８ｇ／ｃｍ３以上、１．３９ｇ／ｃｍ３以上又は１．４０ｇ／ｃｍ３以上であってよく、１．４５ｇ／ｃｍ３以下、１．４４ｇ／ｃｍ３以下、１．４３ｇ／ｃｍ３以下、１．４２ｇ／ｃｍ３以下又は１．４１ｇ／ｃｍ３以下であってよい。改変フィブロイン繊維の密度はＪＩＳＬ １０１３に準拠して測定される値を意味する。 The density of the modified fibroin fiber according to this embodiment is more than 1.34 g / cm 3 . The density of the modified fibroin fiber is 1.35 g / cm 3 or more, 1.36 g / cm 3 or more, 1.37 g / cm 3 or more, 1.38 g / cm 3 or more, 1.39 g / cm 3 or more, or 1.40 g. / may be cm 3 or more, 1.45 g / cm 3 or less, 1.44 g / cm 3 or less, 1.43 g / cm 3 or less, there at 1.42 g / cm 3 or less, or 1.41 g / cm 3 or less May be. The density of the modified fibroin fiber means a value measured according to JISL 1013.
　本実施形態に係る改変フィブロイン繊維の強度は、２００ＭＰａ超、２２０ＭＰａ以上、２４０ＭＰａ以上、２６０ＭＰａ以上、２８０ＭＰａ以上、３００ＭＰａ以上、３２０ＭＰａ以上、３４０ＭＰａ以上又は３５０ＭＰａ以上であってよく、６００ＭＰａ以下、５００ＭＰａ以下、４００ＭＰａ以下又は３８０ＭＰａ以下であってよい。改変フィブロイン繊維の強度は、後述する実施例に記載の方法で測定することができる。 The strength of the modified fibroin fiber according to the present embodiment may be more than 200 MPa, 220 MPa or more, 240 MPa or more, 260 MPa or more, 280 MPa or more, 300 MPa or more, 320 MPa or more, 340 MPa or more, or 350 MPa or more, 600 MPa or less, 500 MPa or less, 400 MPa. Or 380 MPa or less. The strength of the modified fibroin fiber can be measured by the method described in Examples described later.
〔改変フィブロイン繊維の製造方法〕
　本実施形態に係る改変フィブロイン繊維の製造方法は、（ａ）改変フィブロインを含む原料繊維を水中で延伸する第一の延伸工程と、（ｂ）第一の延伸工程を経た原料繊維を加熱して、乾燥させると共に、径方向に熱収縮させる緻密化工程と、を備える。 (Method for producing modified fibroin fiber)
The method for producing a modified fibroin fiber according to the present embodiment comprises: (a) a first drawing step of drawing a raw fiber containing a modified fibroin in water; and (b) heating the raw fiber that has passed through the first drawing step. And a densification step of drying and thermally shrinking in the radial direction.
　本実施形態に係る改変フィブロイン繊維の製造方法によれば、高い強度を有する（例えば、密度が１．３４ｇ／ｃｍ３超である）改変フィブロイン繊維を製造することができる。 According to the method for producing modified fibroin fibers according to the present embodiment, modified fibroin fibers having high strength (for example, having a density of more than 1.34 g / cm 3 ) can be produced.
　本実施形態に係る改変フィブロイン繊維の製造方法は、（ｃ）緻密化工程を経た原料繊維を延伸する第二の延伸工程を更に備えていてよい。この場合、当該製造方法により得られる改変フィブロイン繊維の強度がより一層高くなる。 製造 The method for producing a modified fibroin fiber according to the present embodiment may further include (c) a second drawing step of drawing the raw material fiber after the densification step. In this case, the strength of the modified fibroin fiber obtained by the production method is further increased.
　改変フィブロインを含む原料繊維は、紡糸工程を経て製造することができる。紡糸工程では、溶解用溶媒と、該溶解用溶媒に溶解されている改変フィブロインとを含むドープ液を凝固液に押し出し、未延伸糸として改変フィブロインを含む原料繊維を得る。すなわち、改変フィブロインを含む原料繊維は、紡糸工程により、ドープ液から溶解用溶媒を除去する（脱溶媒又は凝固ともいう。）と共に繊維形成して、得ることができる。 原料 The raw fiber containing the modified fibroin can be produced through a spinning process. In the spinning step, a dope solution containing a solvent for dissolution and modified fibroin dissolved in the solvent for dissolution is extruded into a coagulation liquid to obtain raw fibers containing modified fibroin as undrawn yarn. That is, the raw fiber containing the modified fibroin can be obtained by removing the solvent for dissolution from the dope solution (also referred to as desolvation or coagulation) and forming the fiber by a spinning step.
　溶解用溶媒としては、例えば、ジメチルスルホキシド（ＤＭＳＯ）、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）、ギ酸、又はヘキサフルオロイソプロパノール（ＨＦＩＰ）等が挙げられる。ドープ液は、必要に応じ、無機塩等の溶解促進剤を更に含んでいてもよい。 Examples of the dissolving solvent include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP). The dope solution may further contain a dissolution accelerator such as an inorganic salt, if necessary.
