WO2020158947A1 - Polypeptide - Google Patents

Polypeptide Download PDF

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Publication number
WO2020158947A1
WO2020158947A1 PCT/JP2020/003795 JP2020003795W WO2020158947A1 WO 2020158947 A1 WO2020158947 A1 WO 2020158947A1 JP 2020003795 W JP2020003795 W JP 2020003795W WO 2020158947 A1 WO2020158947 A1 WO 2020158947A1
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Prior art keywords
amino acid
acid sequence
protein
polypeptide
seq
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PCT/JP2020/003795
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French (fr)
Japanese (ja)
Inventor
裕生 上久保
有吾 林
健大 佐藤
中村 浩之
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Spiber株式会社
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Priority to JP2020568644A priority Critical patent/JPWO2020158947A1/en
Publication of WO2020158947A1 publication Critical patent/WO2020158947A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K19/00Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62DNA sequences coding for fusion proteins

Definitions

  • the present invention relates to polypeptides.
  • the present invention also relates to fusion proteins containing the polypeptide.
  • Proteins may form a specific complex through the interaction between protein molecules (protein-protein interaction).
  • the protein-protein interaction can be detected, for example, by the Two-hybrid method.
  • methods for predicting protein-protein interactions have also been reported (for example, Patent Document 1 and Non-Patent Document 1).
  • amino acid sequences involved in protein-protein interactions can be applied. For example, by adding a polypeptide having an amino acid sequence involved in protein-protein interaction to a desired protein, it is considered possible to control the association between the proteins.
  • the higher-order structure of proteins (eg, secondary structure, tertiary structure) is also involved as one factor in the protein-protein interaction.
  • the higher-order structure of a protein is easily affected by, for example, temperature, pH, and the presence of a denaturant, and there is a problem that the proteins cannot stably interact with each other depending on the environment in which the protein is placed.
  • the present invention aims to provide a polypeptide that can selectively interact even under denaturing conditions.
  • ⁇ p i,j (L) and ⁇ a i,j (L) are energies calculated for a pair of amino acid residues.
  • ⁇ i ⁇ k ⁇ i+L is 1 when k belongs to i to i+L ⁇ 1, and is 0 otherwise.
  • ⁇ j ⁇ k ⁇ j+L is 1 when k belongs to j to j+L ⁇ 1, and is 0 otherwise.
  • i and j correspond to the residue numbers in the amino acid sequence with the start point as 1.
  • [7] A nucleic acid encoding the polypeptide according to any of [1] to [6].
  • a fusion protein comprising the amino acid sequence obtained by fusing the polypeptide according to any one of [1] to [6] with a protein.
  • the fusion protein according to [8] which comprises the amino acid sequence of the protein and the amino acid sequence of the polypeptide.
  • the fusion protein according to [8], wherein the amino acid sequence of the protein contains the amino acid sequence of the polypeptide.
  • the fusion protein according to [10] wherein the number of amino acid residues from the N-terminus or C-terminus of the protein to the fusion site is 10% or less with respect to the total number of amino acid residues of the protein.
  • the free energy of aggregation g(k) in the amino acid sequence (#GEN891, SEQ ID NO: 1) in which the tag sequence represented by SEQ ID NO: 17 is added to the N-terminus of the large spit duct dragline protein (ADF4) of Japanese spider Spider is used as a residue number. It is a graph plotted against.
  • the aggregation free energy g(k) in the amino acid sequence (#GEN889, SEQ ID NO: 2) in which the tag sequence represented by SEQ ID NO: 17 is added to the N-terminal of the large spit duct dragline protein (ADF3) of the Japanese spider Spider is designated as the residue number. It is a graph plotted against.
  • FIG. 3 is a photograph showing the results of analysis by SDS-PAGE below.
  • FIG. 3 is a photograph showing the result of SDS-PAGE analysis of each solution in the case of column purification of a polypeptide having the amino acid sequence represented by SEQ ID NO: 20 or 22, in the absence of a denaturing agent.
  • the polypeptide according to this embodiment has an amino acid sequence in which the average value of the aggregation free energy g(k) calculated by the following formula (1) per amino acid residue is ⁇ 4.0 or less.
  • ⁇ p i,j (L) and ⁇ a i,j (L) are energies calculated for a pair of amino acid residues.
  • ⁇ i ⁇ k ⁇ i+L is 1 when k belongs to i to i+L ⁇ 1, and is 0 otherwise.
  • ⁇ j ⁇ k ⁇ j+L is 1 when k belongs to j to j+L ⁇ 1, and is 0 otherwise.
  • i and j correspond to the residue numbers in the amino acid sequence with the start point as 1.
  • is 2.0.
  • the cohesive free energy g(k) is a value calculated for the amino acid residue of residue number k.
  • the average value of aggregation free energy g(k) per amino acid residue is the total value of aggregation free energy g(k) calculated for each amino acid residue in a certain amino acid sequence divided by the number of amino acid residues. Means the value
  • the polypeptide according to this embodiment has such a structure, it can selectively interact (self-associate) even under denaturing conditions.
  • the energies represented by ⁇ p i,j (L) and ⁇ a i,j (L) are calculated by the DSSP algorithm for high-quality globular proteins (top 500) registered in the protein database (PDB). It is a value calculated by the following formula (2) based on the secondary structure to be assigned, and specifically, it is shown in Table 1 (E p ab ) and Table 2 (E a ab ).
  • the polypeptide according to the present embodiment has an amino acid sequence having an average value of aggregation free energy g(k) per amino acid residue of ⁇ 4.0 or less (hereinafter, also referred to as “self-association sequence”). ), and in addition to the self-association sequence, it may have an amino acid sequence having an average value of aggregation free energy g(k) per amino acid residue of more than -4.0.
  • the average value of the aggregation free energy g(k) of the self-association sequence per amino acid residue is preferably ⁇ 4.5 or less, more preferably ⁇ 5.0 or less, and ⁇ 5.5 or less. Is more preferable, it is even more preferably ⁇ 6.0 or less, even more preferably ⁇ 6.5 or less, and particularly preferably ⁇ 7.0 or less.
  • polypeptide As a specific example of the polypeptide according to the present embodiment, an amino acid sequence consisting of the 546th amino acid residue to the 582th amino acid residue of the amino acid sequence shown by SEQ ID NO: 1, the 547th amino acid sequence shown by SEQ ID NO: 2 The amino acid sequence consisting of the 579th amino acid residue from the amino acid residue of, and the amino acid sequences shown in SEQ ID NOs: 3 to 16 are mentioned.
  • amino acid sequences represented by SEQ ID NOs: 3 to 8 are amino acid sequences containing a part or all of the self-association sequence in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
  • a polypeptide according to another embodiment contains an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3 to 16. Since the polypeptide according to the present embodiment has such a constitution, it can selectively interact (self-associate) even in the presence of a denaturant.
  • the amino acid sequence represented by SEQ ID NO: 3 is the amino acid sequence of the non-repetitive region (NRC region) at the C-terminal of the large vesicle guideline protein (ADF4) of the spider (Araneus diadematus).
  • the amino acid sequence represented by SEQ ID NO:4 is a partial sequence of the amino acid sequence represented by SEQ ID NO:3, and is an amino acid sequence corresponding to a portion in which the average value of the aggregation free energy g(k) per amino acid residue is particularly low. is there.
  • the amino acid sequence represented by SEQ ID NO: 5 is the amino acid sequence of the non-repetitive region at the C-terminus of the large vesicle guideline thread protein (ADF3) of the Japanese spider, Spiderwort.
  • the amino acid sequence represented by SEQ ID NO: 6 is a partial sequence of the amino acid sequence represented by SEQ ID NO: 5, and is an amino acid sequence corresponding to a portion in which the average value of the aggregation free energy g(k) per amino acid residue is particularly low. is there.
  • the amino acid sequence represented by SEQ ID NO: 7 is an amino acid sequence in which helix 5 located at the most C-terminal side from the non-repetitive region at the C-terminal of the giant spider duct silkworm thread protein (ADF3) of the armpit spider is deleted.
  • the amino acid sequence represented by SEQ ID NO:8 is a partial sequence of the amino acid sequence represented by SEQ ID NO:7, and is an amino acid sequence corresponding to a portion where the average value of the aggregation free energy g(k) per amino acid residue is particularly low. is there.
  • the amino acid sequences shown in SEQ ID NOs: 10, 12, 14 and 16 are amino acid sequences selected by the following procedure.
  • HMM profile hidden Markov model
  • MaSp large ampullate spidroin
  • An amino acid sequence was generated by executing the hmmemit program using the created HMM as an input value (10000 sequences). With respect to the generated amino acid sequence, the average value of the aggregation free energy g(k) per 1 amino acid residue was calculated for every 5 consecutive amino acid residues. An amino acid sequence having a calculated minimum value of ⁇ 4.5 or less was selected.
  • amino acid sequences shown in SEQ ID NOs: 9, 11, 13 and 15 are amino acid sequences obtained by adding an additional sequence to the N-terminal of the amino acid sequences shown in SEQ ID NOs: 10, 12, 14 and 16, respectively.
  • amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 have at least 60% or more (63%) sequence identity with each other.
  • the polypeptide according to this embodiment may have 65% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3 to 16, or 70% or more sequence identity. May have a sequence identity of 75% or more, may have a sequence identity of 80% or more, and may have a sequence identity of 85% or more. May have a sequence identity of 90% or more, may have a sequence identity of 95% or more, or may have a sequence identity of 98% or more. , 99% or more sequence identity may be sufficient.
  • the polypeptide according to this embodiment may include the amino acid sequence represented by any of SEQ ID NOS: 3 to 16 or may be the amino acid sequence represented by any of SEQ ID NOs: 3 to 16. Good.
  • the polypeptide according to the present embodiment may have a tag sequence added to either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, etc. of the polypeptide according to this embodiment.
  • an affinity tag that utilizes specific affinity (binding, affinity) with another molecule can be mentioned.
  • a specific example of the affinity tag is a histidine tag (His tag).
  • the His tag is a short peptide in which about 4 to 10 histidine residues are lined up and has a property of specifically binding to a metal ion such as nickel. Therefore, the His tag according to this embodiment by metal chelating chromatography It can be used to isolate the polypeptide.
  • Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 17 (amino acid sequence containing 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 be used.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • epitope tag that utilizes the antigen-antibody reaction.
  • a peptide (epitope) showing antigenicity as a tag sequence
  • an antibody against the epitope can be bound.
  • the epitope tag include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, FLAG tag and the like.
  • a tag sequence that can be cleaved with a specific protease can also be used.
  • the polypeptide of the present embodiment adsorbed via the tag sequence can be subjected to a protease treatment.
  • the polypeptide of the present embodiment from which the tag sequence is cleaved can be recovered.
  • polypeptide containing the tag sequence examples include, for example, the polypeptides containing the amino acid sequences represented by any of SEQ ID NOs: 18 to 31.
  • the amino acid sequences represented by SEQ ID NOS: 18 to 31 are obtained by adding the amino acid sequences represented by SEQ ID NO: 17 (amino acid sequences including His tag) to the N-terminals of the amino acid sequences represented by SEQ ID NOS: 3 to 16, respectively.
  • the polypeptide according to one embodiment may include the amino acid sequence shown in any of SEQ ID NOs: 18 to 31, or may consist of the amino acid sequence shown in any of SEQ ID NOs: 18 to 31. Good.
  • the molecular weight of the polypeptide according to this embodiment is not particularly limited, but may be, for example, 1 kDa or more and 300 kDa or less.
  • the molecular weight of the polypeptide according to the present embodiment may be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, or 10 kDa or more, for example, 250 kDa or less, It may be 200 kDa or less, 150 kDa or less, 100 kDa or less, 90 kDa or less, 80 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 20 kDa or less, or 15 kDa or less.
  • the number of amino acid residues of the polypeptide according to this embodiment is not particularly limited, but for example, 150 amino acid residues or less, 120 amino acid residues or less, 100 amino acid residues or less, 80 amino acid residues or less, 60 amino acid residues or less.
  • amino acid residues of the polypeptide according to this embodiment is small (for example, about 20 amino acid residues), for example, when a fusion protein containing the polypeptide according to this embodiment is produced by a microorganism or the like, Since the total number of amino acid residues is not significantly increased, the influence on the productivity can be reduced, and the aggregating property of the fusion protein can be increased or the aggregating property can be added.
  • the polypeptide according to this embodiment may be hydrophilic or hydrophobic.
  • the sum of the hydrophobicity indices (hydropathic index, HI) of all amino acid residues constituting the polypeptide is calculated, and then the sum is divided by the total number of amino acid residues. It is preferable that (average HI, hereinafter also referred to as “hydrophobicity”) be ⁇ 1.0 or more.
  • HI hydrophobicity index
  • a known index Kyte J, & Doolittle R (1982) “A simple method for dissipating the hydropathic character. 105-132).
  • the hydrophobicity index of each amino acid is as shown in Table 3 below.
