WO2021015153A1 - Fibroïne modifiée - Google Patents

Fibroïne modifiée Download PDF

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Publication number
WO2021015153A1
WO2021015153A1 PCT/JP2020/027991 JP2020027991W WO2021015153A1 WO 2021015153 A1 WO2021015153 A1 WO 2021015153A1 JP 2020027991 W JP2020027991 W JP 2020027991W WO 2021015153 A1 WO2021015153 A1 WO 2021015153A1
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amino acid
motif
acid sequence
fibroin
modified fibroin
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PCT/JP2020/027991
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Japanese (ja)
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▲郁▼▲群▼ ▲荘▼
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Ｓｐｉｂｅｒ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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

Definitions

  • the present invention relates to modified fibroin.
  • Fibroin is a kind of fibrous protein and contains up to 90% of glycine residue, alanine residue and serine residue that lead to the formation of ⁇ -pleated sheet (Non-Patent Document 1).
  • proteins silk protein, hornet silk protein, spider silk protein
  • the like that constitute threads produced by insects and spiders are known.
  • Silk protein has excellent mechanical properties, hygroscopic properties and deodorant properties, and is a material widely used as a raw material for clothing.
  • silk thread is an immunologically tolerant natural fiber and has high biocompatibility, so it is also used for surgical sutures and the like.
  • Spider silk proteins are, according to their source organs, a large bottle of spider protein with high toughness (major amplifier spider protein, MaSp), a small bottle of spider protein with high extensibility (minor amplifier spider protein, MiSp), and a whip. They are named spider silk proteins in the form of fragelliform (Flag), tubular (tubularform), aggregate (aggregate), grape-like (aciniform) and pear-like (pyriform).
  • fragelliform Framt
  • tubular tubular
  • aggregate aggregate
  • grape-like aciniform
  • pyriform pear-like
  • Non-Patent Document 2 a structure in which amino acid motifs classified into GPGXX, an extension region rich in alanine residues ((A) n or (GA) n ), GGX, and spacers are repeated is known.
  • Non-Patent Document 2 a structure in which amino acid motifs classified into GPGXX, an extension region rich in alanine residues ((A) n or (GA) n ), GGX, and spacers are repeated is known.
  • Non-Patent Document 2 Further, by replacing the (GA) n motif with the (A) n motif, the elongation is decreased but the tensile strength is increased, the elongation is increased by increasing the number of GPGXX motifs, and the number of GPGXX motifs. It has been reported that the tensile strength is increased by substituting (A) with the n motif (Patent Document 2). Further, the GGX and GPGXX motifs are considered to have a flexible spiral structure that
  • Recombinant spider silk protein and recombinant silk protein are produced in several heterologous protein production systems. For example, a large number of recombinant fibroin productions by recombinant protein production systems using goats, silk moths, plants, mammalian cells, yeasts, molds, gram-negative bacteria, gram-positive bacteria, etc. as hosts have been reported, and certain results have been obtained. (Non-Patent Document 3, Patent Documents 4 and 5).
  • Silk made of fibroin has an elegant texture, beautiful luster, and comfort, but has drawbacks such as shrinkage and yellowing when it gets wet with water, and it is recommended to iron it at a medium temperature of up to 160 ° C. , Generally, it is considered to be a fiber having low heat resistance. In order to use it as a material for a heat-resistant composite material, it is first required to improve the heat resistance of the silk protein itself.
  • Natural spider silk made of fibroin has excellent properties against physical changes such as tensile strength, toughness and extensibility, has high biocompatibility and biodegradability, and is also excellent in heat resistance.
  • silkworms that use natural yarn industrially
  • spiders are difficult to breed in large quantities, so artificial synthetic fibers that imitate spider silk are manufactured, and their properties against physical changes and biocompatibility Attempts have been made to apply it to materials and materials such as yarns and mattresses that have properties and biodegradability, but the production of artificial fibers that are comparable in heat resistance to natural spider silk has not been successful.
  • an object of the present invention is to provide a modified fibroin having improved thermal stability.
  • the present inventors have found that by increasing the alanine content of the alanine continuous sequence in the (A) n motif present in fibroin, fibroin with improved thermostability can be obtained.
  • the present invention is based on this novel finding.
  • the present invention relates to, for example, the following inventions.
  • Formula 1 [(A) n motif-REP] m
  • formula 2 [(A) n motif-REP] m- (A) modified fibroin containing a domain sequence represented by n motif.
  • the total number of alanine residues 6 or more consecutive in the above (A) n motif is 20% or more of the total number of amino acid residues.
  • the hydrophobicity of REP is -1.0 or higher, Modified fibroin in which the total number of amino acid residues is 580 or more.
  • (A) n motifs show an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% of the total number of amino acid residues in n motifs. That is all.
  • REP shows an amino acid sequence consisting of 10-200 amino acid residues.
  • m represents an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • [2] The modified fibroin according to [1], wherein the hydrophobicity of REP is 0 or more.
  • [6] The modified fibroin according to [5], wherein the tag sequence comprises the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • Td pyrolysis temperature
  • [10] It hybridizes with the complementary strand of the nucleic acid according to [9] under stringent conditions, and formula 1: [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m.
  • -(A) A nucleic acid encoding a modified fibroin containing a domain sequence represented by an n motif.
  • (A) n motifs show an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% of the total number of amino acid residues in n motifs. That is all.
  • REP shows an amino acid sequence consisting of 10-200 amino acid residues.
  • m represents an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • [11] It has 90% or more sequence identity with the nucleic acid described in [9], and formula 1: [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m-
  • A) A nucleic acid encoding a modified fibroin containing a domain sequence represented by an n motif.
  • (A) n motifs show an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% of the total number of amino acid residues in n motifs. That is all.
  • REP shows an amino acid sequence consisting of 10-200 amino acid residues.
  • m represents an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • a method for producing modified fibroin Including the step of expressing the nucleic acid by 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.
  • the production method wherein the modified fibroin is the modified fibroin according to any one of [1] to [8].
  • (A) n motifs show an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues is 80% of the total number of amino acid residues in n motifs. That is all.
  • REP shows an amino acid sequence consisting of 10-200 amino acid residues.
  • m represents an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • [14] The method according to [13], wherein the fibroin before modification is naturally derived fibroin.
  • the modified fibroin according to the present invention is represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif.
  • the modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence.
  • the N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.
  • modified fibroin means fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin.
  • naturally-derived fibroin means fibroin whose amino acid sequence is the same as that of fibroin produced by naturally occurring insects, arachnids, and the like.
  • Naturally-derived fibroin is also a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif. Is.
  • modified fibroin is an amino acid sequence modified by relying on naturally occurring fibroin (for example, modified gene sequence of cloned naturally occurring fibroin) as long as it has the amino acid sequence specified in the present invention.
  • the amino acid sequence may be modified by the above (for example, the amino acid sequence is artificially designed and synthesized regardless of naturally occurring fibroin (for example, the nucleic acid encoding the designed amino acid sequence is chemically synthesized). Therefore, it may have a desired amino acid sequence).
  • a modified fibroin amino acid sequence is also included in the modified fibroin if its amino acid sequence is different from that of naturally occurring fibroin.
  • domain sequence refers to a fibroin-specific crystalline region (typically corresponding to (A) n motif of an amino acid sequence) and an amorphous region (typically, REP of an amino acid sequence).
  • An amino acid sequence that produces (corresponding.)) which is represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif.
  • (A) n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 4 to 27.
  • the number of amino acid residues of the n motif is 4 to 20, 8 to 20, 10 to 20, 4 to 16, 5 to 10, 6 to 12, 6 to 18, 7 to 10, 7 to 14, 8 to It may be 16, or 11 to 16.
  • the ratio of the number of alanine residues to the total number of amino acid residues in the n motif is 80% or more.