　ドープ液中の改変フィブロインの濃度は、ドープ液全量を基準として、３質量％以上であることが好ましく、１０質量％以上であることがより好ましく、２０質量％以上であることが更に好ましい。改変フィブロインの溶解性を良好に保つ観点から、ドープ液中の改変フィブロインの濃度は、６０質量％以下であることが好ましく、５０質量％以下であることがより好ましく、４０質量％以下であることが更に好ましく、３０質量％以下であることが更にまた好ましい。 濃度 The concentration of the modified fibroin in the dope solution is preferably 3% by mass or more, more preferably 10% by mass or more, even more preferably 20% by mass or more based on the total amount of the dope solution. From the viewpoint of maintaining good solubility of the modified fibroin, the concentration of the modified fibroin in the dope solution is preferably 60% by mass or less, more preferably 50% by mass or less, and more preferably 40% by mass or less. Is more preferable, and it is still more preferable that it is 30 mass% or less.
　凝固液は、脱溶媒できるものであれば特に制限されず、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、並びにアセトンを使用することができる。凝固液には、適宜水を加えてもよい。凝固液の温度は、例えば、５～３０℃である。凝固液の温度がこの範囲であれば、より安定に紡糸することができる。 The coagulation liquid is not particularly limited as long as it can remove the solvent. For example, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone can be used. Water may be appropriately added to the coagulation liquid. The temperature of the coagulating liquid is, for example, 5 to 30 ° C. If the temperature of the coagulation liquid is within this range, spinning can be performed more stably.
　紡糸工程では、例えば、ドープ液を紡糸口金（口金）から脱溶媒槽の凝固液に押し出すことにより、未延伸糸が得られる。ドープ液を、直径０．１～０．６ｍｍのノズルを有するシリンジポンプから押し出す場合、押し出し速度は、例えば、１ホール当たり０．２～６．０ｍｌ／時間であってよく、１．４～４．０ｍｌ／時間であってもよい。押し出し速度がこの範囲であれば、より安定に紡糸することができる。 In the spinning step, for example, an undrawn yarn is obtained by extruding a dope solution from a spinneret (spinner) into a coagulating solution in a desolvation tank. When the dope is extruded from a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm, the extrusion speed may be, for example, 0.2 to 6.0 ml / hour per hole, and may be 1.4 to 4 ml. 0.0 ml / hour. If the extrusion speed is in this range, spinning can be performed more stably.
＜第一の延伸工程＞
　第一の延伸工程では、改変フィブロインを含む原料繊維を水浴中で延伸（第一延伸）する。第一延伸は、一段延伸で行ってもよいし、二段以上の多段延伸で行ってもよい。改変フィブロインが、改変クモ糸フィブロイン等の分子が配向し難いタンパク質である場合、多段延伸を行うにより、分子を多段で配向させることができ、トータル延伸倍率も高くすることができるため、結果としてタフネスの高い繊維を得ることができる。 <First stretching step>
In the first drawing step, the raw fiber containing the modified fibroin is drawn (first drawing) in a water bath. The first stretching may be performed in one-stage stretching, or may be performed in two or more stages. When the modified fibroin is a protein in which molecules such as the modified spider silk fibroin are difficult to orient, the molecules can be oriented in multiple stages by performing multi-stage stretching, and the total stretching ratio can be increased. Fiber with a high fiber density can be obtained.
　第一延伸は、水浴中で行われる。つまり、第一延伸は、湿熱延伸である。水浴は水以外の溶媒を含んでいてよい。第一の延伸工程における水浴の温度は、５～６０℃、１０～５５℃、１５～５２℃、２０～５０℃、３０～４８℃又は３５～４５℃であってよい。 The first stretching is performed in a water bath. That is, the first stretching is a wet heat stretching. The water bath may contain a solvent other than water. The temperature of the water bath in the first stretching step may be 5-60 ° C, 10-55 ° C, 15-52 ° C, 20-50 ° C, 30-48 ° C or 35-45 ° C.
　第一延伸工程における水浴の温度は、６０℃以下が好ましい。水浴の温度が６０℃以下であると、凝固液が揮発性有機溶媒のときに、延伸する水浴で急激に気化することによる糸切れの発生がより一層抑制されることとなる。また、水浴の温度は、５℃以上が好ましい。水浴の温度が５℃以下であると、過剰に冷却する必要がなく、設備コストの高騰及び工程の煩雑化がより一層抑制されることになる。第一延伸における湿熱延伸倍率は、未延伸糸に対して、例えば、１～３倍であってよい。 水 The temperature of the water bath in the first stretching step is preferably 60 ° C or less. When the temperature of the water bath is 60 ° C. or lower, when the coagulating liquid is a volatile organic solvent, the occurrence of yarn breakage due to rapid vaporization in the stretching water bath is further suppressed. The temperature of the water bath is preferably 5 ° C. or higher. When the temperature of the water bath is 5 ° C. or lower, it is not necessary to cool the water bath excessively, so that a rise in equipment cost and a complicated process are further suppressed. The wet heat draw ratio in the first drawing may be, for example, 1 to 3 times the undrawn yarn.