  • the hydrophobicity of the polypeptide according to this embodiment may be ⁇ 0.5 or more, 0 or more, 1 or more, 5 or more, or 10 or more, and 10 or less, 5 or less, or 1 or less.
  • the polypeptide according to the present embodiment is preferably one in which the polypeptides interact with each other to form a specific complex (that is, one having self-association ability) under denaturing conditions. And more preferably have self-association ability.
  • the polypeptide according to this embodiment may be an artificial polypeptide.
  • artificial polypeptide means an artificially produced polypeptide.
  • Artificial polypeptides include, for example, polypeptides produced using gene recombination technology and chemically synthesized polypeptides.
  • Denaturing conditions are conditions that can destroy the higher-order structure (eg, secondary structure, tertiary structure) of a protein.
  • the denaturing conditions include, for example, the pH of the protein solution (eg, acidic conditions of pH 1 to 4 and alkaline condition of pH 10 to 14), the environmental temperature at which the protein is placed (eg, low temperature condition of ⁇ 20° C. to 5° C., and The high temperature condition of 35° C. or higher), the presence of a modifier, and the like.
  • the denaturing agent includes, for example, a substance that breaks hydrogen bonds, hydrophobic bonds, ionic bonds, and disulfide bonds between protein molecules and/or intramolecularly.
  • Specific examples of the denaturing agent include, for example, urea, guanidine salt (eg, guanidine thiocyanate), sodium dodecyl sulfate (SDS), 2-mercaptoethanol, tetrahydrofuran (THF), and dimethyl sulfoxide (DMSO), acetone, alcohol.
  • polar solvents that are miscible with water such as groups (eg, methanol, ethanol, isopropanol), dioxane, dimethylformamide (DMF), and acetonitrile.
  • the polypeptide according to this embodiment can be produced by a conventional method using a nucleic acid encoding the polypeptide.
  • the nucleic acid encoding the polypeptide may be chemically synthesized based on the nucleotide sequence information, or may be synthesized by utilizing the PCR method or the like.
  • the fusion protein according to this embodiment includes an amino acid sequence in which the polypeptide according to the present invention and the protein are fused.
  • the aspect of fusion in the fusion protein according to the present embodiment is not particularly limited, and includes, for example, a fusion protein containing the amino acid sequence of the protein and the amino acid sequence of the polypeptide of the present invention (for example, the N-terminal of the amino acid sequence of the protein and Fusion protein containing the amino acid sequence of the polypeptide of the present invention in either or both of the C-terminals, and a fusion protein containing the amino acid sequence of the polypeptide of the present invention in the amino acid sequence of the protein (
  • it may be a fusion protein containing the amino acid sequence of the polypeptide of the present invention in the middle of the amino acid sequence of the protein.
  • a fusion site from the N-terminal or C-terminal of the protein (insertion site of the polypeptide of the present invention in the amino acid sequence of the protein) Up to 45%, 40% or less, 35% or less, 30% or less with respect to the total number of amino acid residues of the protein. , 25% or less, 20% or less, preferably 15% or less, preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, It is more preferably 4% or less, 3% or less, 2% or less, or 1% or less.
  • proteins in the fusion protein according to the present embodiment is not particularly limited, and any protein can be used.
  • proteins include proteins that can be used industrially, proteins that can be used medically, and structural proteins.
  • proteins that can be used for industrial or medical purposes include spider silk protein, enzyme, regulatory protein, receptor, peptide hormone, cytokine, membrane or transport protein, antigen used for vaccination, vaccine, antigen-binding protein, It may include immunostimulatory proteins, allergens, and full length antibodies or antibody fragments or derivatives.
  • structural proteins include fibroin (for example, spider silk fibroin (spider silk), silkworm silk, etc.), keratin, collagen, elastin, resilin, fragments of these proteins, and proteins derived therefrom. You can
  • fibroin includes naturally occurring fibroin and modified fibroin.
  • naturally-derived fibroin means a fibroin having the same amino acid sequence as naturally-derived fibroin
  • modified fibroin means a fibroin having an amino acid sequence different from that of naturally-derived fibroin. To do.
  • Fibroin may be spider silk fibroin.
  • "Spider silk fibroin” includes natural spider silk fibroin and modified fibroin derived from natural spider silk fibroin. Examples of the natural spider silk fibroin include spider silk protein (SSP) produced by spiders.
  • SSP spider silk protein
  • Fibroin is, for example, a protein containing a domain sequence represented by the formula 3: [(A) n motif-REP] m or the formula 4: [(A) n motif-REP] m -(A) n motif. May be.
  • the fibroin according to the present embodiment 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 typically, but not limited to, regions having no repeat of the amino acid motif characteristic of fibroin, and consist of about 100 amino acids.
  • domain sequence refers to a crystalline region (typically corresponding to the (A) n motif of an amino acid sequence) and an amorphous region (typically REP of an amino acid sequence) peculiar to fibroin.
  • formula 3 [(A) n motif-REP] m
  • formula 4 [(A) n motif-REP] m -(A) n motif.
  • the (A) n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27.
  • the number of amino acid residues in the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • REP may be an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300, and may be an integer of 10 to 300.
  • the plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • Naturally-occurring fibroin examples include, for example, a domain sequence represented by Formula 3: [(A) n motif-REP] m or Formula 4: [(A) n motif-REP] m -(A) n motif.
  • the proteins included can be mentioned.
  • Specific examples of naturally-derived fibroin include, for example, fibroin produced by insects or arachnids.
  • fibroin produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yam tai rya pynayi, ori peri erygium, Anteraea periyna, Pomegranate (Anteraea periyi), Anteraea periyna, Pomegranate (Anteraea peryny) ), silkworm proteins produced by silkworms such as Anthera apse assap, such as silkworm silkworm (Antheraea assama), such as silkworm silkworms (Samia cynthia), chestnut (Caligura japonica), chusser silkworms (Antheraea mylitta), and mug silkworms (Antheraea assama). Hornet silk protein is mentioned.
  • fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence), and AAA278840.1 (amino acid sequence)).
  • fibroin produced by arachnids examples include spider silk protein produced by arachnids belonging to the order Araneae. More specifically, spiders belonging to the genus Araneus (Araneus) such as spiders, spiders spiders, red spiders, red spiders, and spiders, etc., spiders belonging to the genus Araneus, spider spiders, spider spiders, spider spiders and spiders belonging to the genus Nela spp.
  • spiders belonging to the genus Araneus such as spiders, spiders spiders, red spiders, red spiders, and spiders, etc.
  • spiders belonging to the genus Araneus spider spiders, spider spider spiders, spider spiders and spiders belonging to the genus Nela spp.
  • Spiders belonging to the genus Pronus such as the spider Spider, and spiders belonging to the genus Cyrtarachne, such as Torinofundamasi and Otorinofundamas, and the genus Gasteracanth, such as the spider Spider and the genus Gasteracanthus.
  • Spiders belonging to the genus Ordgarius such as spiders, spiders Mameitaisekimo and Mutsutoeiguisekimo spiders, spiders belonging to the genus Argiope, such as Argiope, spiders belonging to the genus Argiope, such as spiders, Argiope, and Argiope.
  • Spiders belonging to the genus Spider spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, such as spiders, spiders, blue-breasted spiders and spiders belonging to the genus Cytophora, and spiders belonging to the genus Poltys, such as the spider Spider.
  • Spider silk proteins produced by spiders belonging to the genus Cyclosa such as spiders, spider spiders, margot spiders, and black spiders
  • spider silk proteins produced by spiders belonging to the genus Chorizopes such as Yamato kana spiders
  • Spiders that belong to the genus Tetragnatha such as the black-faced spider and the black-legged spider
  • spiders that belong to the genus Leucaug such as the spiders of the genus Leucaug, such as the spiders of the genus Leucaug
  • Spiders belonging to the genus Menosira such as spiders, black spiders, and spiders belonging to the genus Dyschiriognatha, such as the genus Dyschiriognatha, spiders of the genus Genus Lepidoptera, L.
  • Spider silk proteins produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Euprostenops and spiders belonging to the genus Euprosthenops
  • spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, Flag and the like.
  • keratin-derived proteins examples include Capra hircus type I keratin.
  • Examples of the collagen-derived protein include a protein containing a domain sequence represented by Formula 5: [REP2] p (here, p represents an integer of 5 to 300.
  • REP2 is Gly-X- An amino acid sequence composed of Y is shown, and X and Y are arbitrary amino acid residues other than Gly.
  • a plurality of REP2s may be the same amino acid sequence or different amino acid sequences. it can.
  • elastin-derived proteins include proteins having amino acid sequences such as NCBI GenBank Accession Nos. AAC98395 (human), I47076 (sheep), and NP786966 (bovine).
  • Examples of the resilin-derived protein include a protein containing a domain sequence represented by the formula 6: [REP3] q (wherein, q is an integer of 4 to 300 in the formula 6.
  • REP3 is Ser-JJ). Shows an amino acid sequence composed of -Tyr-Gly-U-Pro, J represents an arbitrary amino acid residue, and particularly preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr, U is arbitrary.
  • the amino acid residue is preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser, and a plurality of REP4s may have the same amino acid sequence or different amino acid sequences. ) Can be mentioned.
  • the fusion protein according to this embodiment can be produced by a conventional method using a nucleic acid encoding the fusion protein.
  • the nucleic acid encoding the fusion protein may be chemically synthesized based on the nucleotide sequence information, or may be synthesized by using the PCR method or the like.
  • nucleic acid encodes the polypeptide according to the present invention or the fusion protein according to the present invention.
  • the nucleic acid may be a nucleic acid that hybridizes under stringent conditions with a complementary strand of the nucleic acid encoding the polypeptide of the present invention or the fusion protein of the present invention.
  • the “stringent conditions” refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • the “stringent conditions” may be any of low stringent conditions, medium stringent conditions and high stringent conditions.
  • Low stringency conditions mean that hybridization will occur only when there is at least 85% identity between the sequences, eg, 5 ⁇ SSC containing 0.5% SDS at 42° C.
  • the conditions for hybridizing are mentioned.
  • the moderately stringent condition means that hybridization occurs only when at least 90% identity exists between the sequences, and for example, 5 ⁇ SSC containing 0.5% SDS is used at 50° C.
  • the conditions for hybridizing are mentioned.
  • Highly stringent conditions mean that hybridization occurs only when at least 95% identity exists between the sequences, for example, using 5 ⁇ SSC containing 0.5% SDS at 60° C. The conditions for hybridizing are mentioned.
  • the nucleic acid may be a nucleic acid having 90% or more sequence identity with the nucleic acid encoding the polypeptide of the present invention or the fusion protein of the present invention.
  • the sequence identity is preferably 95% or more.
  • nucleic acid As a specific example of the nucleic acid according to the present embodiment, a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 can be mentioned.
  • the base sequences represented by SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 encode the amino acid sequences represented by SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, respectively.
  • the nucleic acid according to this embodiment may be provided in a form incorporated in an expression vector.
  • the expression vector has the nucleic acid sequence according to the present embodiment 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 according to the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, or an artificial chromosome vector.
  • the present invention can also be regarded as a method for imparting a self-association ability to a protein, which comprises fusing the polypeptide of the present invention with a protein.
  • Specific aspects such as the method for producing the polypeptide, protein, and fusion protein are as described above.
  • ⁇ p i,j (L) and ⁇ a i,j (L) are energies calculated for a pair of amino acid residues (see Table 1 and Table 2).
  • ⁇ i ⁇ k ⁇ i+L is 1 when k belongs to i to i+L ⁇ 1, and 0 otherwise.
  • ⁇ j ⁇ k ⁇ j+L is 1 when k belongs to j to j+L ⁇ 1, and is 0 otherwise.
  • i and j correspond to the residue numbers in the amino acid sequence with the start point as 1.
  • is 2.0.
  • FIG. 1 shows the aggregation free energy g(k) in the amino acid sequence (#GEN891, SEQ ID NO: 1) in which the tag sequence shown in SEQ ID NO: 17 is added to the N-terminus of the large spit duct dragline protein (ADF4) of the Japanese spider. It is the graph which plotted with respect to the residue number.
  • FIG. 2 is an aggregation free energy g(k) in the amino acid sequence (#GEN889, SEQ ID NO: 2) in which the tag sequence represented by SEQ ID NO: 17 is added to the N-terminus of the large spit duct dragline filament protein (ADF3) of the spiderweed spider. It is the graph which plotted with respect to the residue number.
  • the aggregation free energy g(k) of each amino acid residue was ⁇ 4.0 or less from the 546th amino acid residue to the 582nd amino acid residue (FIG. 1). The average value of the aggregation free energy g(k) per amino acid residue in the amino acid sequence composed of these amino acid residues is naturally -4.0 or less.
  • the aggregation free energy g(k) of each amino acid residue was ⁇ 4.0 or less from the 547th amino acid residue to the 579th amino acid residue (FIG. 2).
  • the average value of the aggregation free energy g(k) per amino acid residue in the amino acid sequence composed of these amino acid residues is naturally -4.0 or less. The lower the aggregation free energy g(k), the stronger the aggregation tendency. Therefore, these amino acid sequences are considered to be amino acid sequences having self-association ability.