  • REP shows an amino acid sequence consisting of 10-200 amino acid residues.
  • m represents an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • the number of alanine residues relative to the total number of amino acid residues in the (A) n motif may be 80% or more, preferably 85% or more, and more preferably 90% or more. It is more preferably 95% or more, and even more preferably 100% (meaning that it is composed only of alanine residues). It is preferable that at least seven (A) n motifs present in the domain sequence are composed of only alanine residues.
  • (A) n motif is (Ala) k (Ala indicates alanine residues, k is an integer of 4 to 27, preferably an integer of 4 to 20, more preferably. It means that it has an amino acid sequence represented by (indicating an integer of 4 to 16).
  • the total number of residues of alanine residues 6 or more consecutive in the (A) n motif is 20% or more of the total number of amino acid residues, and the hydrophobicity of REP.
  • the degree is ⁇ 1.0 or higher, and the total number of amino acid residues in total is 580 or higher.
  • the total number of residues of alanine residues 6 or more consecutive in the (A) n motif is 20% or more of the total number of amino acid residues.
  • the total number of alanine residues that are 6 or more consecutive in the n motif is the number of alanine residues when only the number of alanine residues that are 6 or more consecutive is counted. Means the total number of. Therefore, the number of consecutive alanine residues of only 5 residues is not counted.
  • the presence of other amino acid residues between alanine residues means that even if there are no more than 6 consecutive alanine residues, the number of alanine residues remains. Is not counted.
  • the total number of alanine residues 6 or more consecutive in the n motif may be, for example, 21% or more, 22% or more, or 23% or more of the total number of amino acid residues.
  • the total number of alanine residues that are 7 or more consecutive in the (A) n motif is, for example, 20% or more, 21% or more, 22% or more, or 23% or more of the total number of amino acid residues. It may be.
  • the modified fibroin according to this embodiment has a total number of amino acid residues of 580 or more.
  • the total number of amino acid residues in total may be, for example, 590 or more, 600 or more, 610 or more, or 620 or more.
  • the modified fibroin according to the present embodiment is selected from the GGX motif and the GPGXX motif (G indicates a glycine residue, P indicates a phenylalanine residue, and X indicates an amino acid residue other than a glycine residue) in the amino acid sequence of REP. It is preferable that at least one motif is contained. By including these motifs in the REP, the elongation of the modified fibroin can be improved.
  • the content of the GPGXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more. Thereby, the elongation of the modified fibroin can be further improved.
  • the upper limit of the GPGXX motif content is not particularly limited and may be 50% or less, or 30% or less.
  • GPGXX motif content is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m- (A) In a fibroin containing a domain sequence represented by n motif, the most C-terminal side In all REPs included in the sequence excluding the sequence from the located (A) n motif to the C-terminal of the domain sequence from the domain sequence, the total number of GPGXX motifs contained in the region is tripled (that is, that is).
  • Let x be (corresponding to the total number of G and P in the GPGXX motif), remove 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, and further (A) n motif.
  • the GPGXX motif content is calculated as x / y, where y is the total number of amino acid residues in all REPs excluded.
  • the sequence obtained by excluding the sequence from the (A) n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is targeted at "the most C-terminal side".
  • the sequence from (A) n motif to the C end of the domain sequence located in (A) may include a sequence having a low correlation with the sequence characteristic of fibroin, and m is small. In this case (that is, when the domain sequence is short), it affects the calculation result of the GPGXX motif content, and this effect is eliminated.
  • FIG. 1 is a schematic diagram showing a domain sequence of modified fibroin.
  • the modified fibroin according to the present embodiment preferably has a glutamine residue content of 9% or less, more preferably 7% or less, further preferably 4% or less, and preferably 0%. Especially preferable. As a result, the effect of the present invention can be achieved even more remarkably.
  • glucose residue content is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • formula 2 [(A) n motif-REP] m-
  • A) In a fibroin containing a domain sequence represented by n motif, the most C-terminal side (A) All REPs included in the sequence obtained by removing the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "region A" in FIG. 1) are included in that region.
  • Let w be the total number of glutamine residues, and remove the sequence from the (A) n motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence, and further (A) the amino acid residue of all REPs excluding the n motif.
  • the glutamine residue content is calculated as w / y, where y is the total number of groups.
  • the reason why "the sequence from the (A) n motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.
  • the modified fibroin according to the present embodiment has a domain sequence in which one or more glutamine residues in REP have been deleted or replaced with other amino acid residues as compared with the fibroin before modification. It may have a corresponding amino acid sequence.
  • the "other amino acid residue” may be an amino acid residue other than the glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue.
  • a known index Kyte J, & Doolittle R (1982) "A simple method for dispensing the hydropathic character of Protein7, protein7, protein. 105-132) is used.
  • the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
  • 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), glutamine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can.
  • amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferable.
  • Isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are more preferably amino acid residues.
  • the hydrophobicity of REP may be ⁇ 1.0 or higher, preferably ⁇ 0.9 or higher, more preferably ⁇ 0.8 or higher, and ⁇ 0.8 or higher. It is more preferably 0.7 or more, further preferably 0 or more, further preferably 0.2 or more, even more preferably 0.3 or more, and 0.4 or more. Is particularly preferable.
  • the upper limit of the hydrophobicity of REP is not particularly limited, and may be, for example, 1.0 or less, or 0.7 or less.
  • the hydrophobicity of REP may be, for example, ⁇ 1.0 or more and 1.0 or less, and may be 0 or more and 0.7 or less.
  • hydrophilcity of REP is a value calculated by the following method.
  • each amino acid in that region Let z be the sum of the hydrophobicity indexes of the residues, and remove the sequence from the (A) n motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence, and further (A) all REP excluding the n motif.
  • the hydrophobicity of REP is calculated as z / y, where y is the total number of amino acid residues in.
  • the reason why "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 the above-mentioned reason. The same is true.
  • the sum of the hydrophobicity indexes (HI) of all the amino acid residues constituting the modified fibroin is obtained, and then the sum is divided by the total number of amino acid residues (mean HI).
  • HI hydrophobicity indexes
  • -1.0 or more more preferably -0.9 or more, further preferably -0.8 or more, further preferably -0.7 or more, and 0 or more. It is even more preferably 0.2 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more.
  • the upper limit of the average HI of the modified fibroin is not particularly limited, and may be, for example, 1.0 or less, or 0.7 or less.
  • the average HI of the modified fibroin may be, for example, ⁇ 1.0 or more and 1.0 or less, or 0 or more and 0.7 or less.
  • Naturally-derived fibroin is a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif. Yes, specifically, for example, fibroin produced by insects or arachnids.
  • fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea perniya, ⁇ ⁇ (Anteraea pernyi), and tussah. ), Silk moth (Samia synthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama), silk moth (Antheraea assama), silk moth (Antheraea assama), silk moth, silk moth, silk moth, silk moth, silk moth, silk moth.
  • Silk protein can be mentioned.
  • fibroin produced by insects include silk moth fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).
  • fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders,
  • Spiders belonging to the genus Spider spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne) such as Torinofundamashi and Otorinofundamashi, spiders belonging to the genus Cyrtarachne, spiders such as spiders Spiders belonging to (Gasteracantha genus), spiders belonging to the genus Isekigumo (genus Ordgarius) such as Mameitaisekigumo and Mutsutogaysekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc.
  • Trinofundamashi gene Cyrtarachne
  • Torinofundamashi and Otorinofundamashi spiders belonging to the genus Cyrtarachne
  • spiders such as spiders Spiders belonging to (Gasteracantha genus)
  • Spiders belonging to the genus Arachnura spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora ), Spiders belonging to the genus Cyclosa such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders
  • fibroin produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-.
  • fibroin whose sequence information is registered in NCBI GenBank can be mentioned.