＜緻密化工程＞
　緻密化工程では、第一の延伸工程を経た原料繊維（以下「第一延伸糸」ともいう。）を加熱して、乾燥させると共に、径方向（繊維軸直角方向）に熱収縮させる。改変フィブロイン（特に改変クモ糸フィブロイン）を含むドープ液は、脱溶媒して繊維形成される際に、原料繊維内部にボイドが形成される。また、原料繊維内部のボイドには、水等の溶媒が存在する場合がある。本実施形態に係る製造方法では、緻密化工程を行うことにより、第一延伸糸に含まれるボイドに存在する水等の溶媒（例えば水分）を除去すると共に、繊維の径方向への熱収縮に伴ってボイドを扁平化することができる。ボイドが扁平化し、高密度化する結果として、高い強度の改変フィブロイン繊維を得ることができる。緻密化工程は、第一の延伸工程を経た原料繊維（第一延伸糸）に含まれるボイドに存在する水分を除去すると共に、ボイドを扁平化する工程であってよい。 <Densification process>
In the densification step, the raw fiber (hereinafter also referred to as “first drawn yarn”) that has passed through the first drawing step is heated and dried, and is thermally shrunk in the radial direction (the direction perpendicular to the fiber axis). When the dope solution containing the modified fibroin (particularly the modified spider silk fibroin) is desolvated to form fibers, voids are formed inside the raw material fibers. Further, a solvent such as water may be present in the void inside the raw fiber. In the manufacturing method according to the present embodiment, by performing the densification step, a solvent (eg, water) such as water present in the voids included in the first drawn yarn is removed, and the fiber is subjected to heat shrinkage in the radial direction. Accordingly, the void can be flattened. As a result of void flattening and densification, modified fibroin fibers with high strength can be obtained. The densification step may be a step of removing water present in the voids contained in the raw fiber (first drawn yarn) that has passed through the first drawing step and flattening the voids.
　緻密化工程は、第一の延伸工程を経た原料繊維（第一延伸糸）の水分率が１５％以下、１０％以下、又は５％以下となるように実施してよい。ここで、第一の延伸工程を経た原料繊維（第一延伸糸）の水分率とは、第一延伸糸の全質量に占める水分の質量の割合を意味する。第一延伸糸の水分率は、カールフィッシャー法等により測定することができる。 The densification step may be performed such that the moisture content of the raw fiber (first drawn yarn) that has passed through the first drawing step is 15% or less, 10% or less, or 5% or less. Here, the water content of the raw fiber (first drawn yarn) that has passed through the first drawing step means the ratio of the mass of water to the total mass of the first drawn yarn. The moisture content of the first drawn yarn can be measured by a Karl Fischer method or the like.
　緻密化工程は、乾燥装置内で実施してよい。乾燥装置は、ホットローラー、乾熱板等の接触型乾燥装置であってもよいし、熱風乾燥炉等の非接触型乾燥装置であってもよい。乾燥装置は、非接触型乾燥装置が好ましい。この場合、糸切れがより一層抑制されることとなる。 The densification step may be performed in a drying device. The drying device may be a contact type drying device such as a hot roller or a dry heat plate, or may be a non-contact type drying device such as a hot air drying furnace. The drying device is preferably a non-contact type drying device. In this case, the yarn breakage is further suppressed.
　緻密化工程における温度（加熱温度）及び時間（加熱時間）は、第一延伸糸に存在するボイド中の水分除去と繊維の径方向収縮が可能な条件であれば特に限定されない。生産性（作業効率）の観点から、緻密化工程における温度（加熱温度）は、７０～１５０℃、７５～１４０℃、７８～１３５℃、８０～１２０℃、８２～１１０℃又は８５～１００℃であってよい。同様の観点から、緻密化工程における時間（加熱時間）は、５～４０秒、７～３５秒又は８～３０秒であってよい。 温度 The temperature (heating temperature) and the time (heating time) in the densification step are not particularly limited as long as moisture in the voids present in the first drawn yarn can be removed and the fiber can shrink in the radial direction. From the viewpoint of productivity (work efficiency), the temperature (heating temperature) in the densification step is 70 to 150 ° C, 75 to 140 ° C, 78 to 135 ° C, 80 to 120 ° C, 82 to 110 ° C, or 85 to 100 ° C. It may be. From the same viewpoint, the time (heating time) in the densification step may be 5 to 40 seconds, 7 to 35 seconds, or 8 to 30 seconds.
　緻密化工程は、第一延伸糸にテンションをかけて長さ方向での収縮を防止した状態で実施することが好ましい。この場合、第一延伸糸が径方向の収縮量（縮径量）がより大きくなり、ボイドがより扁平化されることとなる。更に、緻密化工程において、ボイドを扁平化することにより、後述する第二の延伸工程での糸切れの発生がより一層抑制されることとなる。 The densification step is preferably performed in a state where tension is applied to the first drawn yarn to prevent shrinkage in the length direction. In this case, the amount of shrinkage (diameter reduction amount) of the first drawn yarn in the radial direction is further increased, and the voids are further flattened. Furthermore, by flattening the voids in the densification step, the occurrence of yarn breakage in the second stretching step described later is further suppressed.