  • the amino acid sequence represented by SEQ ID NO: 20 is obtained by adding the amino acid sequence (tag sequence) shown in SEQ ID NO: 17 to the amino acid sequence of the non-repetitive region at the C-terminal of the giant vesicle guideline thread protein (ADF3) of the Japanese spider. Yes (His-ADF3-NRC, average HI 0.221, molecular weight 13.5 kDa).
  • the amino acid sequence represented by SEQ ID NO: 22 is the amino acid sequence lacking the helix 5 located at the most C-terminal side from the C-terminal non-repetitive region of the giant spider duct silkworm protein of A.
  • the amino acid sequence (tag sequence) shown is added (His-ADF3-shortNRC, average HI 0.777, molecular weight 10.6 kDa).
  • the polypeptide having the amino acid sequence represented by SEQ ID NOs: 18, 20 and 22 is a part of the amino acid sequence consisting of amino acid residues having the aggregation free energy g(k) of #GEN891 and #GEN889 of -4.0 or less, or It includes all.
  • Nucleic acid encoding each polypeptide was cloned into a cloning vector (pUC118). Then, each nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI and excised, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector. Escherichia coli BLR (DE3) was transformed with the obtained expression vector to obtain transformed Escherichia coli (recombinant cell) expressing each polypeptide.
  • the resulting transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 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 5) was added so that the OD 600 was 0.05.
  • the temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • the feed solution (glucose 455 g/1 L, yeast extract 120 g/1 L) was added at a rate of 1 mL/min.
  • the temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was performed for 20 hours.
  • 1 M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce the expression of the desired polypeptide.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the cells prepared from the culture solution before addition of IPTG and after addition of IPTG were ultrasonically disrupted and centrifuged to recover the target polypeptide. SDS-PAGE was performed on the recovered target polypeptide, and it was confirmed that the target polypeptide was expressed as an insoluble substance by the appearance of a band corresponding to the molecular weight of the target polypeptide depending on the addition of IPTG. did.
  • FIG. 3 shows supernatants and precipitates of a polypeptide (His-ADF3-NRC (SEQ ID NO: 20) or His-ADF3-shortNRC (SEQ ID NO: 22)) containing SDS and guanidine thiocyanate, which were treated with a denaturant (2- 1 is a photograph showing the results of analysis by SDS-PAGE in the presence of (mercaptoethanol). Bands corresponding to the molecular weight of each polypeptide (His-ADF3-NRC and His-ADF3-shortNRC) were mainly detected in the supernatant, and each polypeptide was solubilized by the addition of SDS and guanidine thiocyanate. It was shown that
  • FIG. 4 is a photograph showing the results of analyzing each solution at the time of column purification by the SDS-PAGE method in the absence of a denaturing agent.
  • through is a flow-through when the column is loaded with the lysate
  • wash is a flow-through when the column is washed
  • elute1 is an eluate when eluted with the first eluate
  • Elute2 is the eluate when eluted with the second eluate.
  • each polypeptide was detected as a band having a molecular weight corresponding to a dimer. From this result, it was confirmed that each polypeptide dimerizes in the absence of a denaturing agent, and that it has a self-association ability.
  • DTT was added to a His-ADF4-NRC solution (solvent: 5M guanidine thiocyanate, 10 mM Tris hydrochloride, pH 8) so as to be 5 mM.
  • the solution to which DTT was added was transferred to a dialysis tube (BioDesignDiallysisTubing #D100, 8000MWCO, manufactured by BioDesign Inc.), put in a dialysate (5M guanidine thiocyanate, 10 mM tris hydrochloride, pH 8), and dialyzed for 12 hours.
  • the polypeptide having the amino acid sequence represented by SEQ ID NO: 18 was a monomer immediately after the addition of the denaturant, but almost 3 hours after the initiation of dialysis. Was dimerized. On the other hand, it was confirmed that even after 12 hours from the start of dialysis of SNase (K116C), there was a protein that did not dimerize.
  • the polypeptide having the amino acid sequence represented by SEQ ID NO: 18 has a selective self-association ability even in the presence of a denaturing agent.
  • the polypeptide having the amino acid sequence represented by SEQ ID NO: 18 can form a disulfide bond in a short time through selective interaction. Note that it is considered that a part of SNase (K116C) is dimerized by the collision of molecules due to Brownian motion.
  • a polypeptide (His-ADF3-9467) having the amino acid sequence represented by SEQ ID NO: 30 was designed.
  • the amino acid sequence represented by SEQ ID NO: 30 is obtained by adding the amino acid sequence (tag sequence) represented by SEQ ID NO: 17 to the N-terminus of the amino acid sequence represented by SEQ ID NO: 15.
  • the amino acid sequence shown by SEQ ID NO:15 is the amino acid sequence shown by SEQ ID NO:16 with an additional sequence added to the N-terminus.
  • the polypeptide having the amino acid sequence represented by SEQ ID NO: 30 was produced and solubilized by the same procedure as in Test Example 2 except that the nucleic acid encoding the polypeptide was used, and denatured by the same procedure as in Test Example 2.
  • the self-association ability in the presence of the agent was confirmed.
  • a sample was prepared from the polypeptide (His-ADF3-9467) solution immediately after the addition of DTT, 1.5 hours, 3 hours, 6 hours, and 8 hours after the start of dialysis. Collected and analyzed by SDS-PAGE. Results are shown in FIG.
  • FIG. 6 shows SDS-polypeptide (His-ADF3-9467) solutions immediately after the addition of denaturant (DTT) (0 hour), 1.5 hours, 3 hours, 6 hours, and 8 hours after the start of dialysis. It is a photograph which shows the result analyzed by the PAGE method.
  • the polypeptide having the amino acid sequence represented by SEQ ID NO: 30 is a monomer immediately after addition of the denaturant, but 1.5 hours after the initiation of dialysis. It was confirmed that a dimer was formed and that it had selective self-association ability even in the presence of a denaturant.

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Abstract

The present invention pertains to a polypeptide having an amino acid sequence wherein the average aggregation free energy g(k), which is calculated in accordance with formula (1), per amino acid residue is -4.0 or less. [In formula (1): εp i,j(L) and εa i, j(L) represent energy calculated for a pair of amino acid residues; δi≤k<i+L is 1 if k falls within a range of from i to i+L-1, and 0 otherwise; δj≤k<j+L is 1 if k falls within a range of from j to j+L-1, and 0 otherwise; i and j correspond respectively to residue numbers in the amino acid sequence, with the start point being referred to as 1; and λ is 2.0.]

Description

ポリペプチドPolypeptide
 本発明は、ポリペプチドに関する。本発明はまた、当該ポリペプチドを含む融合タンパク質に関する。 The present invention relates to polypeptides. The present invention also relates to fusion proteins containing the polypeptide.
 タンパク質は、タンパク質分子間の相互作用(タンパク質間相互作用)を通じて、特異的複合体を形成することがある。タンパク質間相互作用は、例えば、Two-hybrid法等により検出することができる。また、近年、タンパク質間相互作用を予測する手法も報告されている(例えば、特許文献1及び非特許文献1)。 Proteins may form a specific complex through the interaction between protein molecules (protein-protein interaction). The protein-protein interaction can be detected, for example, by the Two-hybrid method. In recent years, methods for predicting protein-protein interactions have also been reported (for example, Patent Document 1 and Non-Patent Document 1).
特許第3588605号公報Japanese Patent No. 3588605
 タンパク質間相互作用に関与するアミノ酸配列は、種々応用することができる。例えば、所望のタンパク質にタンパク質間相互作用に関与するアミノ酸配列を有するポリペプチドを付加等することにより、当該タンパク質同士の会合を制御することが可能になると考えられる。 A variety of amino acid sequences involved in protein-protein interactions can be applied. For example, by adding a polypeptide having an amino acid sequence involved in protein-protein interaction to a desired protein, it is considered possible to control the association between the proteins.
 タンパク質間相互作用には、タンパク質の高次構造(例えば、二次構造、三次構造)もその一要因として関与していると考えられる。タンパク質の高次構造は、例えば、温度、pH、変性剤の存在により影響を受けやすく、タンパク質が置かれる環境によっては、安定してタンパク質同士を相互作用させることができないという問題があった。 It is considered that the higher-order structure of proteins (eg, secondary structure, tertiary structure) is also involved as one factor in the protein-protein interaction. The higher-order structure of a protein is easily affected by, for example, temperature, pH, and the presence of a denaturant, and there is a problem that the proteins cannot stably interact with each other depending on the environment in which the protein is placed.
 本発明は、変性条件下でも選択的に相互作用させることができるポリペプチドを提供することを目的とする。 The present invention aims to provide a polypeptide that can selectively interact even under denaturing conditions.
 本発明は、例えば、以下の各発明に関する。
[1]
 下記式(1)で算出される凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値が-4.0以下であるアミノ酸配列を有する、ポリペプチド。
Figure JPOXMLDOC01-appb-M000002
[式(1)中、ε i,j(L)及びε i,j(L)は、アミノ酸残基のペアに対して算出されたエネルギーである。δi≦k<i+Lは、kがi~i+L-1に属するときは1であり、それ以外は0である。δj≦k<j+Lは、kがj~j+L-1に属するときは1であり、それ以外は0である。i及びjは、始点を1としたアミノ酸配列中の残基番号に対応する。λは、2.0である。]
[2]
 配列番号3~16のいずれかで示されるアミノ酸配列と60%以上の配列同一性を有するアミノ酸配列を含む、ポリペプチド。
[3]
 N末端及びC末端のいずれか一方又は両方にタグ配列を含む、[1]又は[2]に記載のポリペプチド。
[4]
 上記タグ配列が、配列番号17で示されるアミノ酸配列を含む、[3]に記載のポリペプチド。
[5]
 配列番号3~16のいずれかで示されるアミノ酸配列を含む、ポリペプチド。
[6]
 配列番号18~31のいずれかで示されるアミノ酸配列を含む、ポリペプチド。
[7]
 [1]~[6]のいずれかに記載のポリペプチドをコードする核酸。
[8]
 [1]~[6]のいずれかに記載のポリペプチドと、タンパク質とが融合されたアミノ酸配列を含む、融合タンパク質。
[9]
 上記タンパク質のアミノ酸配列と、上記ポリペプチドのアミノ酸配列とを含む、[8]に記載の融合タンパク質。
[10]
 上記タンパク質のアミノ酸配列中に、上記ポリペプチドのアミノ酸配列を含む、[8]に記載の融合タンパク質。
[11]
 上記タンパク質のN末端又はC末端から融合部位までのアミノ酸残基数が、上記タンパク質の総アミノ酸残基数に対して、10%以下である、[10]に記載の融合タンパク質。
[12]
 上記タンパク質が、構造タンパク質である、[8]~[11]のいずれかに記載の融合タンパク質。
[13]
 上記タンパク質が、クモ糸タンパク質である、[8]~[12]のいずれかに記載の融合タンパク質。
[14]
 [8]~[13]のいずれかに記載の融合タンパク質をコードする核酸。
[15]
 [1]~[6]のいずれかに記載のポリペプチドと、タンパク質とを融合させることを含む、上記タンパク質に自己会合能を付与する方法。
The present invention relates to the following inventions, for example.
[1]
A polypeptide having an amino acid sequence in which the average value of the aggregation free energy g(k) calculated by the following formula (1) per amino acid residue is −4.0 or less.
Figure JPOXMLDOC01-appb-M000002
[In the formula (1), ε p i,j (L) and ε a i,j (L) are energies calculated for a pair of amino acid residues. δ i ≦k<i+L is 1 when k belongs to i to i+L−1, and is 0 otherwise. δ j≦k<j+L is 1 when k belongs to j to j+L−1, and is 0 otherwise. i and j correspond to the residue numbers in the amino acid sequence with the start point as 1. λ is 2.0. ]
[2]
A polypeptide comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 3 to 16.
[3]
The polypeptide according to [1] or [2], which comprises a tag sequence at either or both of the N-terminus and C-terminus.
[4]
The polypeptide according to [3], wherein the tag sequence comprises the amino acid sequence shown by SEQ ID NO:17.
[5]
A polypeptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 3-16.
[6]
A polypeptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 18-31.
[7]
A nucleic acid encoding the polypeptide according to any of [1] to [6].
[8]
A fusion protein comprising the amino acid sequence obtained by fusing the polypeptide according to any one of [1] to [6] with a protein.
[9]
The fusion protein according to [8], which comprises the amino acid sequence of the protein and the amino acid sequence of the polypeptide.
[10]
The fusion protein according to [8], wherein the amino acid sequence of the protein contains the amino acid sequence of the polypeptide.
[11]
The fusion protein according to [10], wherein the number of amino acid residues from the N-terminus or C-terminus of the protein to the fusion site is 10% or less with respect to the total number of amino acid residues of the protein.
[12]
The fusion protein according to any of [8] to [11], wherein the protein is a structural protein.