  • sequence information registered in NCBI GenBank among the sequences containing INV as DIVISION, spidroin, complete, fibroin, "silk and protein", or “silk and protein” are described as keywords in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.
  • the modified fibroin according to the present embodiment is, for example, in the domain sequence of fibroin before modification, (A) inserting one or more alanine residues into the n motif or REP, or other amino acids adjacent to the alanine residues. It can be obtained by adding to the modification corresponding to the substitution of the residue with alanine. Further, there may be modification of the amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Energy, 100, 448 (1983).
  • one or more alanine residues are inserted into (A) n motif or REP with respect to the gene sequence of cloned naturally occurring fibroin, or adjacent to the alanine residue. It can be obtained by adding a mutation equivalent to substituting alanine for another matching amino acid residue. Also, for example, for the amino acid sequence of naturally occurring fibroin, (A) inserting one or more alanine residues in the n motif or REP, or replacing other amino acid residues adjacent to the alanine residue with alanine. 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.
  • the molecular weight of the modified fibroin according to the present invention is not particularly limited, but may be, for example, 40 kDa or more and 700 kDa or less.
  • the molecular weight of the modified fibroin according to the present invention may be, for example, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, 600 kDa or less, 500 kDa or less, 400 kDa or less, 300 kDa or less, Alternatively, it may be 200 kDa or less.
  • modified fibroin As a more specific example of the modified fibroin according to the present invention, (i) the modified fibroin containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or (ii) SEQ ID NO: 1, SEQ ID NO: 2 Alternatively, a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 can be mentioned.
  • Amino acid sequence shown in SEQ ID NO: 1 (Met-PRT1069) is a fibroin naturally occurring Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415) based on the nucleotide sequence and amino acid sequence of, (A) n Amino acid sequence in which alanine residues are continuous in the motif is changed to 6 consecutive alanine residues, all QQs are replaced with VF, and the remaining Qs are replaced with I.
  • the hydrophobicity of REP is 0.21.
  • the amino acid sequence (Met-PRT1070) shown in SEQ ID NO: 2 is the number of consecutive alanine residues by inserting one alanine residue in each (A) n motif of the amino acid sequence shown in SEQ ID NO: 1.
  • the hydrophobicity of REP is 0.21.
  • the amino acid sequence (Met-PRT1076) shown in SEQ ID NO: 3 is a REP of the amino acid sequence shown in SEQ ID NO: 2 in which some amino acid residues are deleted, and the hydrophobicity of the REP is 0. 21.
  • amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 all have a content of 6 or more consecutive alanines of 20% or more (Table 3).
  • the modified fibroin of (ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the modified fibroin of (ii) also has a domain sequence represented by formula 1: [(A) n motif-REP] m or formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein contained.
  • the sequence identity may be 80% or more, 85% or more, and preferably 95% or more.
  • modified fibroin described above may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization and the like of modified fibroin.
  • tag sequence examples include affinity tags that utilize specific affinity (binding, affinity) with other molecules.
  • affinity tag is a histidine tag (His tag).
  • His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chromatography) is performed. Can be used for.
  • Specific examples of the tag sequence include the amino acid sequence shown by SEQ ID NO: 4 or SEQ ID NO: 5 (amino acid sequence including His tag).
  • tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose-binding protein (MBP) that specifically binds to maltose can also be used.
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • an "epitope tag” utilizing an antigen-antibody reaction can also be used.
  • an antigenic peptide (epitope) as a tag sequence
  • an antibody against the epitope can be bound.
  • the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like.
  • a tag sequence in which the tag sequence can be separated by a specific protease can also be used.
  • the modified fibroin from which the tag sequence has been separated can also be recovered.
  • the modified fibroin containing the tag sequence As a more specific example of the modified fibroin containing the tag sequence, the modified fibroin containing the amino acid sequence set forth in (iii) SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or (iv) SEQ ID NO: 6, SEQ ID NO: 7 Alternatively, a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8 can be mentioned.
  • amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 is the amino acid represented by SEQ ID NO: 4 or SEQ ID NO: 5 at the N-terminal of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively. It has an amino acid sequence with an additional sequence (including His tag).
  • the modified fibroin of (iii) may consist of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the modified fibroin of (iv) 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 or SEQ ID NO: 8.
  • the modified fibroin of (iv) also has a domain sequence represented by formula 1: [(A) n motif-REP] m or formula 2: [(A) n motif-REP] m- (A) n motif. It is a protein contained.
  • the sequence identity may be 80% or more, 85% or more, and preferably 95% or more.
  • the amino acid sequence shown by SEQ ID NO: 6 (PRT1069), the amino acid sequence shown by SEQ ID NO: 7 (PRT1070), and the amino acid sequence shown by SEQ ID NO: 8 (PRT1076) are all six or more consecutive alanins.
  • the content of is 20% or more (Table 4). In each case, the hydrophobicity of REP is 0.21.
  • the modified fibroin described above may contain a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretory signal can be appropriately set according to the type of host.
  • thermal decomposition temperature Td
  • glass transition temperature Tg
  • the thermal decomposition temperature of a protein such as modified fibroin of the present invention can be measured, for example, by thermogravimetric analysis, which observes the mass change of the sample with heating.
  • the glass transition point of a protein such as modified fibroin of the present invention can be measured by differential scanning calorimetry, which is an analytical method for detecting the glass transition, crystallization, and melting phenomenon of a polymer as a heat flow change. it can.
  • the thermal decomposition temperature and / or the glass transition point can be measured by using, for example, a differential thermal-thermogravimetric simultaneous measuring device (DTG-60H, manufactured by Shimadzu Corporation) or the like. For example, general characteristics are maintained even at high temperatures and general characteristics are maintained even when exposed to high temperatures for a long time.
  • DTG-60H differential thermal-thermogravimetric simultaneous measuring device
  • the thermal stability of protein fibers such as the modified fibroin of the present invention is measured, for example, by observing changes in the physical properties of a sample due to long-term high-temperature treatment, and measuring the magnitude of resistance when a test piece is given a certain elongation. Can perform thermomechanical analysis (TMA).
  • TMA thermomechanical analysis
  • the modified fibroin in the present embodiment may have a thermal decomposition temperature of 268 ° C or higher, 270 ° C or higher, 272 ° C or higher, 280 ° C or 290 ° C or higher. Further, the thermal decomposition temperature of the modified fibroin in the present embodiment may be 5 ° C. or higher, 8 ° C. or higher, 10 ° C. or higher, or 12 ° C. or higher as compared with the fibroin before modification.
  • the modified fibroin in the present embodiment may have a glass transition point (Tg) of 185 ° C. or higher, 190 ° C. or higher, 192 ° C. or higher, or 195 ° C. or higher. Further, the glass transition point of the modified fibroin in the present embodiment may be 5 ° C. or higher, 8 ° C. or higher, 10 ° C. or higher, or 12 ° C. or higher as compared with the fibroin before modification.
  • Tg glass transition point
  • the nucleic acid according to the present invention encodes the modified fibroin according to the present invention.
  • a modified fibroin containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or SEQ ID NO: 4 or a sequence at either or both of the N-terminal and C-terminal of these amino acid sequences.
  • examples thereof include nucleic acids encoding modified fibroin or the like to which the amino acid sequence (tag sequence) represented by No. 5 is bound.
  • the nucleic acid hybridizes with the complementary strand of the nucleic acid encoding the modified fibroin according to the present invention under stringent conditions, and formula 1: [(A) n motif-REP] m or formula 2. : [(A) n motif-REP] m- (A) A nucleic acid encoding a modified fibroin containing a domain sequence represented by the n motif.
  • the total number of residues of alanine residues 6 or more consecutive in the above (A) n motif is preferably 20% or more of the total number of amino acid residues. ..
  • the modified fibroin encoded by the nucleic acid preferably has a REP hydrophobicity of ⁇ 1.0 or higher. Also, the modified fibroin encoded by the nucleic acid has a total number of amino acid residues of 580 or more. Is preferable.