　緻密化工程は、例えば、第一延伸糸の端部が固定された状態で実施してよい。第一延伸糸の固定は、長さ方向の収縮を低減できるものであってよい。第一延伸糸の固定は、例えば、所定長さの延伸糸を外力のかからない状態（自然状態）で緩みなく伸ばし、この状態で繊維の両端部を固定することで行ってよい。この第一延伸糸の端部の固定は、第一延伸糸の少なくとも端部が固定されていればよく、原料繊維全体を二枚の金属板に挟み込むことにより全体を固定するような態様をとってもよい。原料繊維の固定手段は、特に制限されるものではなく、任意の固定手段を用いてよい。固定手段としては、着脱可能なものであることが好ましく、例えば、粘着テープ、接着剤、クランプ等が挙げられる。 The densification step may be performed, for example, with the end of the first drawn yarn fixed. The fixing of the first drawn yarn may be capable of reducing the shrinkage in the length direction. The first drawn yarn may be fixed by, for example, stretching a drawn yarn of a predetermined length without looseness in a state where no external force is applied (natural state), and fixing both ends of the fiber in this state. The end portion of the first drawn yarn may be fixed at least at the end portion of the first drawn yarn, and the whole raw material fiber may be fixed by sandwiching the whole raw material fiber between two metal plates. Good. The means for fixing the raw fibers is not particularly limited, and any fixing means may be used. The fixing means is preferably a detachable one, and examples thereof include an adhesive tape, an adhesive, and a clamp.
　緻密化工程は、第一の延伸工程と第二の延伸工程の間で実施されてよい。緻密化工程は、一回で行ってもよく、乾燥装置を多段設けて、複数回に分けて行ってもよいが、本発明の効果がより顕著に発揮されることから、一回で行うことが好ましい。 The densification step may be performed between the first stretching step and the second stretching step. The densification step may be performed once, or may be performed in multiple stages by providing a drying device in multiple stages, but since the effect of the present invention is more remarkably exhibited, it is preferable to perform the process once. Is preferred.
＜第二の延伸工程＞
　第二の延伸工程では、緻密化工程を経た原料繊維を延伸（第二延伸）する。第二の延伸工程により、緻密化工程で扁平化したボイドを更に扁平化することが可能となる。これにより、得られる改変フィブロイン繊維がより高密度になり、結果として、より一層高い強度を有する改変フィブロインが得られることとなる。緻密化工程で、ボイドを扁平化した後に、第二の延伸工程を実施する場合、第二の延伸工程での糸切れの発生がより一層抑制されることとなる。 <Second stretching step>
In the second drawing step, the raw fibers that have undergone the densification step are drawn (second drawing). By the second stretching step, the voids flattened in the densification step can be further flattened. As a result, the resulting modified fibroin fiber has a higher density, and as a result, a modified fibroin having higher strength is obtained. In the case where the second stretching step is performed after flattening the voids in the densification step, the occurrence of yarn breakage in the second stretching step is further suppressed.
　第二延伸は、一段延伸で行ってもよいし、二段以上の多段延伸で行ってもよい。第二延伸は、湿熱延伸であってもよいし、乾熱延伸であってもよい。第二の延伸における湿熱延伸は、温水中、温水に有機溶剤等を加えた溶液中、又はスチーム加熱中で行うことができる。なお、スチーム加熱中で行う湿熱延伸をスチーム延伸という。第二の延伸は、好ましくはスチーム延伸又は乾熱延伸であり、特に好ましくはスチーム延伸である。 The second stretching may be performed in one-stage stretching or in two or more stages. The second stretching may be wet heat stretching or dry heat stretching. The wet heat stretching in the second stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating. Note that the wet heat stretching performed during the steam heating is referred to as steam stretching. The second stretching is preferably a steam stretching or a dry heat stretching, particularly preferably a steam stretching.
　第二の延伸工程は、好ましくはスチーム延伸を行うことを含む。スチーム延伸は熱伝導率が高いため、或いは繊維内部に浸透した水分が可塑剤等として機能することで、高延伸をより確実に実現し得ることが期待される。 The second stretching step preferably includes performing steam stretching. It is expected that high stretching can be achieved more reliably by steam stretching because of high thermal conductivity or by the moisture permeated into the fiber functioning as a plasticizer or the like.
　スチーム延伸以外の湿熱延伸を行う際の温度としては、例えば、５０～９０℃であってよく、７５～８５℃が好ましい。スチーム延伸を行う際の温度としては、例えば、１００～２００℃、１００～１５０℃又は１００～１２０℃であってよい。第二延伸における湿熱延伸倍率は、緻密化工程を経た原料繊維に対して、１～１０倍又は２～８倍であってよい。 温度 The temperature for performing the wet heat stretching other than the steam stretching may be, for example, 50 to 90 ° C, and preferably 75 to 85 ° C. The temperature for performing the steam stretching may be, for example, 100 to 200 ° C., 100 to 150 ° C., or 100 to 120 ° C. The wet heat stretching ratio in the second stretching may be 1 to 10 times or 2 to 8 times with respect to the raw material fiber that has passed through the densification step.