[13]
The fusion protein according to any of [8] to [12], wherein the protein is a spider silk protein.
[14]
A nucleic acid encoding the fusion protein according to any of [8] to [13].
[15]
A method for imparting a self-association ability to the above protein, which comprises fusing the protein according to any one of [1] to [6] with a protein.
 本発明によれば、変性条件下でも選択的に相互作用させることができるポリペプチドの提供が可能となる。 According to the present invention, it is possible to provide a polypeptide that can selectively interact even under denaturing conditions.
ニワオニグモの大吐糸管しおり糸タンパク質(ADF4)のN末端に配列番号17で示されるタグ配列を付加したアミノ酸配列(#GEN891,配列番号1)における凝集自由エネルギーg(k)を残基番号に対してプロットしたグラフである。The free energy of aggregation g(k) in the amino acid sequence (#GEN891, SEQ ID NO: 1) in which the tag sequence represented by SEQ ID NO: 17 is added to the N-terminus of the large spit duct dragline protein (ADF4) of Japanese spider Spider is used as a residue number. It is a graph plotted against. ニワオニグモの大吐糸管しおり糸タンパク質(ADF3)のN末端に配列番号17で示されるタグ配列を付加したアミノ酸配列(#GEN889,配列番号2)における凝集自由エネルギーg(k)を残基番号に対してプロットしたグラフである。The aggregation free energy g(k) in the amino acid sequence (#GEN889, SEQ ID NO: 2) in which the tag sequence represented by SEQ ID NO: 17 is added to the N-terminal of the large spit duct dragline protein (ADF3) of the Japanese spider Spider is designated as the residue number. It is a graph plotted against. SDS及びグアニジンチオシアン酸塩を含むポリペプチド(His-ADF3-NRC(配列番号20)又はHis-ADF3-shortNRC(配列番号22))液の上清及び沈殿を、変性剤(2-メルカプトエタノール)存在下でSDS-PAGE法により分析した結果を示す写真である。The supernatant and precipitate of the polypeptide containing SDS and guanidine thiocyanate (His-ADF3-NRC (SEQ ID NO: 20) or His-ADF3-shortNRC (SEQ ID NO: 22)) were treated with the presence of a denaturing agent (2-mercaptoethanol). 3 is a photograph showing the results of analysis by SDS-PAGE below. 配列番号20又は22で示されるアミノ酸配列を有するポリペプチドのカラム精製の際の各溶液を変性剤非存在下でSDS-PAGE法により分析した結果を示す写真である。FIG. 3 is a photograph showing the result of SDS-PAGE analysis of each solution in the case of column purification of a polypeptide having the amino acid sequence represented by SEQ ID NO: 20 or 22, in the absence of a denaturing agent. 変性剤添加前、変性剤(DTT)添加後、透析開始から3時間後、6時間後及び12時間後のポリペプチド(His-ADF4-NRC(配列番号18))溶液をSDS-PAGE法により分析した結果を示す写真である。SDS-PAGE analysis of polypeptide (His-ADF4-NRC (SEQ ID NO: 18)) solutions before addition of denaturant, after addition of denaturant (DTT), 3 hours, 6 hours and 12 hours after the start of dialysis It is a photograph which shows the result. 変性剤(DTT)添加直後(0時間)、透析開始から1.5時間後、3時間後、6時間後及び8時間後のポリペプチド(His-ADF3-9467(配列番号30))溶液をSDS-PAGE法により分析した結果を示す写真である。Immediately after the addition of the denaturant (DTT) (0 hour), 1.5 hours, 3 hours, 6 hours, and 8 hours after the start of dialysis, the polypeptide (His-ADF3-9467 (SEQ ID NO: 30)) solution was added to SDS. -A photograph showing the result of analysis by the PAGE method.
 以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
 本実施形態に係るポリペプチドは、下記式(1)で算出される凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値が-4.0以下であるアミノ酸配列を有する。
Figure JPOXMLDOC01-appb-M000003
[式(1)中、ε i,j(L)及びε i,j(L)は、アミノ酸残基のペアに対して算出されたエネルギーである。δi≦k<i+Lは、kがi~i+L-1に属するときは1であり、それ以外は0である。δj≦k<j+Lは、kがj~j+L-1に属するときは1であり、それ以外は0である。i及びjは、始点を1としたアミノ酸配列中の残基番号に対応する。λは、2.0である。]
The polypeptide according to this embodiment has an amino acid sequence in which the average value of the aggregation free energy g(k) calculated by the following formula (1) per amino acid residue is −4.0 or less.
Figure JPOXMLDOC01-appb-M000003
[In the formula (1), ε p i,j (L) and ε a i,j (L) are energies calculated for a pair of amino acid residues. δ i ≦k<i+L is 1 when k belongs to i to i+L−1, and is 0 otherwise. δ j≦k<j+L is 1 when k belongs to j to j+L−1, and is 0 otherwise. i and j correspond to the residue numbers in the amino acid sequence with the start point as 1. λ is 2.0. ]
 なお、凝集自由エネルギーg(k)は、残基番号kのアミノ酸残基に対して算出される値である。凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値とは、あるアミノ酸配列における各アミノ酸残基に対して算出された凝集自由エネルギーg(k)の合計値をアミノ酸残基数で除した値を意味する。 The cohesive free energy g(k) is a value calculated for the amino acid residue of residue number k. The average value of aggregation free energy g(k) per amino acid residue is the total value of aggregation free energy g(k) calculated for each amino acid residue in a certain amino acid sequence divided by the number of amino acid residues. Means the value
 本実施形態に係るポリペプチドは、このような構成を有することから、変性条件下でも選択的に相互作用(自己会合)することができる。 Since the polypeptide according to this embodiment has such a structure, it can selectively interact (self-associate) even under denaturing conditions.
 ε i,j(L)及びε i,j(L)で表されるエネルギーは、タンパク質データベース(PDB)に登録されている高品質の球状タンパク質(上位500)に対して、DSSPアルゴリズムにより帰属される二次構造を元に下記式(2)で算出した値であり、具体的には表1(E ab)及び表2(E ab)に示すものである。
Figure JPOXMLDOC01-appb-M000004
The energies represented by ε p i,j (L) and ε a i,j (L) are calculated by the DSSP algorithm for high-quality globular proteins (top 500) registered in the protein database (PDB). It is a value calculated by the following formula (2) based on the secondary structure to be assigned, and specifically, it is shown in Table 1 (E p ab ) and Table 2 (E a ab ).
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
 表1及び表2中、第1列及び第1行のアルファベットは、アミノ酸残基の一文字表記である。 In Tables 1 and 2, the alphabets in the first column and the first row are the one-letter codes of amino acid residues.
 本実施形態に係るポリペプチドは、少なくとも一部に凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値が-4.0以下であるアミノ酸配列(以下、「自己会合配列」ともいう。)を有すればよく、当該自己会合配列に加えて、凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値が-4.0超であるアミノ酸配列を有していてもよい。 The polypeptide according to the present embodiment has an amino acid sequence having an average value of aggregation free energy g(k) per amino acid residue of −4.0 or less (hereinafter, also referred to as “self-association sequence”). ), and in addition to the self-association sequence, it may have an amino acid sequence having an average value of aggregation free energy g(k) per amino acid residue of more than -4.0.
 自己会合配列の凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値は、-4.5以下であることが好ましく、-5.0以下であることがより好ましく、-5.5以下であることが更に好ましく、-6.0以下であることが更により好ましく、-6.5以下であることが更により一層好ましく、-7.0以下であることが特に好ましい。 The average value of the aggregation free energy g(k) of the self-association sequence per amino acid residue is preferably −4.5 or less, more preferably −5.0 or less, and −5.5 or less. Is more preferable, it is even more preferably −6.0 or less, even more preferably −6.5 or less, and particularly preferably −7.0 or less.
 本実施形態に係るポリペプチドの具体例として、配列番号1で示されるアミノ酸配列の546番目のアミノ酸残基から582番目のアミノ酸残基からなるアミノ酸配列、配列番号2で示されるアミノ酸配列の547番目のアミノ酸残基から579番目のアミノ酸残基からなるアミノ酸配列、配列番号3~16で示されるアミノ酸配列が挙げられる。 As a specific example of the polypeptide according to the present embodiment, an amino acid sequence consisting of the 546th amino acid residue to the 582th amino acid residue of the amino acid sequence shown by SEQ ID NO: 1, the 547th amino acid sequence shown by SEQ ID NO: 2 The amino acid sequence consisting of the 579th amino acid residue from the amino acid residue of, and the amino acid sequences shown in SEQ ID NOs: 3 to 16 are mentioned.
 配列番号3~8で示されるアミノ酸配列は、配列番号1又は配列番号2で示されるアミノ酸配列中の自己会合配列の一部又は全部を含むアミノ酸配列である。 The amino acid sequences represented by SEQ ID NOs: 3 to 8 are amino acid sequences containing a part or all of the self-association sequence in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
 他の実施形態に係るポリペプチドは、配列番号3~16のいずれかで示されるアミノ酸配列と60%以上の配列同一性を有するアミノ酸配列を含む。本実施形態に係るポリペプチドは、このような構成を有することから、変性剤存在下でも選択的に相互作用(自己会合)することができる。 A polypeptide according to another embodiment contains an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3 to 16. Since the polypeptide according to the present embodiment has such a constitution, it can selectively interact (self-associate) even in the presence of a denaturant.
 配列番号3で示されるアミノ酸配列は、二ワオニグモ(Araneus diadematus)の大吐糸管しおり糸タンパク質(ADF4)のC末端の非繰り返し領域(NRC領域)のアミノ酸配列である。配列番号4で示されるアミノ酸配列は、配列番号3で示されるアミノ酸配列の部分配列であり、凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値が特に低い部分に対応するアミノ酸配列である。 The amino acid sequence represented by SEQ ID NO: 3 is the amino acid sequence of the non-repetitive region (NRC region) at the C-terminal of the large vesicle guideline protein (ADF4) of the spider (Araneus diadematus). The amino acid sequence represented by SEQ ID NO:4 is a partial sequence of the amino acid sequence represented by SEQ ID NO:3, and is an amino acid sequence corresponding to a portion in which the average value of the aggregation free energy g(k) per amino acid residue is particularly low. is there.
 配列番号5で示されるアミノ酸配列は、二ワオニグモの大吐糸管しおり糸タンパク質(ADF3)のC末端の非繰り返し領域のアミノ酸配列である。配列番号6で示されるアミノ酸配列は、配列番号5で示されるアミノ酸配列の部分配列であり、凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値が特に低い部分に対応するアミノ酸配列である。 The amino acid sequence represented by SEQ ID NO: 5 is the amino acid sequence of the non-repetitive region at the C-terminus of the large vesicle guideline thread protein (ADF3) of the Japanese spider, Spiderwort. The amino acid sequence represented by SEQ ID NO: 6 is a partial sequence of the amino acid sequence represented by SEQ ID NO: 5, and is an amino acid sequence corresponding to a portion in which the average value of the aggregation free energy g(k) per amino acid residue is particularly low. is there.
 配列番号7で示されるアミノ酸配列は、二ワオニグモの大吐糸管しおり糸タンパク質(ADF3)のC末端の非繰り返し領域から最もC末端側に位置するヘリックス5が欠損したアミノ酸配列である。配列番号8で示されるアミノ酸配列は、配列番号7で示されるアミノ酸配列の部分配列であり、凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値が特に低い部分に対応するアミノ酸配列である。 The amino acid sequence represented by SEQ ID NO: 7 is an amino acid sequence in which helix 5 located at the most C-terminal side from the non-repetitive region at the C-terminal of the giant spider duct silkworm thread protein (ADF3) of the armpit spider is deleted. The amino acid sequence represented by SEQ ID NO:8 is a partial sequence of the amino acid sequence represented by SEQ ID NO:7, and is an amino acid sequence corresponding to a portion where the average value of the aggregation free energy g(k) per amino acid residue is particularly low. is there.
 配列番号10、12、14及び16で示されるアミノ酸配列は、以下の手順で選出したアミノ酸配列である。まず、既知の大瓶状腺スピドロイン(MaSp)のC末端のアミノ酸配列(具体的には、配列番号27~191で示されるアミノ酸配列)からプロファイル隠れマルコフモデル(HMM)を作成した。作成したHMMを入力値としてhmmemitプログラムを実行することにより、アミノ酸配列を生成した(10000配列)。生成したアミノ酸配列に対して、連続する5アミノ酸残基ごとに凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値を算出した。算出した平均値の最小値が-4.5以下になるアミノ酸配列を選出した。 The amino acid sequences shown in SEQ ID NOs: 10, 12, 14 and 16 are amino acid sequences selected by the following procedure. First, a profile hidden Markov model (HMM) was created from the C-terminal amino acid sequence of a known large ampullate spidroin (MaSp) (specifically, the amino acid sequences represented by SEQ ID NOs: 27 to 191). An amino acid sequence was generated by executing the hmmemit program using the created HMM as an input value (10000 sequences). With respect to the generated amino acid sequence, the average value of the aggregation free energy g(k) per 1 amino acid residue was calculated for every 5 consecutive amino acid residues. An amino acid sequence having a calculated minimum value of −4.5 or less was selected.