  • the "stringent condition” refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • the “stringent condition” may be any of a low stringent condition, a medium stringent condition and a high stringent condition.
  • Low stringent conditions mean that hybridization occurs only when at least 85% or more identity is present between the sequences, eg, using a 5 ⁇ SSC containing 0.5% SDS at 42 ° C.
  • Conditions for hybridization can be mentioned.
  • Medium stringent conditions mean that hybridization occurs only when at least 90% or more identity is present between the sequences, eg, using a 5 ⁇ SSC containing 0.5% SDS at 50 ° C.
  • Conditions for hybridization can be mentioned.
  • High stringent conditions mean that hybridization occurs only when at least 95% or more identity is present between the sequences, eg, using a 5 ⁇ SSC containing 0.5% SDS at 60 ° C.
  • Conditions for hybridization can be mentioned.
  • the nucleic acid according to another embodiment has 90% or more sequence identity with the nucleic acid encoding the modified fibroin according to the present invention, and has formula 1: [(A) n motif-REP] m , or formula 2: [(A) n motif-REP] m- (A) A nucleic acid encoding modified fibroin containing a domain sequence represented by the n motif.
  • the total number of residues of alanine residues 6 or more consecutive in the above (A) n motif is preferably 20% or more of the total number of amino acid residues. ..
  • the modified fibroin encoded by the nucleic acid preferably has a REP hydrophobicity of ⁇ 1.0 or higher.
  • the total number of amino acid residues in the modified fibroin encoded by the nucleic acid is preferably 580 or more.
  • the sequence identity may be 80% or more, 85% or more, and preferably 95% or more.
  • the expression vector has a nucleic acid sequence according to the present invention and one or more regulatory sequences operably linked to the nucleic acid sequence.
  • the regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host.
  • the type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector.
  • the host according to the present invention is transformed with the expression vector according to the present invention.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • a vector that can be autonomously replicated in a host cell or can be integrated into the chromosome of the host and contains a promoter at a position where the nucleic acid according to the present invention can be transcribed is preferably used.
  • the expression vector according to the present invention can replicate autonomously in the prokaryote and at the same time contains a promoter, a ribosome binding sequence, a nucleic acid according to the present invention, and a transcription termination sequence. It is preferably a vector. It may contain genes that control promoters.
  • prokaryotes examples include microorganisms belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • Microorganisms belonging to the genus Escherichia include, for example, Escherichia coli BL21 (Novagen), Escherichia coli BL21 (DE3) (Life Technologies), Escherichia coli BLR (DE3) (Merck Millipore), Escherichia coli DH1, Escherichia. ⁇ Kori GI698, Escherichia Kori HB101, Escherichia Kori JM109, Escherichia Kori K5 (ATCC 23506), Escherichia Kori KY3276, Escherichia Kori MC1000, Escherichia Kori MG1655 (ATCC 47076), Escherichia Kori MG1655 (ATCC 47076) 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherich
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus bolsterensis, Brevibacillus centroporus Brevibacillus formosas, Brevibacillus invocatus, Brevibacillus lachilosporus, Brevibacillus limnofilus, Brevibacillus parabis.
  • Brevibacillus Reus Zeri Brevibacillus Thermolver, Brevibacillus Brevis 47 (FERM BP-1223), Brevibacillus Brevis 47K (FERM BP-2308), Brevibacillus Brevis 47-5 (FERM BP-1664), Brevi Brevibacillus chocinensis 47-5Q (JCM8975), Brevibacillus chocinensis HPD31 (FERM BP-1087), Brevibacillus chocinensis HPD31-S (FERM BP-6623), Brevibacillus chocinensis HPD31-OK (FERM BP-) 4573), Brevibacillus chocinensis SP3 strain (manufactured by Takara), and the like.
  • microorganisms belonging to the genus Serratia include, for example, Serratia liquefacience ATCC14460, Serratia entomophila, Serratia ficaria, Serratia ficiaria, Serratia serracia, Serratia serracia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia, Serratia. (Serratia grimesii), Serratia proteamaculans, Serratia odorifera, Serratia primushika (Serratia primusica, etc.), Serratia primusica, Serratia, etc.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis, Bacillus amyloliquefaciens, and the like.
  • microorganisms belonging to the genus Microbacterium include Microbacterium Ammonia Philum ATCC15354 and the like.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corinebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corinebacterium glutamicum ATCC14067) ATCC13826, ATCC14067, and Brevibacterium immariophyllum.
  • Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC6781, ATCC6782, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC140.
  • Pseudomonas for example, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brushcaseram, Pseudomonas pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas. (Pseudomonas sp.) D-0110 and the like can be mentioned.
  • any method for introducing DNA into the host cell can be used.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • the protoplast method Japanese Patent Laid-Open No. 63-248394
  • the method described in Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979), etc. can be mentioned.
  • Transformation of microorganisms belonging to the genus Brevibacillus is carried out, for example, by the method of Takahashi et al. (J. Bacteriol., 1983, 156: 1130-1134) or the method of Takagi et al. (Agric. Biol. Chem., 1989, 53:30). -3100), or by the method of Okamoto et al. (Bioscii. Biotechnol. Biochem., 1997, 61: 202-203).
  • vector into which the nucleic acid according to the present invention is introduced (hereinafter, simply referred to as “vector”) include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), and pSE280 (manufactured by Pharmacia). Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem.
  • pUC18 When Escherichia coli is used as a host, pUC18, pBluescriptII, pSupex, pET22b, pCold and the like can be mentioned as suitable vectors.
  • vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (Japanese Patent Laid-Open No. 2-31682), pNY700 (Japanese Patent Laid-Open No. 4-278901), pHY4831 (J. .Vector., 1987, 1239-1245), pNU200 (Shigezo Utaka, Journal of the Japanese Society of Agricultural Chemistry 1987, 61: 669-676), pNU100 (Appl. Microbial. Biotechnol., 1989, 30: 75-80), pNU211 (J.
  • the promoter is not limited as long as it functions in the host cell.
  • promoters derived from Escherichia coli or phages such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter can be mentioned.
  • an artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series (Ptrp ⁇ 2), a tac promoter, a lacT7 promoter, and a let I promoter can also be used.
  • the transcription termination sequence is not always necessary for the expression of the nucleic acid according to the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.
  • Eukaryotic hosts include, for example, yeast, filamentous fungi (molds, etc.) and insect cells.
  • yeast examples include the genus Saccharomyces, the genus Schizoccharomyces, the genus Kluyveromyces, the genus Trichosporon, the genus Trichosporon, the genus Trichosporon, the genus Pichia chan, the genus Pichia, and the genus Pichia. , Yeasts belonging to the genus Pichia, the genus Pichia, and the like.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Schizosaccharomyces pombe Schizosaccharomyces pombe
  • Kluyveromyces lactis Kluyveromyces lactis
  • Kluyveromyces marxianus Kluyveromyces marxianus
  • Trichosporon pullulans Trichosporon pullulans
  • Shiwaniomaisesu - Albius (Schwanniomyces alluvius), Swanniomyces occidentalis
  • Candida utilis Pichia pastris (Pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pi
  • Expression vectors when yeast is used as a host cell are usually the origin of replication (when amplification in the host is required) and a selection marker for vector growth in E. coli, a promoter for recombinant protein expression in yeast and It preferably contains a terminator, as well as a selection marker for yeast.
  • the expression vector When the expression vector is a non-integrated vector, it preferably further contains a self-replicating sequence (ARS). Thereby, the stability of the expression vector in the cell can be improved (Myers, AM, et al. (1986) Gene 45: 299-310).
  • ARS self-replicating sequence
  • Vectors for using yeast as a host include, for example, YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZ ⁇ , pGAPZ ⁇ , pGAPZ ⁇ And so on.