　乾熱延伸は、電気管状炉、乾熱板等を使用して行うことができる。第二の延伸工程は、乾熱板上での延伸操作を含んでいることが好ましい。乾熱延伸を行う際の温度（乾熱延伸温度）は、例えば、１４０℃～２７０℃であってよく、１５０～２６０℃であってよい。第二延伸における乾熱延伸倍率は、緻密化工程を経た原料繊維に対して、例えば、０．５～８倍又は１～６倍であってよい。第二の延伸工程を乾熱延伸で実施すれば、緻密化工程で水等の溶媒が除去されたボイド内への水分の再浸入が阻止されることが期待される。 Dry heat drawing can be performed using an electric tube furnace, a dry heat plate or the like. The second stretching step preferably includes a stretching operation on a dry heat plate. The temperature at which the dry heat stretching is performed (dry heat drawing temperature) may be, for example, 140 ° C. to 270 ° C., or may be 150 ° C. to 260 ° C. The dry heat drawing ratio in the second drawing may be, for example, 0.5 to 8 times or 1 to 6 times with respect to the raw material fiber that has passed through the densification step. If the second stretching step is performed by dry heat stretching, it is expected that re-entry of water into the void from which the solvent such as water has been removed in the densification step is prevented.
　湿熱延伸及び乾熱延伸はそれぞれ単独で行ってもよく、またこれらを多段で、又は組み合わせて行ってもよい。すなわち、一段目延伸を湿熱延伸で行い、二段目延伸を乾熱延伸で行う、又は一段目延伸を湿熱延伸行い、二段目延伸を湿熱延伸行い、更に三段目延伸を乾熱延伸で行う等、湿熱延伸及び乾熱延伸を適宜組み合わせて行うことができる。 (4) The wet heat stretching and the dry heat stretching may each be performed alone, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and further the third stage stretching is performed by dry heat stretching. For example, wet heat stretching and dry heat stretching can be performed in an appropriate combination.
　総延伸倍率（最終的な延伸倍率）は、その下限値が、未延伸糸に対して、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 total draw ratio (final draw ratio) is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more of the undrawn yarn. , 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less, It is 12 times or less, 11 times or less, and 10 times or less.
　改変フィブロインの製造方法において、第一の延伸工程、緻密化工程及び第二の延伸工程は、連続で実施してもよいし、連続で実施しなくてもよい。連続で実施しない場合、例えば、第一の延伸工程及び緻密化工程を連続で実施した後、緻密化工程後の原料繊維を、巻糸体とし、この巻糸体を巻出しつつ、第二の延伸工程を実施してもよい。 に お い て In the method for producing modified fibroin, the first stretching step, the densification step, and the second stretching step may or may not be performed continuously. When not performed continuously, for example, after the first stretching step and the densification step are continuously performed, the raw material fiber after the densification step is used as a wound body, and while the wound body is unwound, the second fiber is unwound. A stretching step may be performed.
　図６及び図７は、改変フィブロイン繊維の製造装置の一例を示す説明図である。図６に示す製造装置は、紡糸工程と第一の延伸工程を連続して行う紡糸延伸装置である。紡糸延伸装置１００は、押し出し装置１と、未延伸糸製造装置２と、湿熱延伸装置３と、乾燥装置１０と、乾熱延伸装置４を備える。ドープ液６は、貯槽７に貯蔵され、ギアポンプ８により口金９から押し出される。ラボスケールにおいては、ドープ液をシリンダーに充填し、シリンジポンプを用いてノズルから押し出してもよい。押し出されたドープ液６は、エアギャップ１９を介して、又はエアギャップ１９を介さずに直接、凝固液槽２０の凝固液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、未延伸糸（原料繊維）が形成される。次いで、未延伸糸は、延伸浴槽２１内の温水１２内に供給され、１段目延伸が行われる。延伸倍率は、供給ニップローラ１３と引き取りニップローラ１４との速度比によって決まる。１段目延伸を経た延伸糸（第一延伸糸）は、次いで乾燥装置１０に供給され、糸道２２ａ内で加熱される。加熱された第一延伸糸は、乾熱延伸装置１７に供給され、糸道２２ｂ内で２段目延伸が行われる。２段目延伸を経た延伸糸（第二延伸糸）は、巻き取られて巻糸体５となる。２段目延伸における延伸倍率は供給ニップローラ１５と引き取りニップローラ１６との速度比によって決まる。１８ａ～１８ｇは糸ガイドである。 FIGS. 6 and 7 are explanatory diagrams showing an example of an apparatus for producing a modified fibroin fiber. The manufacturing apparatus shown in FIG. 6 is a spin stretching apparatus that continuously performs a spinning step and a first stretching step. The spinning and drawing apparatus 100 includes an extruder 1, an undrawn yarn manufacturing apparatus 2, a wet heat drawing apparatus 3, a drying apparatus 10, and a dry heat drawing apparatus 4. The dope solution 6 is stored in a storage tank 7 and pushed out of a base 9 by a gear pump 8. In a laboratory scale, a dope solution may be filled in a cylinder and extruded from a nozzle using a syringe pump. The extruded dope liquid 6 is supplied into the coagulation liquid 11 of the coagulation liquid tank 20 via the air gap 19 or directly without passing through the air gap 19. The solvent is removed from the dope solution 6 in the coagulation solution 11, and an undrawn yarn (raw material fiber) is formed. Next, the undrawn yarn is supplied into the hot water 12 in the drawing bath 21 and the first-stage drawing is performed. The stretching ratio is determined by the speed ratio between the supply nip roller 13 and the take-off nip roller 14. The drawn yarn (first drawn yarn) that has passed through the first-stage drawing is then supplied to the drying device 10 and heated in the yarn path 22a. The heated first drawn yarn is supplied to the dry heat drawing device 17, and the second stage drawing is performed in the yarn path 22b. The drawn yarn (second drawn yarn) that has passed through the second-stage drawing is wound up to form a wound body 5. The stretching ratio in the second stage stretching is determined by the speed ratio between the supply nip roller 15 and the take-off nip roller 16. 18a to 18g are yarn guides.