 配列番号9、11、13及び15で示されるアミノ酸配列は、それぞれ配列番号10、12、14及び16で示されるアミノ酸配列のN末端に付加配列を付加したアミノ酸配列である。 The amino acid sequences shown in SEQ ID NOs: 9, 11, 13 and 15 are amino acid sequences obtained by adding an additional sequence to the N-terminal of the amino acid sequences shown in SEQ ID NOs: 10, 12, 14 and 16, respectively.
 配列番号3、配列番号5及び配列番号7で示されるアミノ酸配列は、相互に少なくとも60%以上(63%)の配列同一性を有する。 The amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 have at least 60% or more (63%) sequence identity with each other.
 本実施形態に係るポリペプチドは、配列番号3~16のいずれかで示されるアミノ酸配列と65%以上の配列同一性を有するものであってもよく、70%以上の配列同一性を有するものであってもよく、75%以上の配列同一性を有するものであってもよく、80%以上の配列同一性を有するものであってもよく、85%以上の配列同一性を有するものであってもよく、90%以上の配列同一性を有するものであってもよく、95%以上の配列同一性を有するものであってもよく、98%以上の配列同一性を有するものであってもよく、99%以上の配列同一性を有するものであってもよい。 The polypeptide according to this embodiment may have 65% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3 to 16, or 70% or more sequence identity. May have a sequence identity of 75% or more, may have a sequence identity of 80% or more, and may have a sequence identity of 85% or more. May have a sequence identity of 90% or more, may have a sequence identity of 95% or more, or may have a sequence identity of 98% or more. , 99% or more sequence identity may be sufficient.
 本実施形態に係るポリペプチドは、配列番号3~16のいずれかで示されるアミノ酸配列を含むものであってよく、配列番号3~16のいずれかで示されるアミノ酸配列からなるものであってもよい。 The polypeptide according to this embodiment may include the amino acid sequence represented by any of SEQ ID NOS: 3 to 16 or may be the amino acid sequence represented by any of SEQ ID NOs: 3 to 16. Good.
 本実施形態に係るポリペプチドは、N末端及びC末端のいずれか一方又は両方にタグ配列が付加されたものであってよい。これにより、本実施形態に係るポリペプチドの単離、固定化、検出及び可視化等が可能となる。 The polypeptide according to the present embodiment may have a tag sequence added to either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, etc. of the polypeptide according to this embodiment.
 タグ配列として、例えば、他の分子との特異的親和性(結合性、アフィニティ)を利用したアフィニティタグを挙げることができる。アフィニティタグの具体例として、ヒスチジンタグ(Hisタグ)を挙げることができる。Hisタグは、ヒスチジン残基が4から10個程度並んだ短いペプチドで、ニッケル等の金属イオンと特異的に結合する性質があるため、金属キレートクロマトグラフィー(chelating metal chromatography)による本実施形態に係るポリペプチドの単離に利用することができる。タグ配列の具体例として、例えば、配列番号17で示されるアミノ酸配列(Hisタグを含むアミノ酸配列)が挙げられる。 As the tag sequence, for example, an affinity tag that utilizes specific affinity (binding, affinity) with another molecule can be mentioned. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are lined up and has a property of specifically binding to a metal ion such as nickel. Therefore, the His tag according to this embodiment by metal chelating chromatography It can be used to isolate the polypeptide. Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 17 (amino acid sequence containing His tag).
 また、グルタチオンに特異的に結合するグルタチオン-S-トランスフェラーゼ(GST)、マルトースに特異的に結合するマルトース結合タンパク質(MBP)等のタグ配列を利用することもできる。 Alternatively, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can be used.
 さらに、抗原抗体反応を利用した「エピトープタグ」を利用することもできる。抗原性を示すペプチド(エピトープ)をタグ配列として付加することにより、当該エピトープに対する抗体を結合させることができる。エピトープタグとして、HA(インフルエンザウイルスのヘマグルチニンのペプチド配列)タグ、mycタグ、FLAGタグ等を挙げることができる。エピトープタグを利用することにより、高い特異性で容易に本実施形態に係るポリペプチドを精製することができる。 Furthermore, you can also use an "epitope tag" that utilizes the antigen-antibody reaction. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, FLAG tag and the like. By using the epitope tag, the polypeptide according to this embodiment can be easily purified with high specificity.
 さらにタグ配列を特定のプロテアーゼで切り離せるようにしたものも使用することができる。当該タグ配列を介して吸着した本実施形態に係るポリペプチドをプロテアーゼ処理することにより、タグ配列を切り離した本実施形態に係るポリペプチドを回収することもできる。 Furthermore, a tag sequence that can be cleaved with a specific protease can also be used. By subjecting the polypeptide of the present embodiment adsorbed via the tag sequence to a protease treatment, the polypeptide of the present embodiment from which the tag sequence is cleaved can be recovered.
 タグ配列を含むポリペプチドの具体例として、例えば、配列番号18~31のいずれかで示されるアミノ酸配列を含むポリペプチドを挙げることができる。配列番号18~31で示されるアミノ酸配列は、それぞれ配列番号3~16で示されるアミノ酸配列のN末端に配列番号17で示されるアミノ酸配列(Hisタグを含むアミノ酸配列)を付加したものである。 Specific examples of the polypeptide containing the tag sequence include, for example, the polypeptides containing the amino acid sequences represented by any of SEQ ID NOs: 18 to 31. The amino acid sequences represented by SEQ ID NOS: 18 to 31 are obtained by adding the amino acid sequences represented by SEQ ID NO: 17 (amino acid sequences including His tag) to the N-terminals of the amino acid sequences represented by SEQ ID NOS: 3 to 16, respectively.
 一実施形態に係るポリペプチドは、配列番号18~31のいずれかで示されるアミノ酸配列を含むものであってよく、配列番号18~31のいずれかで示されるアミノ酸配列からなるものであってもよい。 The polypeptide according to one embodiment may include the amino acid sequence shown in any of SEQ ID NOs: 18 to 31, or may consist of the amino acid sequence shown in any of SEQ ID NOs: 18 to 31. Good.
 本実施形態に係るポリペプチドの分子量は、特に限定されないが、例えば、1kDa以上300kDa以下であってよい。本実施形態に係るポリペプチドの分子量は、例えば、2kDa以上、3kDa以上、4kDa以上、5kDa以上、6kDa以上、7kDa以上、8kDa以上、9kDa以上、又は10kDa以上であってよく、例えば、250kDa以下、200kDa以下、150kDa以下、100kDa以下、90kDa以下、80kDa以下、70kDa以下、60kDa以下、50kDa以下、40kDa以下、30kDa以下、20kDa以下、又は15kDa以下であってよい。 The molecular weight of the polypeptide according to this embodiment is not particularly limited, but may be, for example, 1 kDa or more and 300 kDa or less. The molecular weight of the polypeptide according to the present embodiment may be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, or 10 kDa or more, for example, 250 kDa or less, It may be 200 kDa or less, 150 kDa or less, 100 kDa or less, 90 kDa or less, 80 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 20 kDa or less, or 15 kDa or less.
 本実施形態に係るポリペプチドのアミノ酸残基数は、特に限定されないが、例えば、150アミノ酸残基以下、120アミノ酸残基以下、100アミノ酸残基以下、80アミノ酸残基以下、60アミノ酸残基以下、40アミノ酸残基以下、30アミノ酸残基以下、25アミノ酸残基以下、又は20アミノ酸残基以下であってよく、例えば、5アミノ酸残基以上、10アミノ酸残基以上、又は15アミノ酸残基以上であってよい。本実施形態に係るポリペプチドのアミノ酸残基数が少ない場合(例えば、20アミノ酸残基程度)、例えば、微生物等で本実施形態に係るポリペプチドを含む融合タンパク質を生産させる場合に、融合タンパク質のアミノ酸残基総数を大きく増やさないため生産性に与える影響を低くすることができ、また融合タンパク質の凝集性を高める、又は凝集性を付加することができる。 The number of amino acid residues of the polypeptide according to this embodiment is not particularly limited, but for example, 150 amino acid residues or less, 120 amino acid residues or less, 100 amino acid residues or less, 80 amino acid residues or less, 60 amino acid residues or less. , 40 amino acid residues or less, 30 amino acid residues or less, 25 amino acid residues or less, or 20 amino acid residues or less, for example, 5 amino acid residues or more, 10 amino acid residues or more, or 15 amino acid residues or more May be When the number of amino acid residues of the polypeptide according to this embodiment is small (for example, about 20 amino acid residues), for example, when a fusion protein containing the polypeptide according to this embodiment is produced by a microorganism or the like, Since the total number of amino acid residues is not significantly increased, the influence on the productivity can be reduced, and the aggregating property of the fusion protein can be increased or the aggregating property can be added.
 本実施形態に係るポリペプチドは、親水性であってもよく、疎水性であってもよい。本実施形態に係るポリペプチドは、ポリペプチドを構成する全てのアミノ酸残基の疎水性指標(ハイドロパシー・インデックス、HI)の総和を求め、次にその総和を全アミノ酸残基数で除した値(平均HI、以下「疎水度」とも表す。)が-1.0以上であるものが好ましい。アミノ酸残基の疎水性指標については、公知の指標(Hydropathy index:Kyte J,&Doolittle R(1982)“A simple method for displaying the hydropathic character of a protein”,J.Mol.Biol.,157,pp.105-132)を使用する。具体的には、各アミノ酸の疎水性指標は、下記表3に示すとおりである。 The polypeptide according to this embodiment may be hydrophilic or hydrophobic. For the polypeptide according to the present embodiment, the sum of the hydrophobicity indices (hydropathic index, HI) of all amino acid residues constituting the polypeptide is calculated, and then the sum is divided by the total number of amino acid residues. It is preferable that (average HI, hereinafter also referred to as “hydrophobicity”) be −1.0 or more. Regarding the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for dissipating the hydropathic character. 105-132). Specifically, the hydrophobicity index of each amino acid is as shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
 本実施形態に係るポリペプチドの疎水度は、-0.5以上、0以上、1以上、5以上又は10以上であってよく、また、10以下、5以下又は1以下であってよい。 The hydrophobicity of the polypeptide according to this embodiment may be −0.5 or more, 0 or more, 1 or more, 5 or more, or 10 or more, and 10 or less, 5 or less, or 1 or less.
 本実施形態に係るポリペプチドは、当該ポリペプチド同士でタンパク質間相互作用し、特異的複合体を形成するものであること(すなわち、自己会合能を有するものであること)が好ましく、変性条件下で自己会合能を有するものであることがより好ましい。 The polypeptide according to the present embodiment is preferably one in which the polypeptides interact with each other to form a specific complex (that is, one having self-association ability) under denaturing conditions. And more preferably have self-association ability.
 本実施形態に係るポリペプチドは、人工ポリペプチドであってよい。本明細書において「人工ポリペプチド」は、人為的に製造されたポリペプチドを意味する。人工ポリペプチドは、例えば、遺伝子組換え技術を使用して製造されたポリペプチド、化学合成されたポリペプチドを含む。 The polypeptide according to this embodiment may be an artificial polypeptide. As used herein, the term "artificial polypeptide" means an artificially produced polypeptide. Artificial polypeptides include, for example, polypeptides produced using gene recombination technology and chemically synthesized polypeptides.
(変性条件)
 変性条件は、タンパク質の高次構造(例えば、二次構造、三次構造)を破壊し得る条件である。変性条件としては、例えば、タンパク質溶液のpH(例えば、pH1~4の酸性条件、及びpH10~14のアルカリ性条件)、タンパク質がおかれる環境温度(例えば、-20℃~5℃の低温条件、及び35℃以上の高温条件)、変性剤の存在等が挙げられる。
(Denaturation conditions)
Denaturing conditions are conditions that can destroy the higher-order structure (eg, secondary structure, tertiary structure) of a protein. The denaturing conditions include, for example, the pH of the protein solution (eg, acidic conditions of pH 1 to 4 and alkaline condition of pH 10 to 14), the environmental temperature at which the protein is placed (eg, low temperature condition of −20° C. to 5° C., and The high temperature condition of 35° C. or higher), the presence of a modifier, and the like.
 変性剤としては、例えば、タンパク質分子間及び/又は分子内の水素結合、疎水結合、イオン結合、ジスルフィド結合を破壊する物質が挙げられる。変性剤の具体例としては、例えば、尿素、グアニジン塩(例えば、グアニジンチオシアン酸塩)、ドデシル硫酸ナトリウム(SDS)、2-メルカプトエタノール、テトラヒドロフラン(THF)、並びにジメチルスルホキシド(DMSO)、アセトン、アルコール類(例えば、メタノール、エタノール、イソプロパノール)、ジオキサン、ジメチルホルムアミド(DMF)及びアセトニトリル等の水と混和する極性溶媒が挙げられる。 The denaturing agent includes, for example, a substance that breaks hydrogen bonds, hydrophobic bonds, ionic bonds, and disulfide bonds between protein molecules and/or intramolecularly. Specific examples of the denaturing agent include, for example, urea, guanidine salt (eg, guanidine thiocyanate), sodium dodecyl sulfate (SDS), 2-mercaptoethanol, tetrahydrofuran (THF), and dimethyl sulfoxide (DMSO), acetone, alcohol. Examples include polar solvents that are miscible with water, such as groups (eg, methanol, ethanol, isopropanol), dioxane, dimethylformamide (DMF), and acetonitrile.