  • the promoter is not limited as long as it can be expressed in yeast.
  • promoters of glycolytic genes such as hexsource kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MF ⁇ 1 promoter, CUP 1 promoter, pGAP promoter, Examples thereof include the pGCW14 promoter, the AOX1 promoter, and the MOX promoter.
  • any method for introducing DNA into yeast can be used.
  • an electroporation method Metals Enzymol., 194,182 (1990)
  • spheroplast. Method Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)
  • Lithium acetate method J. Vector., 153, 163 (1983)
  • Proc. Natl. Acad. Sci. The method described in USA, 75, 1929 (1978) and the like can be mentioned.
  • Filamentous fungi include, for example, the genus Acremonium, the genus Aspergillus, the genus Ustirago, the genus Trichoderma, the genus Neurospora, the genus Fusalium, the genus Fusaria, Penicillium, Myceliophtra, Botryts, Magnaporthe, Mucor, Metalithium, Rhizopus, Monascus, Monascus , And fungi belonging to the genus Rhizopus.
  • filamentous fungi include Aspergillus aspergillus, Aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus asper
  • Penicillium penicillium Penicillium penicillium, Penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium pe
  • the promoter may be any of a gene related to glycolysis, a gene related to constitutive expression, an enzyme gene related to hydrolysis, and the like, and specifically, amyB, glaA, agdA, glaB, TEF1. , XynF1 tannessgene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, catB and the like can be mentioned.
  • the introduction of the expression vector into the filamentous fungus can be carried out by using a conventionally known method.
  • a conventionally known method for example, the method of Cohen et al. (Calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)], Protoplast method [Mol. Gen. Genet. , 168: 111 (1979)], Competent Method [J. et al. Mol. Biol. , 56: 209 (1971)], electroporation method and the like.
  • insect cell examples include a winged insect cell, more specifically, an insect cell derived from Prodenia frugiperda such as Sf9 and Sf21, and a cabbage looper (Trichoplus) such as High 5. ) Derived insect cells and the like.
  • baculoviridae virus Baculoviridae virus
  • Augurafa californica nuclear polyhedrosis virus which is a virus that infects night-stealing insects.
  • Examples of the method for co-introducing a recombinant gene transfer vector into insect cells and baculovirus for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075) and a lipofection method (Proc. Natl. Acad). .Sci.USA, 84,7413 (1987)) and the like.
  • the recombinant vector according to the present invention preferably further contains a selectable marker gene for transformant selection.
  • a selectable marker gene for example, in Escherichia coli, as a selectable marker gene, a resistance gene to various drugs such as tetracycline, ampicillin, and kanamycin can be used. Recessive selectable markers that can complement gene mutations involved in auxotrophy can also be used.
  • yeast a resistance gene to genetisin can be used as the selectable marker gene, and selectable markers such as LEU2, URA3, TRP1 and HIS3, which complement gene mutations involved in auxotrophy, can also be used.
  • selectable marker genes such as selectable marker genes, niaD (Biosci. Biotechnol.
  • the host transformed with the expression vector according to the present invention can be selected by plaque hybridization, colony hybridization or the like using a probe that selectively binds to the nucleic acid according to the present invention.
  • a probe that selectively binds to the nucleic acid according to the present invention.
  • a partial DNA fragment amplified by the PCR method modified with radioisotope or digoxigenin based on the sequence information of the nucleic acid according to the present invention can be used.
  • the modified fibroin according to the present invention can be produced by a method including a step of expressing the nucleic acid according to the present invention by a host transformed with the expression vector according to the present embodiment.
  • the method for producing a modified fibroin according to an embodiment is transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin according to the present invention and one or more regulatory sequences operably linked to the nucleic acid sequence. This includes the step of expressing the nucleic acid by the host.
  • modified fibroin can be obtained as a polypeptide to which sugar or sugar chain is added.
  • a host transformed with the expression vector according to the present invention is cultured in a culture medium, and the modified fibroin according to the present invention is produced and accumulated in the culture medium, and collected from the culture medium. It can be manufactured by doing so.
  • the method for culturing the host according to the present invention in a culture medium can be carried out according to the method usually used for culturing the host.
  • the culture medium of the host according to the present invention contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host.
  • a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the host.
  • the carbon source may be any assimilated by the host, for example, glucose, fructose, sucrose, carbohydrates containing them such as molasses, starch and starch hydrolyzate, and organic acids such as acetic acid and propionic acid. , And alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include, for example, ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, etc. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and their digests can be used.
  • ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, etc.
  • Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and their digests can be used.
  • inorganic salt for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is, for example, 15-40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during culturing is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during the culture.
  • an inducer may be added to the medium as needed.
  • isopropyl- ⁇ -D-thiogalactopyranoside and the like are used when culturing microorganisms transformed with an expression vector using the lac promoter
  • indol acrylic is used when culturing microorganisms transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.
  • TNM-FH medium manufactured by Harmingen
  • Sf-900 II SFM medium manufactured by Life Technologies
  • ExCell400 manufactured by ExCell405
  • Race' s Insect Medium Race' s Insect Medium (Nature, 195,788 (1962)) or the like
  • Insect cells can be cultured under conditions such as a culture medium pH of 6 to 7 and a culture temperature of 25 to 30 ° C. for a culture time of 1 to 5 days.
  • an antibiotic such as gentamicin may be added to the culture medium as needed during the culture.
  • the transformed plant cell When the host is a plant cell, the transformed plant cell may be cultured as it is, or it can be differentiated into a plant organ and cultured.
  • a commonly used Murashige and Scoog (MS) medium, White medium, or a medium obtained by adding plant hormones such as auxin and cytokinin to these media is used. be able to.
  • the culture of animal cells can be carried out under conditions such as a culture medium pH of 5 to 9 and a culture temperature of 20 to 40 ° C. for a culture time of 3 to 60 days.
  • antibiotics such as kanamycin and hygromycin may be added to the medium as needed during culturing.
  • a method for producing a modified fibroin using a host transformed with the expression vector As a method for producing a modified fibroin using a host transformed with the expression vector according to the present embodiment, a method for producing the modified fibroin inside the host cell, a method for secreting the modified fibroin outside the host cell, and a host cell outer membrane There is a way to produce it above. Each of these methods can be selected by varying the structure of the host cell used and the modified fibroin produced.
  • modified fibroin when modified fibroin is produced in the host cell or on the outer membrane of the host cell, the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Rowe et al. (Proc. Natl. Acad).
  • the modified fibroin can be modified to be actively secreted outside the host cell.
  • the modified fibroin can be actively secreted outside the host cell by expressing the modified fibroin in the form of adding a signal peptide to the polypeptide containing the active site of the modified fibroin by using a gene recombination method.
  • the modified fibroin produced by the host transformed with the expression vector according to the present embodiment can be isolated and purified by the method usually used for protein isolation and purification.
  • the modified fibroin when expressed in a lysed state in cells, after the culture is completed, the host cells are collected by centrifugation, turbid in an aqueous buffer solution, and then an ultrasonic crusher, a French press, or manton gaulin. Crush the host cells with a homogenizer, dynomil, or the like to obtain a cell-free extract.
  • a method usually used for isolating and purifying a protein that is, a solvent extraction method, a salting-out method using sulfuric acid, a desalting method, or an organic solvent is used.
  • Precipitation method anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -cepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia) Ion exchange chromatography method, hydrophobic chromatography method using resin such as butyl Sepharose, phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis Purified preparations can be obtained by using the above methods alone or in combination.
  • a resin such as diethylaminoethyl (DEAE) -cepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei)
  • S-Sepharose FF manufactured by Pharmacia
  • Ion exchange chromatography method hydrophobic chromatography method using resin such as but
  • the insoluble matter of the modified fibroin is recovered as a precipitate fraction by similarly collecting the host cell, crushing it, and centrifuging it.