　図７に示す製造装置は、紡糸工程と延伸工程（２段延伸）を行う紡糸延伸装置である。図７（Ａ）は、未延伸糸製造装置３０、１段目延伸装置４０及び乾燥装置６０を示し、図７（Ｂ）は、２段目延伸装置５０を示す。それぞれの装置ごとに糸を巻き取るか又は巻き取らずに容器に溜めてもよい。未延伸糸製造装置３０においては、押し出し装置（マイクロシリンジ）３１内にドープ液３２を入れておき、シリンジポンプを用いて矢印Ｐ方向に移動させ、ノズル３３からドープ液３２を押し出し、凝固液槽３４内の凝固液３５にドープ液３２を供給し、未延伸糸３６（原料繊維）とする。続いて、１段目延伸装置４０においては、未延伸糸３６を延伸浴槽３７内の温水３８内に供給し、１段目延伸する。１段目延伸糸（第一延伸糸）は、乾燥装置６０に供給され、糸道６１内で加熱される。加熱後の１段目延伸糸は、巻き取られて巻糸体３９となる。延伸倍率は供給ニップローラ４１と引き取りニップローラ４２との速度比によって決まる。次いで巻糸体３９から１段目延伸糸を引き出し、乾熱延伸装置４３に供給し、糸道４７内で２段目延伸する。延伸倍率は供給ニップローラ４５と引き取りニップローラ４６との速度比によって決まる。次いで二段目延伸糸（第二延伸糸）を巻糸体４４に巻き取る。 製造 The production apparatus shown in FIG. 7 is a spinning and stretching apparatus that performs a spinning step and a stretching step (two-stage stretching). FIG. 7A shows an undrawn yarn production device 30, a first-stage drawing device 40, and a drying device 60, and FIG. 7B shows a second-stage drawing device 50. The yarn may be wound in each device or stored in a container without being wound. In the undrawn yarn producing apparatus 30, the dope liquid 32 is put in an extruder (micro syringe) 31, and is moved in the direction of arrow P using a syringe pump, and the dope liquid 32 is extruded from the nozzle 33 to form a coagulating liquid tank. The dope liquid 32 is supplied to the coagulating liquid 35 in 34 to make an undrawn yarn 36 (raw fiber). Subsequently, in the first-stage drawing device 40, the undrawn yarn 36 is supplied into the hot water 38 in the drawing bath 37 to perform the first-stage drawing. The first-stage drawn yarn (first drawn yarn) is supplied to the drying device 60 and heated in the yarn path 61. The first-stage drawn yarn after heating is wound into a wound body 39. The stretching ratio is determined by the speed ratio between the supply nip roller 41 and the take-off nip roller 42. Next, the first-stage drawn yarn is drawn from the wound body 39, supplied to the dry heat drawing device 43, and subjected to the second-stage drawing in the yarn path 47. The stretching ratio is determined by the speed ratio between the supply nip roller 45 and the take-off nip roller 46. Next, the second-stage drawn yarn (second drawn yarn) is wound around the wound body 44.
　以下、実施例等に基づいて本発明をより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples and the like. However, the present invention is not limited to the following examples.
〔（１）改変フィブロイン（人造フィブロイン）の製造〕
（改変フィブロインをコードする核酸の合成、及び発現ベクターの構築）
　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（ＰＲＴ７９９）を設計した。設計した改変フィブロイン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、終止コドン下流にＥｃｏＲＩサイトを付加した。この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。 [(1) Production of modified fibroin (artificial fibroin)]
(Synthesis of nucleic acid encoding modified fibroin and construction of expression vector)
A modified fibroin (PRT799) having the amino acid sequence represented by SEQ ID NO: 15 was designed. A nucleic acid encoding the designed modified fibroin was synthesized. An NdeI site was added to the 5 'end of the nucleic acid, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was digested with NdeI and EcoRI and cut out, followed by recombination into a protein expression vector pET-22b (+) to obtain an expression vector.
（改変フィブロインの発現）
　得られたｐＥＴ－２２ｂ（＋）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。 (Expression of modified fibroin)
Escherichia coli BLR (DE3) was transformed with the obtained pET-22b (+) expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 became 0.005. The temperature of the culture was maintained at 30 ° C., and the flask was cultured until the OD 600 reached 5 (about 15 hours) to obtain a seed culture.
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％に維持した。 The seed culture solution was added to a jar fermenter to which 500 mL of a production medium (Table 5) had been added so that the OD 600 was 0.05. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. The concentration of dissolved oxygen in the culture was maintained at 20% of the saturated concentration of dissolved oxygen.
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。培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持しながら、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、目的とする改変フィブロインを発現誘導させた。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改変フィブロインのサイズに相当するバンドの出現により、目的とする改変フィブロインの発現を確認した。 フ ィ ー ド Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g / 1 L of glucose, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Culture was performed 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 the expression of the desired modified fibroin. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of a band corresponding to the size of the target modified fibroin depending on the addition of IPTG revealed the presence of the target modified fibroin. Expression was confirmed.