(ポリペプチドの製造方法)
 本実施形態に係るポリペプチドは、当該ポリペプチドをコードする核酸を使用して、常法により製造することができる。当該ポリペプチドをコードする核酸は、塩基配列情報に基づいて、化学合成してもよく、PCR法等を利用して合成してもよい。
(Method for producing polypeptide)
The polypeptide according to this embodiment can be produced by a conventional method using a nucleic acid encoding the polypeptide. The nucleic acid encoding the polypeptide may be chemically synthesized based on the nucleotide sequence information, or may be synthesized by utilizing the PCR method or the like.
〔融合タンパク質〕
 本実施形態に係る融合タンパク質は、本発明に係るポリペプチドと、タンパク質とが融合されたアミノ酸配列を含む。
[Fusion protein]
The fusion protein according to this embodiment includes an amino acid sequence in which the polypeptide according to the present invention and the protein are fused.
 本実施形態に係る融合タンパク質における融合の態様は特に限定されず、例えば、タンパク質のアミノ酸配列と、本発明に係るポリペプチドのアミノ酸配列とを含む融合タンパク質(例えば、タンパク質のアミノ酸配列のN末端及びC末端のいずれか一方又は両方に本発明に係るポリペプチドのアミノ酸配列を含む融合タンパク質)であってもよく、タンパク質のアミノ酸配列中に、本発明に係るポリペプチドのアミノ酸配列を含む融合タンパク質(例えば、タンパク質のアミノ酸配列の途中に本発明に係るポリペプチドのアミノ酸配列を含む融合タンパク質)であってもよい。 The aspect of fusion in the fusion protein according to the present embodiment is not particularly limited, and includes, for example, a fusion protein containing the amino acid sequence of the protein and the amino acid sequence of the polypeptide of the present invention (for example, the N-terminal of the amino acid sequence of the protein and Fusion protein containing the amino acid sequence of the polypeptide of the present invention in either or both of the C-terminals, and a fusion protein containing the amino acid sequence of the polypeptide of the present invention in the amino acid sequence of the protein ( For example, it may be a fusion protein containing the amino acid sequence of the polypeptide of the present invention in the middle of the amino acid sequence of the protein.
 タンパク質のアミノ酸配列中に、本発明に係るポリペプチドのアミノ酸配列を含む融合タンパク質においては、タンパク質のN末端又はC末端から融合部位(タンパク質のアミノ酸配列中の本発明に係るポリペプチドの挿入部位)までのアミノ酸残基数が、タンパク質の総アミノ酸残基数に対して、45%以下であってよく、40%以下であってよく、35%以下であってよく、30%以下であってよく、25%以下であってよく、20%以下であってよく、15%以下であることが好ましく、10%以下、9%以下、8%以下、7%以下、6%以下、5%以下、4%以下、3%以下、2%以下、又は1%以下であることがより好ましい。当該比率が低いほど、本発明に係るポリペプチドが、タンパク質のN末端又はC末端により近い位置に融合されていることになり、選択的な相互作用(自己会合)をより生じやすくなる。 In a fusion protein containing the amino acid sequence of the polypeptide of the present invention in the amino acid sequence of the protein, a fusion site from the N-terminal or C-terminal of the protein (insertion site of the polypeptide of the present invention in the amino acid sequence of the protein) Up to 45%, 40% or less, 35% or less, 30% or less with respect to the total number of amino acid residues of the protein. , 25% or less, 20% or less, preferably 15% or less, preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, It is more preferably 4% or less, 3% or less, 2% or less, or 1% or less. The lower the ratio, the more likely the polypeptide of the present invention is fused to a position closer to the N-terminal or C-terminal of the protein, and more likely to cause selective interaction (self-association).
(タンパク質)
 本実施形態に係る融合タンパク質におけるタンパク質には、特に制限はなく、任意のタンパク質を使用することができる。タンパク質の具体例として、例えば、工業用に利用できるタンパク質、医療用に利用できるタンパク質、及び構造タンパク質等を挙げることができる。工業用又は医療用に利用できるタンパク質の具体例としては、クモ糸タンパク質、酵素、制御タンパク質、受容体、ペプチドホルモン、サイトカイン、膜又は輸送タンパク質、予防接種に使用する抗原、ワクチン、抗原結合タンパク質、免疫刺激タンパク質、アレルゲン、及び完全長抗体又は抗体フラグメント若しくは誘導体を挙げることができる。構造タンパク質の具体例としては、フィブロイン(例えば、クモ糸フィブロイン(スパイダーシルク)、カイコシルク等)、ケラチン、コラ-ゲン、エラスチン、レシリン、及びこれらタンパク質の断片、並びにこれら由来のタンパク質等を挙げることができる。
(protein)
The protein in the fusion protein according to the present embodiment is not particularly limited, and any protein can be used. Specific examples of proteins include proteins that can be used industrially, proteins that can be used medically, and structural proteins. Specific examples of proteins that can be used for industrial or medical purposes include spider silk protein, enzyme, regulatory protein, receptor, peptide hormone, cytokine, membrane or transport protein, antigen used for vaccination, vaccine, antigen-binding protein, It may include immunostimulatory proteins, allergens, and full length antibodies or antibody fragments or derivatives. Specific examples of structural proteins include fibroin (for example, spider silk fibroin (spider silk), silkworm silk, etc.), keratin, collagen, elastin, resilin, fragments of these proteins, and proteins derived therefrom. You can
 本明細書においてフィブロインは、天然由来のフィブロインと改変フィブロインとを含む。本明細書において「天然由来のフィブロイン」とは、天然由来のフィブロインと同一のアミノ酸配列を有するフィブロインを意味し、「改変フィブロイン」とは、天然由来のフィブロインとは異なるアミノ酸配列を有するフィブロインを意味する。 In the present specification, fibroin includes naturally occurring fibroin and modified fibroin. In the present specification, "naturally-derived fibroin" means a fibroin having the same amino acid sequence as naturally-derived fibroin, and "modified fibroin" means a fibroin having an amino acid sequence different from that of naturally-derived fibroin. To do.
 フィブロインは、クモ糸フィブロインであってよい。「クモ糸フィブロイン」には、天然クモ糸フィブロイン、及び天然クモ糸フィブロインに由来する改変フィブロインが含まれる。天然クモ糸フィブロインとしては、例えば、クモ類が産生するスパイダーシルクタンパク質(SSP)が挙げられる。 Fibroin may be spider silk fibroin. "Spider silk fibroin" includes natural spider silk fibroin and modified fibroin derived from natural spider silk fibroin. Examples of the natural spider silk fibroin include spider silk protein (SSP) produced by spiders.
 フィブロインは、例えば、式3:[(A)モチーフ-REP]、又は式4:[(A)モチーフ-REP]-(A)モチーフで表されるドメイン配列を含むタンパク質であってもよい。本実施形態に係るフィブロインは、ドメイン配列のN末端側及びC末端側のいずれか一方又は両方に更にアミノ酸配列(N末端配列及びC末端配列)が付加されていてもよい。N末端配列及びC末端配列は、これに限定されるものではないが、典型的には、フィブロインに特徴的なアミノ酸モチーフの反復を有さない領域であり、100残基程度のアミノ酸からなる。 Fibroin is, for example, a protein containing a domain sequence represented by the formula 3: [(A) n motif-REP] m or the formula 4: [(A) n motif-REP] m -(A) n motif. May be. The fibroin according to the present embodiment 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 typically, but not limited to, regions having no repeat of the amino acid motif characteristic of fibroin, and consist of about 100 amino acids.
 本明細書において「ドメイン配列」とは、フィブロイン特有の結晶領域(典型的には、アミノ酸配列の(A)モチーフに相当する。)と非晶領域(典型的には、アミノ酸配列のREPに相当する。)を生じるアミノ酸配列であり、式3:[(A)モチーフ-REP]、又は式4:[(A)モチーフ-REP]-(A)モチーフで表されるアミノ酸配列を意味する。ここで、(A)モチーフは、アラニン残基を主とするアミノ酸配列を示し、アミノ酸残基数は2~27である。(A)モチーフのアミノ酸残基数は、2~20、4~27、4~20、8~20、10~20、4~16、8~16、又は10~16の整数であってよい。また、(A)モチーフ中の全アミノ酸残基数に対するアラニン残基数の割合は40%以上であればよく、60%以上、70%以上、80%以上、83%以上、85%以上、86%以上、90%以上、95%以上、又は100%(アラニン残基のみで構成されることを意味する。)であってもよい。ドメイン配列中に複数存在する(A)モチーフは、少なくとも7つがアラニン残基のみで構成されてもよい。REPは2~200アミノ酸残基から構成されるアミノ酸配列を示す。REPは、10~200アミノ酸残基から構成されるアミノ酸配列であってもよい。mは2~300の整数を示し、10~300の整数であってもよい。複数存在する(A)モチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するREPは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。 As used herein, the term “domain sequence” refers to a crystalline region (typically corresponding to the (A) n motif of an amino acid sequence) and an amorphous region (typically REP of an amino acid sequence) peculiar to fibroin. Corresponding to the amino acid sequence represented by formula 3: [(A) n motif-REP] m or formula 4: [(A) n motif-REP] m -(A) n motif. Means an array. Here, the (A) n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27. (A) The number of amino acid residues in the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. The plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.
 天然由来のフィブロインとしては、例えば、式3:[(A)モチーフ-REP]、又は式4:[(A)モチーフ-REP]-(A)モチーフで表されるドメイン配列を含むタンパク質を挙げることができる。天然由来のフィブロインの具体例としては、例えば、昆虫又はクモ類が産生するフィブロインが挙げられる。 Examples of naturally-occurring fibroin include, for example, a domain sequence represented by Formula 3: [(A) n motif-REP] m or Formula 4: [(A) n motif-REP] m -(A) n motif. The proteins included can be mentioned. Specific examples of naturally-derived fibroin include, for example, fibroin produced by insects or arachnids.
 昆虫が産生するフィブロインとしては、例えば、ボンビックス・モリ(Bombyx mori)、クワコ(Bombyx mandarina)、天蚕(Antheraea yamamai)、柞蚕(Anteraea pernyi)、楓蚕(Eriogyna pyretorum)、蓖蚕(Pilosamia Cynthia ricini)、樗蚕(Samia cynthia)、栗虫(Caligura japonica)、チュッサー蚕(Antheraea mylitta)、ムガ蚕(Antheraea assama)等のカイコが産生する絹タンパク質、及びスズメバチ(Vespa simillima xanthoptera)の幼虫が吐出するホーネットシルクタンパク質が挙げられる。 Examples of the fibroin produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yam tai rya pynayi, ori peri erygium, Anteraea periyna, Pomegranate (Anteraea periyi), Anteraea periyna, Pomegranate (Anteraea peryny) ), silkworm proteins produced by silkworms such as Anthera apse assap, such as silkworm silkworm (Antheraea assama), such as silkworm silkworms (Samia cynthia), chestnut (Caligura japonica), chusser silkworms (Antheraea mylitta), and mug silkworms (Antheraea assama). Hornet silk protein is mentioned.
 昆虫が産生するフィブロインのより具体的な例としては、例えば、カイコ・フィブロインL鎖(GenBankアクセッション番号M76430(塩基配列)、及びAAA27840.1(アミノ酸配列))が挙げられる。 More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence), and AAA278840.1 (amino acid sequence)).