  • the insoluble form of the recovered modified fibroin can be solubilized with a protein denaturing agent.
  • a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.
  • the modified fibroin or a derivative having a sugar chain added to the modified fibroin can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant and using the same isolation and purification method as described above from the culture supernatant.
  • the artificially modified fibroin composition according to the present embodiment contains at least the modified fibroin according to the present invention.
  • the content of the modified fibroin in the artificially modified fibroin composition may be 30 to 100% by mass, preferably 35 to 100% by mass, preferably 40 to 100% by mass, based on the total amount of the artificially modified fibroin composition. Is more preferable.
  • the artificially modified fibroin composition according to the present embodiment may further contain other additives depending on its form, use and the like.
  • Additives include, for example, plasticizers, leveling agents, cross-linking agents, crystal nucleating agents, antioxidants, UV absorbers, colorants, fillers, and synthetic resins.
  • the content of the additive may be 50 parts by mass or less with respect to 100 parts by mass of the total amount of the modified fibroin.
  • the artificially modified fibroin composition according to the present embodiment may be in any form of powder, paste, or liquid (for example, suspension or solution).
  • the artificially modified fibroin composition according to the present embodiment contains the artificially modified fibroin composition in addition to the form of the raw material composition (for example, protein powder, dope solution), or is molded from the artificially modified fibroin composition. It may be in the form of a body (eg, fiber, thread, film, foam, granule, molded article).
  • the artificially modified fibroin composition according to the present embodiment may be in the form of a doping liquid.
  • the doping solution according to this embodiment contains at least a modified fibroin and a solvent.
  • the doping solution according to this embodiment may further contain a dissolution accelerator.
  • the doping solution according to this embodiment may further contain a protein other than modified fibroin.
  • Solvents include, for example, hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and urea, guanidine, sodium dodecyl sulfate (SDS), Examples thereof include an aqueous solution containing lithium bromide, calcium chloride, lithium thiocyanate and the like. These solvents may be used alone or in combination of two or more.
  • HFIP hexafluoroisopropanol
  • HFA hexafluoroacetone
  • DMSO dimethyl sulfoxide
  • DMF N-dimethylformamide
  • SDS sodium dodecyl sulfate
  • solvents may be used alone or in combination of two or more.
  • the content of the modified fibroin in the doping solution may be 15% by mass or more, 30% by mass or more, 40% by mass or more, or 50% by mass or more based on the total mass of the doping solution.
  • the content of the modified fibroin may be 70% by mass or less, 65% by mass or less, or 60% by mass or less based on the total mass of the doping solution from the viewpoint of the production efficiency of the doping solution.
  • Examples of the dissolution accelerator include the following inorganic salts composed of Lewis acid and Lewis base.
  • Examples of the Lewis base include oxoacid ion (nitrate ion, perchlorate ion, etc.), metal oxoacid ion (permanganate ion, etc.), halide ion, thiocyanate ion, cyanate ion, and the like.
  • Examples of the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions.
  • the inorganic salt composed of Lewis acid and Lewis base include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate, calcium chloride, and calcium bromide.
  • Calcium salts such as calcium iodide, calcium nitrate, calcium perchlorate, and calcium thiocyanate, iron chlorides, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron salts such as iron thiocyanate,
  • Aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate, potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and potassium thiocyanate.
  • Potassium salts sodium chloride, sodium bromide, sodium iodide, sodium nitrate, sodium perchlorate, and sodium salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, perchloric acid, etc.
  • Zinc and zinc salts such as zinc thiocyanate, magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium salts such as magnesium thiocyanate, barium chloride, barium bromide, barium iodide, Examples include barium salts such as barium nitrate, barium perchlorate, and barium thiocyanate, and strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate.
  • the content of the dissolution accelerator is 1.0 part by mass or more, 5.0 part by mass or more, 9.0 part by mass or more, 15 part by mass or more, or 20.0 part by mass with respect to 100 parts by mass of the total amount of modified fibroin. That may be the above.
  • the content of the dissolution accelerator may be 40 parts by mass or less, 35 parts by mass or less, or 30 parts by mass or less with respect to 100 parts by mass of the total amount of the modified fibroin.
  • the doping solution may be heated to 30 to 90 ° C.
  • the meltable temperature may be set in a timely manner according to the solvent used, the type of modified fibroin, and the like. It may be shaken and stirred to promote dissolution.
  • the viscosity of the doping solution according to the present embodiment may be appropriately set according to the use of the doping solution and the like.
  • its viscosity may be appropriately set according to the spinning method, for example, 100 to 15,000 cP (centipoise) at 35 ° C. and 100 at 40 ° C. It may be set to ⁇ 30,000 cP (centipoise) or the like.
  • the viscosity of the spinning stock solution can be measured using, for example, the trade name "EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.
  • the artificially modified fibroin composition according to the present embodiment may be in the form of protein fiber.
  • Protein fibers can be obtained, for example, by spinning the above-mentioned doping solution (spinning solution) by a method usually used for spinning fibroin.
  • the spinning method is not particularly limited as long as it can spin the modified fibroin according to the present invention, and examples thereof include dry spinning, melt spinning, and wet spinning. Wet spinning can be mentioned as a preferable spinning method.
  • the dope liquid is extruded from the spinneret (nozzle) into the coagulation liquid (coagulation liquid tank), and the modified fibroin is solidified in the coagulation liquid to obtain an undrawn yarn in the shape of a yarn.
  • the coagulant may be any solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. Water may be added to the coagulating liquid as appropriate.
  • the temperature of the coagulating liquid is preferably 0 to 30 ° C.
  • the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole, and 1.4 to 4.0 ml / hour. More preferably, it is time.
  • the length of the coagulation liquid tank may be such that the solvent can be efficiently removed, for example, 200 to 500 mm.
  • the take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min.
  • the residence time may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes.
  • the coagulation liquid may be kept at a low temperature and taken in the state of undrawn yarn.
  • the coagulation liquid tank may be provided in multiple stages, and stretching may be performed in each stage or in a specific stage, if necessary.
  • the undrawn yarn (or pre-drawn yarn) obtained by the above method can be made into a drawn yarn through a drawing step.
  • the stretching method include moist heat stretching and dry heat stretching.
  • Moist heat stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating.
  • the temperature may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C.
  • the undrawn yarn (or the pre-drawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.
  • Dry heat stretching can be performed using an electric tube furnace, a dry heat plate, or the like.
  • the temperature may be, for example, 140 ° C. to 270 ° C., preferably 160 ° C. to 230 ° C.
  • the undrawn yarn (or the pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
  • Wet heat stretching and dry heat stretching may be performed individually, or they may be performed in multiple stages or in combination. That is, the first-stage stretching is performed by moist heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by moist heat stretching, the second stage stretching is performed by moist heat stretching, and the third stage stretching is performed by dry heat stretching. Wet heat stretching and dry heat stretching can be appropriately combined.
  • the final draw ratio in the drawing step is, for example, 5 to 20 times, preferably 6 to 11 times, that of the undrawn yarn (or the pre-drawn yarn).
  • the protein fibers may be stretched and then chemically crosslinked between the polypeptide molecules in the protein fibers.
  • the functional group that can be crosslinked include an amino group, a carboxyl group, a thiol group and a hydroxy group.
  • the amino group of the lysine side chain contained in the polypeptide can be crosslinked with an amide bond by dehydration condensation with the carboxyl group of the glutamic acid or aspartic acid side chain. It may be crosslinked by performing a dehydration condensation reaction under vacuum heating, or may be crosslinked with a dehydration condensation agent such as carbodiimide.
  • Cross-linking between polypeptide molecules may be carried out using a cross-linking agent such as carbodiimide or glutaraldehyde, or may be carried out using an enzyme such as transglutaminase.