（改変フィブロインの精製）
　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２０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）に懸濁させ、高圧ホモジナイザー（ＧＥＡ　Ｎｉｒｏ　Ｓｏａｖｉ社）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２０ｍＭ　Ｔｒｉｓ－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、２０ｍＭ　ＮａＣｌ、１ｍＭ　Ｔｒｉｓ－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た。回収した凝集タンパク質から凍結乾燥機で水分を除き、目的とする改変フィブロインの凍結乾燥粉末を得た。 (Purification of modified fibroin)
Two hours after the addition of IPTG, the collected cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 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 obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing 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) so as to have a concentration of 100 mg / mL, and then suspended at 60 ° C. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation. Water was removed from the collected aggregated protein using a freeze dryer to obtain a freeze-dried powder of the desired modified fibroin.
〔（２）人造フィブロイン繊維の製造〕
（ドープ液の調製）
　４．０質量％になるようにＬｉＣｌを溶解させたジメチルスルホキシド（ＤＭＳＯ）を溶媒として用意し、そこに改変フィブロインの凍結乾燥粉末を、濃度２４質量％質量％となるよう添加し、シェーカーを使用して３時間溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液を得た。 [(2) Manufacture of artificial fibroin fiber]
(Preparation of dope solution)
Prepare dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to have a concentration of 4.0% by mass as a solvent, and add a freeze-dried powder of modified fibroin to the solution to a concentration of 24% by mass, and use a shaker. And dissolved for 3 hours. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution.
（紡糸）
　得られた改変フィブロイン溶液をドープ液（紡糸原液）として、実施例１～４及び比較例１の改変フィブロイン繊維を製造した。実施例１～３の改変フィブロイン繊維は、図６に示す紡糸延伸装置１００を用いて、紡糸及び延伸を行ったものである。実施例４の改変フィブロイン繊維は、図６に示す紡糸延伸装置１００において、乾熱延伸装置の代わりに湿熱延伸装置（蒸気延伸（スチーム延伸）装置）を備える紡糸延伸装置を用いて紡糸及び延伸を行ったものである。比較例１の改変フィブロイン繊維は、図６の紡糸延伸装置１００において、乾燥装置１０を備えていない紡糸延伸装置を用いて紡糸及び延伸を行ったものである。製造条件は以下のとおりである。実施例１～４での乾熱処理は、原料繊維を、長さ方向に収縮しないように、弛みない状態で二枚の金属板に挟むことにより固定した状態で実施した。
　　押出しノズル直径：０．１ｍｍ
　　凝固液（メタノール）温度：１０℃
　　洗浄浴（湿熱延伸）温度：４０℃
　　乾燥（緻密化）温度：９０℃（熱ロール）
　　乾燥（緻密化）時間：２０秒
　　乾熱延伸温度：２５０℃（乾熱板）
　　蒸気延伸温度：１１０℃
　　湿熱延伸倍率、乾熱処理の有無、乾熱延伸倍率、蒸気延伸倍率及び総延伸倍率：表６参照 (spinning)
Using the obtained modified fibroin solution as a dope solution (spinning stock solution), modified fibroin fibers of Examples 1 to 4 and Comparative Example 1 were produced. The modified fibroin fibers of Examples 1 to 3 were obtained by spinning and drawing using the spinning and drawing apparatus 100 shown in FIG. The modified fibroin fiber of Example 4 was spun and drawn by using a spin drawing apparatus provided with a wet heat drawing apparatus (steam drawing (steam drawing) apparatus) instead of the dry heat drawing apparatus in the spin drawing apparatus 100 shown in FIG. It is what went. The modified fibroin fiber of Comparative Example 1 was obtained by spinning and drawing using a spinning and drawing apparatus without the drying apparatus 10 in the spinning and drawing apparatus 100 of FIG. The manufacturing conditions are as follows. The dry heat treatment in Examples 1 to 4 was performed in a state where the raw material fibers were fixed by being sandwiched between two metal plates without being loosened so as not to shrink in the length direction.
Extrusion nozzle diameter: 0.1mm
Coagulation liquid (methanol) temperature: 10 ° C
Washing bath (wet heat stretching) temperature: 40 ° C
Drying (densification) temperature: 90 ° C (hot roll)
Drying (densification) time: 20 seconds Dry heat stretching temperature: 250 ° C (dry heat plate)
Steam stretching temperature: 110 ° C
Wet heat draw ratio, presence or absence of dry heat treatment, dry heat draw ratio, steam draw ratio, and total draw ratio: See Table 6.
＜密度（繊維密度）の測定＞
　上記のようにして得られた実施例１～４と比較例１の改変フィブロイン繊維のそれぞれ約１gの束を、島津製作所社製　乾式密度計アキュピック１３３０にセットして密度を計測した。 <Measurement of density (fiber density)>
A bundle of about 1 g of each of the modified fibroin fibers of Examples 1 to 4 and Comparative Example 1 obtained as described above was set on a dry density meter Acupic 1330 manufactured by Shimadzu Corporation, and the density was measured.