 クモ類が産生するフィブロインとしては、例えば、クモ目(Araneae)に属するクモが産生するスパイダーシルクタンパク質が挙げられる。より具体的には、オニグモ、ニワオニグモ、アカオニグモ、アオオニグモ及びマメオニグモ等のオニグモ属(Araneus属)に属するクモ、ヤマシロオニグモ、イエオニグモ、ドヨウオニグモ及びサツマノミダマシ等のヒメオニグモ属(Neoscona属)に属するクモ、コオニグモモドキ等のコオニグモモドキ属(Pronus属)に属するクモ、トリノフンダマシ及びオオトリノフンダマシ等のトリノフンダマシ属(Cyrtarachne属)に属するクモ、トゲグモ及びチブサトゲグモ等のトゲグモ属(Gasteracantha属)に属するクモ、マメイタイセキグモ及びムツトゲイセキグモ等のイセキグモ属(Ordgarius属)に属するクモ、コガネグモ、コガタコガネグモ及びナガコガネグモ等のコガネグモ属(Argiope属)に属するクモ、キジロオヒキグモ等のオヒキグモ属(Arachnura属)に属するクモ、ハツリグモ等のハツリグモ属(Acusilas属)に属するクモ、スズミグモ、キヌアミグモ及びハラビロスズミグモ等のスズミグモ属(Cytophora属)に属するクモ、ゲホウグモ等のゲホウグモ属(Poltys属)に属するクモ、ゴミグモ、ヨツデゴミグモ、マルゴミグモ及びカラスゴミグモ等のゴミグモ属(Cyclosa属)に属するクモ、及びヤマトカナエグモ等のカナエグモ属(Chorizopes属)に属するクモが産生するスパイダーシルクタンパク質、並びにアシナガグモ、ヤサガタアシナガグモ、ハラビロアシダカグモ及びウロコアシナガグモ等のアシナガグモ属(Tetragnatha属)に属するクモ、オオシロカネグモ、チュウガタシロカネグモ及びコシロカネグモ等のシロカネグモ属(Leucauge属)に属するクモ、ジョロウグモ及びオオジョロウグモ等のジョロウグモ属(Nephila属)に属するクモ、キンヨウグモ等のアズミグモ属(Menosira属)に属するクモ、ヒメアシナガグモ等のヒメアシナガグモ属(Dyschiriognatha属)に属するクモ、クロゴケグモ、セアカゴケグモ、ハイイロゴケグモ及びジュウサンボシゴケグモ等のゴケグモ属(Latrodectus属)に属するクモ、及びユープロステノプス属(Euprosthenops属)に属するクモ等のアシナガグモ科(Tetragnathidae科)に属するクモが産生するスパイダーシルクタンパク質が挙げられる。スパイダーシルクタンパク質としては、例えば、MaSp(MaSp1及びMaSp2)、ADF(ADF3及びADF4)等の牽引糸タンパク質、MiSp(MiSp1及びMiSp2)、AcSp、PySp、Flag等が挙げられる。 Examples of fibroin produced by arachnids include spider silk protein produced by arachnids belonging to the order Araneae. More specifically, spiders belonging to the genus Araneus (Araneus) such as spiders, spiders spiders, red spiders, red spiders, and spiders, etc., spiders belonging to the genus Araneus, spider spiders, spider spiders, spider spiders and spiders belonging to the genus Nela spp. Spiders belonging to the genus Pronus, such as the spider Spider, and spiders belonging to the genus Cyrtarachne, such as Torinofundamasi and Otorinofundamas, and the genus Gasteracanth, such as the spider Spider and the genus Gasteracanthus. Spiders belonging to the genus Ordgarius (genus Ordgarius) such as spiders, spiders Mameitaisekimo and Mutsutoeiguisekimo spiders, spiders belonging to the genus Argiope, such as Argiope, spiders belonging to the genus Argiope, such as spiders, Argiope, and Argiope. Spiders belonging to the genus Spider, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, such as spiders, spiders, blue-breasted spiders and spiders belonging to the genus Cytophora, and spiders belonging to the genus Poltys, such as the spider Spider. Spider silk proteins produced by spiders belonging to the genus Cyclosa (genus Cyclosa), such as spiders, spider spiders, margot spiders, and black spiders, and spider silk proteins produced by spiders belonging to the genus Chorizopes, such as Yamato kana spiders, Spiders that belong to the genus Tetragnatha, such as the black-faced spider and the black-legged spider, spiders that belong to the genus Leucaug, such as the spiders of the genus Leucaug, such as the spiders of the genus Leucaug Spiders belonging to the genus Menosira, such as spiders, black spiders, and spiders belonging to the genus Dyschiriognatha, such as the genus Dyschiriognatha, spiders of the genus Genus Lepidoptera, L. sp. Spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprostenops and spiders belonging to the genus Euprosthenops To be Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, Flag and the like.
 ケラチン由来のタンパク質として、例えば、カプラ・ヒルクス(Capra hircus)のタイプIケラチン等を挙げることができる。 Examples of keratin-derived proteins include Capra hircus type I keratin.
 コラーゲン由来のタンパク質としては、例えば、式5:[REP2]で表されるドメイン配列を含むタンパク質(ここで、式5中、pは5~300の整数を示す。REP2は、Gly-X-Yから構成されるアミノ酸配列を示し、X及びYはGly以外の任意のアミノ酸残基を示す。複数存在するREP2は、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。)を挙げることができる。 Examples of the collagen-derived protein include a protein containing a domain sequence represented by Formula 5: [REP2] p (here, p represents an integer of 5 to 300. REP2 is Gly-X- An amino acid sequence composed of Y is shown, and X and Y are arbitrary amino acid residues other than Gly. A plurality of REP2s may be the same amino acid sequence or different amino acid sequences. it can.
 エラスチン由来のタンパク質としては、例えば、NCBIのGenBankのアクセッション番号AAC98395(ヒト)、I47076(ヒツジ)、NP786966(ウシ)等のアミノ酸配列を有するタンパク質を挙げることができる。 Examples of elastin-derived proteins include proteins having amino acid sequences such as NCBI GenBank Accession Nos. AAC98395 (human), I47076 (sheep), and NP786966 (bovine).
 レシリン由来のタンパク質としては、例えば、式6:[REP3]で表されるドメイン配列を含むタンパク質(ここで、式6中、qは4~300の整数を示す。REP3はSer-J-J-Tyr-Gly-U-Proから構成されるアミノ酸配列を示す。Jは任意アミノ酸残基を示し、特にAsp、Ser及びThrからなる群から選ばれるアミノ酸残基であることが好ましい。Uは任意のアミノ酸残基を示し、特にPro、Ala、Thr及びSerからなる群から選ばれるアミノ酸残基であることが好ましい。複数存在するREP4は、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。)を挙げることができる。 Examples of the resilin-derived protein include a protein containing a domain sequence represented by the formula 6: [REP3] q (wherein, q is an integer of 4 to 300 in the formula 6. REP3 is Ser-JJ). Shows an amino acid sequence composed of -Tyr-Gly-U-Pro, J represents an arbitrary amino acid residue, and particularly preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr, U is arbitrary. The amino acid residue is preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser, and a plurality of REP4s may have the same amino acid sequence or different amino acid sequences. ) Can be mentioned.
(融合タンパク質の製造方法)
 本実施形態に係る融合タンパク質は、当該融合タンパク質をコードする核酸を使用して、常法により製造することができる。当該融合タンパク質をコードする核酸は、塩基配列情報に基づいて、化学合成してもよく、PCR法等を利用して合成してもよい。
(Method for producing fusion protein)
The fusion protein according to this embodiment can be produced by a conventional method using a nucleic acid encoding the fusion protein. The nucleic acid encoding the fusion protein may be chemically synthesized based on the nucleotide sequence information, or may be synthesized by using the PCR method or the like.
〔核酸〕
 本実施形態に係る核酸は、本発明に係るポリペプチド、又は本発明に係る融合タンパク質をコードする。
[Nucleic acid]
The nucleic acid according to the present embodiment encodes the polypeptide according to the present invention or the fusion protein according to the present invention.
 一実施形態に係る核酸は、本発明に係るポリペプチド、又は本発明に係る融合タンパク質をコードする核酸の相補鎖とストリンジェントな条件下でハイブリダイズする核酸であってもよい。 The nucleic acid according to one embodiment may be a nucleic acid that hybridizes under stringent conditions with a complementary strand of the nucleic acid encoding the polypeptide of the present invention or the fusion protein of the present invention.
 「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。「ストリンジェントな条件」は、低ストリンジェントな条件、中ストリンジェントな条件及び高ストリンジェントな条件のいずれでもよい。低ストリンジェントな条件とは、少なくとも85%以上の同一性が配列間に存在する時のみハイブリダイゼーションが起こることを意味し、例えば、0.5%SDSを含む5×SSCを用い、42℃でハイブリダイズする条件が挙げられる。中ストリンジェントな条件とは、少なくとも90%以上の同一性が配列間に存在する時のみハイブリダイゼーションが起こることを意味し、例えば、0.5%SDSを含む5×SSCを用い、50℃でハイブリダイズする条件が挙げられる。高ストリンジェントな条件とは、少なくとも95%以上の同一性が配列間に存在する時のみハイブリダイゼーションが起こることを意味し、例えば、0.5%SDSを含む5×SSCを用い、60℃でハイブリダイズする条件が挙げられる。 ”The “stringent conditions” refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. The “stringent conditions” may be any of low stringent conditions, medium stringent conditions and high stringent conditions. Low stringency conditions mean that hybridization will occur only when there is at least 85% identity between the sequences, eg, 5×SSC containing 0.5% SDS at 42° C. The conditions for hybridizing are mentioned. The moderately stringent condition means that hybridization occurs only when at least 90% identity exists between the sequences, and for example, 5×SSC containing 0.5% SDS is used at 50° C. The conditions for hybridizing are mentioned. Highly stringent conditions mean that hybridization occurs only when at least 95% identity exists between the sequences, for example, using 5×SSC containing 0.5% SDS at 60° C. The conditions for hybridizing are mentioned.
 一実施形態に係る核酸は、本発明に係るポリペプチド、又は本発明に係る融合タンパク質をコードする核酸と90%以上の配列同一性を有する核酸であってもよい。配列同一性は、95%以上であることが好ましい。 The nucleic acid according to one embodiment may be a nucleic acid having 90% or more sequence identity with the nucleic acid encoding the polypeptide of the present invention or the fusion protein of the present invention. The sequence identity is preferably 95% or more.
 本実施形態に係る核酸の具体例として、配列番号32、配列番号33又は配列番号34で示される塩基配列を有する核酸を挙げることができる。配列番号32、配列番号33又は配列番号34で示される塩基配列は、それぞれ配列番号3、配列番号5及び配列番号7で示されるアミノ酸配列をコードするものである。 As a specific example of the nucleic acid according to the present embodiment, a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 can be mentioned. The base sequences represented by SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 encode the amino acid sequences represented by SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, respectively.
 本実施形態に係る核酸は、発現ベクターに組み込まれた形態で提供されてもよい。当該発現ベクターは、本実施形態に係る核酸配列と、当該核酸配列に作動可能に連結された1又は複数の調節配列とを有する。調節配列は、宿主における組換えタンパク質の発現を制御する配列(例えば、プロモーター、エンハンサー、リボソーム結合配列、転写終結配列等)であり、宿主の種類に応じて適宜選択することができる。発現ベクターの種類は、プラスミドベクター、ウイルスベクター、コスミドベクター、フォスミドベクター、人工染色体ベクター等、宿主の種類に応じて適宜選択することができる。 The nucleic acid according to this embodiment may be provided in a form incorporated in an expression vector. The expression vector has the nucleic acid sequence according to the present embodiment 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 according to the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, or an artificial chromosome vector.
〔タンパク質に自己会合能を付与する方法〕
 本発明はまた、本発明に係るポリペプチドと、タンパク質とを融合させることを含む、タンパク質に自己会合能を付与する方法と捉えることもできる。ポリペプチド、タンパク質、及び融合タンパク質の製造方法等の具体的な態様等は、上述したとおりである。
[Method of giving protein self-association ability]
The present invention can also be regarded as a method for imparting a self-association ability to a protein, which comprises fusing the polypeptide of the present invention with a protein. Specific aspects such as the method for producing the polypeptide, protein, and fusion protein are as described above.
 以下、実施例に基づいて本発明をより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
[試験例1:凝集自由エネルギーg(k)の評価]
 二ワオニグモの大吐糸管しおり糸タンパク質(ADF4)のN末端に配列番号17で示されるタグ配列を付加したアミノ酸配列(#GEN891,配列番号1)及び二ワオニグモの大吐糸管しおり糸タンパク質(ADF3)のN末端に配列番号17で示されるタグ配列を付加したアミノ酸配列(#GEN889,配列番号2)の凝集自由エネルギーg(k)を下記式(1)に従って算出した。
Figure JPOXMLDOC01-appb-M000008
[Test Example 1: Evaluation of cohesive free energy g(k)]
The amino acid sequence (#GEN891, SEQ ID NO: 1) in which the tag sequence represented by SEQ ID NO: 17 is added to the N-terminal of the giant spider canal silkworm protein (ADF4) of the spiderweed spider, and the large spitrel bookmark silkworm protein of the spiderweed spider, The aggregation free energy g(k) of the amino acid sequence (#GEN889, SEQ ID NO: 2) in which the tag sequence represented by SEQ ID NO: 17 was added to the N-terminus of ADF3) was calculated according to the following formula (1).
Figure JPOXMLDOC01-appb-M000008
 式(1)中、ε i,j(L)及びε i,j(L)は、アミノ酸残基のペアに対して算出されたエネルギーである(表1及び表2参照)。 In the formula (1), ε p i,j (L) and ε a i,j (L) are energies calculated for a pair of amino acid residues (see Table 1 and Table 2).
 また、式(1)中、δi≦k<i+Lは、kがi~i+L-1に属するときは1であり、それ以外は0である。δj≦k<j+Lは、kがj~j+L-1に属するときは1であり、それ以外は0である。i及びjは、始点を1としたアミノ酸配列中の残基番号に対応する。λは、2.0である。 Further, in the formula (1), δ i ≦k<i+L is 1 when k belongs to i to i+L−1, and 0 otherwise. δ j≦k<j+L is 1 when k belongs to j to j+L−1, and is 0 otherwise. i and j correspond to the residue numbers in the amino acid sequence with the start point as 1. λ is 2.0.