  • carbodiimides include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N, N'-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide. , Diisopropylcarbodiimide (DIC) and the like.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • DCC N, N'-dicyclohexylcarbodiimide
  • DIC Diisopropylcarbodiimide
  • EDC and DIC are preferable because they have a high ability to form an amide bond between polypeptide molecules and easily undergo a cross-linking reaction.
  • the cross-linking treatment it is preferable to impart a cross-linking agent to the protein fibers and carry out cross-linking by vacuum heating and drying.
  • a cross-linking agent a pure product may be added to the protein fiber, or a product diluted to a concentration of 0.005 to 10% by mass with a lower alcohol having 1 to 5 carbon atoms and a buffer solution or the like may be added to the protein fiber. Good.
  • the cross-linking treatment is preferably carried out at a temperature of 20 to 45 ° C. for 3 to 42 hours. By the cross-linking treatment, a higher stress (strength) can be applied to the protein fiber.
  • the artificially modified fibroin composition according to the present embodiment may be in the form of a film.
  • the film can be obtained, for example, by casting the above-mentioned doping solution onto the surface of the substrate, drying and / or dessolving the solvent.
  • the viscosity of the dope solution is preferably 15 to 80 cP (centipores), more preferably 20 to 70 cP.
  • the concentration of the modified fibroin according to the present invention is preferably 3 to 50% by mass, more preferably 3.5 to 35% by mass, and 4.2 to 15. It is more preferably 8% by mass.
  • the dope solution When preparing the dope solution, it may be heated to 30 to 60 ° C. It may be shaken and stirred to promote dissolution.
  • the base material may be a resin substrate, a glass substrate, a metal substrate, or the like.
  • the base material is preferably a resin substrate from the viewpoint that the film after cast molding can be easily peeled off.
  • the resin substrate may be, for example, a polyethylene terephthalate (PET) film, a fluororesin film such as polytetrafluoroethylene, a polypropylene (PP) film, or a release film in which a silicone compound is immobilized on the surface of these films.
  • PET polyethylene terephthalate
  • PP polypropylene
  • the base material is stable against HFIP, DMSO solvent, etc., the dope solution can be stably cast molded, and the film after molding can be easily peeled off. From the viewpoint, the silicone compound is immobilized on the PET film or the surface of the PET film. It is more preferable that the release film is made.
  • the doping solution is poured on the surface of the base material, and a predetermined thickness (for example, drying and / or removal) is performed using a film thickness controlling means such as an applicator, a knife coater, and a bar coater.
  • a film thickness controlling means such as an applicator, a knife coater, and a bar coater.
  • a wet film having a thickness of 1 to 1000 ⁇ m after the solvent is prepared.
  • Drying and / or desolvation can be carried out dry or wet.
  • Examples of the dry method include vacuum drying, hot air drying, and air drying.
  • Examples of the wet method include a method of immersing the cast film in a desolvent solution (also referred to as a coagulation solution) to desorb the solvent.
  • Examples of the desolvation solution include an alcohol solution such as water, methanol, ethanol, 2-propanol and other lower alcohols having 1 to 5 carbon atoms, and a mixed solution of water and alcohol.
  • the temperature of the desolvent solution (coagulant solution) is preferably 0 to 90 ° C.
  • the unstretched film after drying and / or desolvation can be uniaxially stretched or biaxially stretched in water.
  • the biaxial stretching may be sequential stretching or simultaneous biaxial stretching. Two or more stages of multi-stage stretching may be performed.
  • the draw ratio is preferably 1.01 to 6 times, more preferably 1.05 to 4 times in both the vertical and horizontal directions. Within this range, stress-strain balance is easy to achieve.
  • the underwater stretching is preferably carried out at a water temperature of 20 to 90 ° C.
  • the stretched film is preferably heat-fixed at 50 to 200 ° C. for 5 to 600 seconds. By this heat fixing, dimensional stability at room temperature can be obtained.
  • the uniaxially stretched film becomes a uniaxially oriented film
  • the biaxially stretched film becomes a biaxially oriented film.
  • the artificially modified fibroin composition according to the present invention can be produced by a method including a step of preparing the modified fibroin according to the present invention.
  • the method for producing an artificially modified fibroin composition according to the present invention may further include a step of preparing a modified fibroin solution (for example, a doping solution) containing the modified fibroin according to the present invention.
  • a method for improving the thermal stability of modified fibroin containing a domain sequence represented by n motifs is to insert one or more alanine residues into at least one (A) n motif or REP in unmodified fibroin. , Or by substituting alanine for another amino acid residue adjacent to the alanine residue, it comprises increasing the number of consecutive alanine residues in at least one (A) n motif.
  • the thermal decomposition temperature (Td) of fibroin after modification is 5 ° C. or higher.
  • the unmodified fibroin includes naturally occurring fibroin and modified fibroin.
  • (A) n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 4 to 27.
  • the number of amino acid residues of the n motif is 4 to 20, 8 to 20, 10 to 20, 4 to 16, 5 to 10, 6 to 12, 6 to 18, 7 to 10, 7 to 14, 8 to It may be 16, or 11 to 16.
  • the ratio of the number of alanine residues to the total number of amino acid residues in the n motif is 80% or more.
  • REP shows an amino acid sequence consisting of 10-200 amino acid residues.
  • m represents an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • the number of alanine residues relative to the total number of amino acid residues in the (A) n motif may be 80% or more, preferably 85% or more, and more preferably 90% or more. It is more preferably 95% or more, and even more preferably 100% (meaning that it is composed only of alanine residues). It is preferable that at least seven (A) n motifs present in the domain sequence are composed of only alanine residues.
  • (A) n motif is (Ala) k (Ala indicates alanine residues, k is an integer of 4 to 27, preferably an integer of 4 to 20, more preferably. It means that it has an amino acid sequence represented by (indicating an integer of 4 to 16).
  • the method according to the present embodiment comprises increasing the number of consecutive alanine residues in at least one (A) n motif in unmodified fibroin.
  • the method according to the present embodiment may increase the number of consecutive alanine residues in at least two or more (A) n motifs in the fibroin before modification, and may increase the number of alanine residues before modification. It may increase the number of consecutive alanine residues in at least 5 or more (A) n motifs in fibroin, and in at least 6 or more (A) n motifs in unmodified fibroin.
  • the fibroin before modification may be naturally-derived fibroin or modified fibroin.
  • the method according to this embodiment is to insert one or more alanine residues into at least one (A) n motif or REP in unmodified fibroin, or to insert other amino acid residues adjacent to the alanine residues.
  • it may further include the modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues.
  • Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Energy, 100, 448 (1983).
  • the method according to the present embodiment is, for example, inserting one or more alanine residues into (A) n motif or REP for the cloned naturally occurring fibroin gene sequence, or adjoining the alanine residue. It can include making a mutation equivalent to replacing the amino acid residue of alanine with alanine.
  • the modified fibroin can be, for example, by inserting one or more alanine residues into (A) n motif or REP with respect to the amino acid sequence of naturally occurring fibroin, or other amino acid residues adjacent to the alanine residue. It can also be obtained by designing an amino acid sequence corresponding to substituting alanine with alanine and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • the total number of alanine residues 6 or more consecutive in the (A) n motif is 20% or more of the total number of amino acid residues. Is preferable.
  • the total number of alanine residues that are 6 or more consecutive in the n motif is the number of alanine residues when only the number of alanine residues that are 6 or more consecutive is counted. Means the total number of. Therefore, the number of consecutive alanine residues of only 5 residues is not counted.
  • the presence of other amino acid residues between alanine residues means that even if there are no more than 6 consecutive alanine residues, the number of alanine residues remains. Is not counted.
  • the total number of alanine residues 6 or more consecutive in the n motif is, for example, 20% or more, 21% or more, 22% or more, or 23% or more of the total number of amino acid residues. You may.
  • the total number of alanine residues that are 7 or more consecutive in the n motif is, for example, 20% or more of the total number of amino acid residues, 2 It may be 1% or more, 22% or more, or 23% or more.