＜強度（引張強度）の測定＞
　上記のようして得られた実施例１～４と比較例１の改変フィブロイン繊維のそれぞれを用い、ＩＮＳＴＲＯＮ３３４２引張試験機により、試験片長３００ｍｍ、引張り速度３００ｍｍ／ｍｉｎで１０回測定して、その平均値を算出した。 <Measurement of strength (tensile strength)>
Using each of the modified fibroin fibers of Examples 1 to 4 and Comparative Example 1 obtained as described above, an INSTRON 3342 tensile tester was used to measure 10 times at a test piece length of 300 mm and a tensile speed of 300 mm / min, and the average was measured. Values were calculated.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　密度が１．３４ｇ／ｃｍ３超である実施例１～４の改変フィブロイン繊維は、密度が１．３４ｇ／ｃｍ３以下である比較例１の改変フィブロイン繊維と比べて、強度が高かった。 Modified fibroin fibers density 1.34 g / cm 3 greater than in an exemplary 1-4, density compared to the modified fibroin fibers of Comparative Example 1 is 1.34 g / cm 3 or less, the strength was high.
　１，３１…押し出し装置、２，３０…未延伸糸製造装置、３，４０…湿熱延伸装置（１段目延伸装置）、４，５０…乾熱延伸装置（２段目延伸装置）、５，３９，４４…巻糸体、６，３２…ドープ液、７…貯槽、８…ギアポンプ、９…口金、６０，１００…紡糸延伸装置、１１，３５…凝固液、３６…未延伸糸、１２，３８…温水、１３，１５，４１，４５，…供給ニップローラ、１４，１６，４２，４６…引き取りニップローラ、１７，４３…乾熱延伸装置、１８ａ～１８ｇ…糸ガイド、１９…エアギャップ、２０，３４…凝固液槽、２１，３７…延伸浴槽、２２，４７…糸道。 1,31: Extruder, 2,30: Undrawn yarn manufacturing device, 3,40: Wet heat drawing device (first stage drawing device), 4,50: Dry heat drawing device (second stage drawing device), 5, 39, 44: wound body, 6, 32: dope solution, 7: storage tank, 8: gear pump, 9: spinneret, 60, 100: spinning and drawing device, 11, 35: solidified solution, 36: undrawn yarn, 12, 38, hot water, 13, 15, 41, 45, supply nip roller, 14, 16, 42, 46, take-off nip roller, 17, 43, dry heat drawing device, 18a to 18g, thread guide, 19, air gap, 20, 34: coagulation bath, 21, 37: stretching bath, 22, 47: yarn path.

Claims (9)

  1. 　改変フィブロインを含み、密度が１．３４ｇ／ｃｍ３超である、改変フィブロイン繊維。 A modified fibroin fiber comprising the modified fibroin and having a density greater than 1.34 g / cm 3 .
  2. 　前記改変フィブロインが改変クモ糸フィブロインである、請求項１に記載の改変フィブロイン繊維。 改 変 The modified fibroin fiber according to claim 1, wherein the modified fibroin is a modified spider silk fibroin.
  3. 　（ａ）改変フィブロインを含む原料繊維を水浴中で延伸する第一の延伸工程と、
    　（ｂ）前記第一の延伸工程を経た前記原料繊維を加熱して、乾燥させると共に、径方向に熱収縮させる緻密化工程と、
    を備える、改変フィブロイン繊維の製造方法。     (A) a first stretching step of stretching a raw fiber containing the modified fibroin in a water bath;
    (B) heating and drying the raw material fiber after the first drawing step, and a densification step of thermally shrinking in a radial direction;
    A method for producing a modified fibroin fiber, comprising:
  4. 　前記緻密化工程が、前記第一の延伸工程を経た前記原料繊維に含まれるボイドに存在する水分を除去すると共に、該ボイドを扁平化する工程である、請求項３に記載の改変フィブロイン繊維の製造方法。 4. The modified fibroin fiber according to claim 3, wherein the densification step is a step of removing water present in voids contained in the raw material fiber that has passed through the first drawing step and flattening the void. 5. Production method.
  5. 　前記水浴の温度が５～６０℃である、請求項３又は４に記載の改変フィブロイン繊維の製造方法。 方法 The method for producing modified fibroin fiber according to claim 3, wherein the temperature of the water bath is 5 to 60 ° C.
  6. 　（ｃ）前記緻密化工程を経た前記原料繊維を延伸する第二の延伸工程を更に備える、請求項３～５のいずれか一項に改変フィブロイン繊維の製造方法。 方法 (c) The method for producing modified fibroin fibers according to any one of claims 3 to 5, further comprising a second drawing step of drawing the raw material fibers that have passed through the densification step.
  7. 　前記第二の延伸工程が、スチーム延伸を行うことを含む、請求項６に記載の改変フィブロイン繊維の製造方法。 The method for producing modified fibroin fibers according to claim 6, wherein the second drawing step includes performing steam drawing.
  8. 　前記第二の延伸工程が、乾熱延伸を行うことを含む、請求項６又は７に記載の改変フィブロイン繊維の製造方法。 方法 The method for producing a modified fibroin fiber according to claim 6 or 7, wherein the second drawing step includes performing dry heat drawing.
  9. 　前記改変フィブロインが改変クモ糸フィブロインである、請求項３～８のいずれか一項に記載の改変フィブロイン繊維の製造方法。 方法 The method for producing a modified fibroin fiber according to any one of claims 3 to 8, wherein the modified fibroin is a modified spider silk fibroin.
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