  • the modified fibroin in the method according to this embodiment preferably has a total number of amino acid residues of 580 or more.
  • the total number of amino acid residues in total may be, for example, 580 or more, 590 or more, 600 or more, 610 or more, or 620 or more.
  • the modified fibroin in the method according to the present embodiment preferably has a REP hydrophobicity of ⁇ 1.0 or higher, more preferably ⁇ 0.9 or higher, and more preferably ⁇ 0.8 or higher. More preferably, it is more preferably ⁇ 0.7 or more, further preferably 0 or more, further preferably 0.2 or more, further preferably 0.3 or more, and 0. 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, for example, 1.0 or less, or 0.7 or less.
  • the hydrophobicity of REP may be, for example, ⁇ 1.0 or more and 1.0 or less, and may be 0 or more and 0.7 or less.
  • the thermal decomposition temperature (Td) of the modified fibroin may be higher by 5 ° C. or higher as compared with the fibroin before modification, for example, 8 ° C. or higher, 10 ° C. or higher, or 12 ° C. or higher. It may be a thing.
  • the fibroin before modification is preferably naturally derived fibroin, more preferably fibroin derived from insects or arachnids, and is a large bottle-shaped spider protein (MaSp) or a small bottle-shaped spider protein (MiSp) of arachnids. Is even more preferable.
  • the glass transition point (Tg) of the modified fibroin is, for example, 5 ° C. or higher, 8 ° C. or higher, 10 ° C. or higher, or 12 ° C. or higher, as compared with the fibroin before modification. It may be a thing.
  • Modification of the fibroin protein in the method according to the present embodiment can be carried out so as to be in the preferred embodiment described as the embodiment of the modified fibroin.
  • the modified fibroin or artificially modified fibroin composition according to the present invention includes fibers (long fibers, short fibers, multifilaments, monofilaments, etc.) or yarns (spun yarns, twisted yarns, false twisted yarns, processed yarns, mixed yarns, etc. As a blended yarn, etc.), it can be applied to textiles, knitted fabrics, braids, non-woven fabrics, etc. It can also be applied to high-strength applications such as ropes, surgical sutures, flexible fasteners for electrical components, and bioactive materials for transplantation (eg, artificial ligaments and aortic bands).
  • the modified fibril or artificially modified fibril composition according to the present invention includes foams, granules (spheres or non-spheres, etc.), nanofibrils, gels (hydrogels, etc.), resins and their equivalents. These can also be applied to, and these can be produced according to the methods described in JP-A-2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735 and the like.
  • the product according to this embodiment contains the modified fibroin of the present invention and is selected from the group consisting of fibers, threads, films, foams, granules, nanofibrils, gels and resins.
  • amino acid sequence (PRT380) shown in SEQ ID NO: 9 is deleted so that the number of consecutive alanine residues in each (A) n motif of the naturally occurring fibroin is 5, and all GGX in REP are deleted. Is replaced with GQX, and the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 4 is added to the N-terminal.
  • the amino acid sequence (PRT525) shown in SEQ ID NO: 10 is obtained by deleting every other (A) n motif ((A) 5 ) from the N-terminal side to the C-terminal side of the naturally occurring fibroin. Insert one [(A) n motif-REP] in front of the C-terminal sequence, replace all GGX in the REP with GQX, and place two alanine residues on the C-terminal side of each (A) n motif. Insert it, replace some glutamine (Q) residues with serine (S) residues, delete some amino acids on the C-terminal side so that the molecular weight is almost the same as PRT380, and make it N-terminal.
  • the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 4 is added.
  • the amino acid sequence shown by SEQ ID NO: 11 is based on the amino acid sequence shown by SEQ ID NO: 12 (PRT564) by deleting one alanine residue on the C-terminal side of each (A) n motif and REP. The Q inside is replaced with VFI.
  • the amino acid sequence (PRT1069) shown in SEQ ID NO: 6 is obtained by inserting one alanine residue on the C-terminal side of each (A) n motif of PRT1068.
  • the amino acid sequence (PRT1070) shown in SEQ ID NO: 7 is obtained by inserting two alanine residues on the C-terminal side of each (A) n motif of PRT1068.
  • Nucleic acids encoding proteins having the designed amino acid sequences shown in SEQ ID NOs: 6 to 12 were synthesized. An NdeI site was added to the nucleic acid at the 5'end, and an EcoRI site was added downstream of the stop codon. These five types of nucleic acids were cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the protein expression vector pET-22b (+) to obtain an expression vector.
  • Escherichia coli BLR (DE3) was transformed with a pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequences shown in SEQ ID NOs: 6 to 12.
  • the transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of seed culture medium (Table 5) containing ampicillin so that OD 600 was 0.005.
  • the temperature of the culture solution was kept at 30 ° C., and flask culture was carried out until the OD 600 reached 5, (about 15 hours) to obtain a seed culture solution.
  • the seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 6) was added so that the OD 600 was 0.05, and transformed Escherichia coli was inoculated.
  • the temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the target protein.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the SDS washed granules were suspended in a DMSO solution containing 1 M lithium chloride so as to have a concentration of 100 mg / mL, and heat-treated at 80 ° C. for 1 hour. Then, it was centrifuged (11,000 g, 30 minutes, room temperature) with the Kubota centrifuge, and the supernatant was collected.
  • Td thermal decomposition temperature
  • Tg glass transition point
  • the thermal decomposition temperature (Td) was measured by tangential intersection analysis of the differential thermal analysis (DTA) curve.
  • the glass transition point (Tg) was measured by tangential intersection analysis of the DTA curve.
  • Table 7 shows the characteristics of the amino acid sequences of PRT380 and PRT525. The results of comparing the thermal decomposition temperatures and the glass transition points of PRT380 and PRT525 are shown in FIGS. 2 and 3, respectively. PRT525 showed higher pyrolysis temperature and glass transition point than PRT380.
  • the characteristics of the amino acid sequences of PRT1068, PRT1069 and PRT1070 are shown in Table 8.
  • the results of comparing the thermal decomposition temperatures of PRT1068, PRT1069 and PRT1070 are shown in FIG. By increasing the number of consecutive alanine residues in each (A) n motif, the thermal decomposition temperature tended to increase.
  • FIG. 5 shows the results of comparing the thermal decomposition temperatures (Td) of PRT525 (SEQ ID NO: 10) and PRT1070 (SEQ ID NO: 7) in which the number of consecutive alanine residues in each (A) n motif is 7.
  • the hydrophobicity of REP of PRT525 is ⁇ 1.32, and the hydrophobicity of REP of PRT1070 is 0.21.
  • PRT1070 which has a higher degree of hydrophobicity in REP, showed a higher pyrolysis temperature than PRT525.

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Abstract

La présente invention concerne une fibroïne modifiée qui comprend une séquence de domaine représentée par la formule 1, [(A)n <sb />motif-REP]m, ou la formule 2, [(A)n <sb />motif-REP]m-(A)n <sb />motif. Dans le (A)n motif, le nombre total de résidus d'alanine dans des répétitions de six résidus ou plus est au moins égal à 20 % du nombre total de résidus d'acides aminés dans le motif entier, l'hydrophobicité de REP est supérieure ou égale à -1,0, et le nombre total de résidus d'acides aminés dans le motif entier est supérieur ou égal à 580.
PCT/JP2020/027991 2019-07-19 2020-07-20 Fibroïne modifiée WO2021015153A1 (fr)

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WO2017200659A2 (fr) * 2016-04-08 2017-11-23 Cornell University Procédé pour améliorer la cicatrisation de plaies à l'aide de protéines dérivées de la soie
WO2019022163A1 (fr) * 2017-07-26 2019-01-31 Ｓｐｉｂｅｒ株式会社 Fibroïne modifiée

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