 図1は、二ワオニグモの大吐糸管しおり糸タンパク質(ADF4)のN末端に配列番号17で示されるタグ配列を付加したアミノ酸配列(#GEN891,配列番号1)における凝集自由エネルギーg(k)を残基番号に対してプロットしたグラフである。図2は、二ワオニグモの大吐糸管しおり糸タンパク質(ADF3)のN末端に配列番号17で示されるタグ配列を付加したアミノ酸配列(#GEN889,配列番号2)における凝集自由エネルギーg(k)を残基番号に対してプロットしたグラフである。#GEN891は、546番目のアミノ酸残基から582番目のアミノ酸残基において、各アミノ酸残基の凝集自由エネルギーg(k)が-4.0以下となった(図1)。なお、これらのアミノ酸残基からなるアミノ酸配列における凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値も当然-4.0以下である。#GEN889は、547番目のアミノ酸残基から579番目のアミノ酸残基において、各アミノ酸残基の凝集自由エネルギーg(k)が-4.0以下となった(図2)。なお、これらのアミノ酸残基からなるアミノ酸配列における凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値も当然-4.0以下である。凝集自由エネルギーg(k)が低い程、凝集傾向は強くなるため、これらのアミノ酸配列は、自己会合能を有するアミノ酸配列であると考えられる。 FIG. 1 shows the aggregation free energy g(k) in the amino acid sequence (#GEN891, SEQ ID NO: 1) in which the tag sequence shown in SEQ ID NO: 17 is added to the N-terminus of the large spit duct dragline protein (ADF4) of the Japanese spider. It is the graph which plotted with respect to the residue number. FIG. 2 is an aggregation free energy g(k) in the amino acid sequence (#GEN889, SEQ ID NO: 2) in which the tag sequence represented by SEQ ID NO: 17 is added to the N-terminus of the large spit duct dragline filament protein (ADF3) of the spiderweed spider. It is the graph which plotted with respect to the residue number. In #GEN891, the aggregation free energy g(k) of each amino acid residue was −4.0 or less from the 546th amino acid residue to the 582nd amino acid residue (FIG. 1). The average value of the aggregation free energy g(k) per amino acid residue in the amino acid sequence composed of these amino acid residues is naturally -4.0 or less. In #GEN889, the aggregation free energy g(k) of each amino acid residue was −4.0 or less from the 547th amino acid residue to the 579th amino acid residue (FIG. 2). The average value of the aggregation free energy g(k) per amino acid residue in the amino acid sequence composed of these amino acid residues is naturally -4.0 or less. The lower the aggregation free energy g(k), the stronger the aggregation tendency. Therefore, these amino acid sequences are considered to be amino acid sequences having self-association ability.
[試験例2:自己会合能を有するポリペプチドの製造及び評価]
(1)自己会合能を有するポリペプチドの製造
 配列番号18、20及び22で示されるアミノ酸配列を有するポリペプチドを設計した。配列番号18で示されるアミノ酸配列は、二ワオニグモ(Araneus diadematus)の大吐糸管しおり糸タンパク質(ADF4)のC末端の非繰り返し領域(NRC領域)のアミノ酸配列に配列番号17で示すアミノ酸配列(タグ配列)を付加したものである(His-ADF4-NRC,平均HI 0.297,分子量12.9kDa)。配列番号20で示されるアミノ酸配列は、二ワオニグモの大吐糸管しおり糸タンパク質(ADF3)のC末端の非繰り返し領域のアミノ酸配列に配列番号17で示すアミノ酸配列(タグ配列)を付加したものである(His-ADF3-NRC,平均HI 0.221,分子量13.5kDa)。配列番号22で示されるアミノ酸配列は、二ワオニグモの大吐糸管しおり糸タンパク質(ADF3)のC末端の非繰り返し領域から最もC末端側に位置するヘリックス5が欠損したアミノ酸配列に配列番号17で示すアミノ酸配列(タグ配列)を付加したものである(His-ADF3-shortNRC,平均HI 0.777,分子量10.6kDa)。配列番号18、20及び22で示されるアミノ酸配列を有するポリペプチドは、#GEN891及び#GEN889の凝集自由エネルギーg(k)が-4.0以下であるアミノ酸残基からなるアミノ酸配列の一部又は全部を含むものである。
[Test Example 2: Production and evaluation of polypeptide having self-association ability]
(1) Production of polypeptide having self-association ability Polypeptides having the amino acid sequences shown in SEQ ID NOs: 18, 20 and 22 were designed. The amino acid sequence represented by SEQ ID NO: 18 is the amino acid sequence represented by SEQ ID NO: 17 in the amino acid sequence of the non-repetitive region (NRC region) at the C-terminal of the large vesicular duct dragline protein (ADF4) of the spider (Araneus diadematus). Tag sequence) (His-ADF4-NRC, average HI 0.297, molecular weight 12.9 kDa). The amino acid sequence represented by SEQ ID NO: 20 is obtained by adding the amino acid sequence (tag sequence) shown in SEQ ID NO: 17 to the amino acid sequence of the non-repetitive region at the C-terminal of the giant vesicle guideline thread protein (ADF3) of the Japanese spider. Yes (His-ADF3-NRC, average HI 0.221, molecular weight 13.5 kDa). The amino acid sequence represented by SEQ ID NO: 22 is the amino acid sequence lacking the helix 5 located at the most C-terminal side from the C-terminal non-repetitive region of the giant spider duct silkworm protein of A. The amino acid sequence (tag sequence) shown is added (His-ADF3-shortNRC, average HI 0.777, molecular weight 10.6 kDa). The polypeptide having the amino acid sequence represented by SEQ ID NOs: 18, 20 and 22 is a part of the amino acid sequence consisting of amino acid residues having the aggregation free energy g(k) of #GEN891 and #GEN889 of -4.0 or less, or It includes all.
 各ポリペプチドをコードする核酸をそれぞれクローニングベクター(pUC118)にクローニングした。その後、各核酸をNdeI及びEcoRIで制限酵素処理して切り出した後、それぞれタンパク質発現ベクターpET-22b(+)に組換えて発現ベクターを得た。得られた発現ベクターで、大腸菌BLR(DE3)をそれぞれ形質転換して、各ポリペプチドを発現する形質転換大腸菌(組換え細胞)を得た。 Nucleic acid encoding each polypeptide was cloned into a cloning vector (pUC118). Then, each nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI and excised, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector. Escherichia coli BLR (DE3) was transformed with the obtained expression vector to obtain transformed Escherichia coli (recombinant cell) expressing each polypeptide.
 得られた形質転換大腸菌を、アンピシリンを含む2mLのLB培地で15時間培養した。当該培養液を、アンピシリンを含む100mLのシード培養用培地(表4)にOD600が0.005となるように添加した。培養液温度を30℃に保ち、OD600が5になるまでフラスコ培養を行い(約15時間)、シード培養液を得た。 The resulting transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 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.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
 500mLの生産培地(表5)を添加したジャーファーメンターにOD600が0.05となるように当該シード培養液を添加した。培養液温度を37℃に保ち、pH6.9で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の20%に維持した。 The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 5) was added so that the OD 600 was 0.05. The temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
 生産培地中のグルコースが完全に消費された直後に、フィード液(グルコース455g/1L、酵母エキス120g/1L)を1mL/分の速度で添加した。培養液温度を37℃に保ち、pH6.9で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の20%に維持し、20時間培養を行った。その後、1Mのイソプロピル-β-チオガラクトピラノシド(IPTG)を培養液に対して終濃度1mMになるよう添加し、目的のポリペプチドを発現誘導させた。IPTG添加後20時間経過した時点で、培養液を遠心分離し、菌体を回収した。IPTG添加前とIPTG添加後の培養液から調製した菌体を超音波破砕し、遠心分離を行い、目的とするポリペプチドを回収した。回収した目的とするポリペプチドをSDS-PAGEを行い、IPTG添加に依存した目的とするポリペプチドの分子量に対応するバンドの出現により、目的とするポリペプチドが不溶体として発現されていることを確認した。 Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, yeast extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture solution was maintained at 37° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was performed for 20 hours. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce the expression of the desired polypeptide. At 20 hours after the addition of IPTG, the culture solution was centrifuged to collect the bacterial cells. The cells prepared from the culture solution before addition of IPTG and after addition of IPTG were ultrasonically disrupted and centrifuged to recover the target polypeptide. SDS-PAGE was performed on the recovered target polypeptide, and it was confirmed that the target polypeptide was expressed as an insoluble substance by the appearance of a band corresponding to the molecular weight of the target polypeptide depending on the addition of IPTG. did.
(2)自己会合能を有するポリペプチドの可溶化
 目的とするポリペプチドをエッペンチューブに分取し、ドデシル硫酸ナトリウム(SDS)(2%)(富士フィルム和光純薬社製、生化学用)及びグアニジンチオシアン酸塩(富士フィルム和光純薬社製、生化学用)を添加し、目的とする各ポリペプチドを溶解させた。図3は、SDS及びグアニジンチオシアン酸塩を含むポリペプチド(His-ADF3-NRC(配列番号20)又はHis-ADF3-shortNRC(配列番号22))液の上清及び沈殿を、変性剤(2-メルカプトエタノール)存在下でSDS-PAGE法により分析した結果を示す写真である。各ポリペプチド(His-ADF3-NRC及びHis-ADF3-shortNRC)の分子量に対応するバンドが主に上清から検出されており、SDS及びグアニジンチオシアン酸塩の添加により、各ポリペプチドを可溶化できたことが示された。
(2) Solubilization of Polypeptide Having Self-Association Ability The desired polypeptide is collected in an Eppendorf tube and sodium dodecyl sulfate (SDS) (2%) (Fuji Film Wako Pure Chemical Industries, for biochemistry) and Guanidine thiocyanate (manufactured by Fuji Film Wako Pure Chemical Industries, for biochemistry) was added to dissolve each target polypeptide. FIG. 3 shows supernatants and precipitates of a polypeptide (His-ADF3-NRC (SEQ ID NO: 20) or His-ADF3-shortNRC (SEQ ID NO: 22)) containing SDS and guanidine thiocyanate, which were treated with a denaturant (2- 1 is a photograph showing the results of analysis by SDS-PAGE in the presence of (mercaptoethanol). Bands corresponding to the molecular weight of each polypeptide (His-ADF3-NRC and His-ADF3-shortNRC) were mainly detected in the supernatant, and each polypeptide was solubilized by the addition of SDS and guanidine thiocyanate. It was shown that
(3)自己会合能の確認
 各ポリペプチドをSDSを含む緩衝液(1%SDS、5mMトリス塩酸塩、2mM DTT、pH8.0)に溶解し、透析によって溶媒を尿素を含む緩衝液(8M尿素、25mMトリス塩酸塩、pH8.0)に交換し、ヒスチジンタグを利用して以下に示す条件でカラム精製を行った。
(3) Confirmation of self-association ability Each polypeptide was dissolved in a buffer solution containing SDS (1% SDS, 5 mM Tris hydrochloride, 2 mM DTT, pH 8.0), and the solvent was diluted by dialysis with a buffer solution containing urea (8 M urea). , 25 mM Tris hydrochloride, pH 8.0) and column purification was performed under the following conditions using a histidine tag.
(カラム精製)
 平衡化溶液(8M尿素、100mM塩化ナトリウム、25mMトリス塩酸塩、10mMイミダゾール、pH8.0)で平衡化したHis Gravi Trapカラム(ベッド体積1mL、GEヘルスケア社製)に各ポリペプチドを含む溶液を通し、各ポリペプチドをカラムに吸着させた。カラムを4mLの洗浄液(8M尿素、100mM塩化ナトリウム、25mMトリス塩酸塩、10mMイミダゾール、pH8.0)で洗浄した。次にカラムに第1の溶出液(8M尿素、100mM塩化ナトリウム、25mMトリス塩酸塩、300mMイミダゾール、pH8.0)を3mL流し、ポリペプチドを溶出させた。さらに第2の溶出液(8M尿素、100mM塩化ナトリウム、25mMトリス塩酸塩、500mMイミダゾール、pH8.0)を3mL流し、ポリペプチドを溶出させた。
(Column purification)
A solution containing each polypeptide was added to a His Gravi Trap column (bed volume: 1 mL, manufactured by GE Healthcare) equilibrated with an equilibration solution (8 M urea, 100 mM sodium chloride, 25 mM Tris hydrochloride, 10 mM imidazole, pH 8.0). Each polypeptide was adsorbed on the column. The column was washed with 4 mL of washing solution (8 M urea, 100 mM sodium chloride, 25 mM Tris hydrochloride, 10 mM imidazole, pH 8.0). Next, 3 mL of the first eluate (8 M urea, 100 mM sodium chloride, 25 mM Tris hydrochloride, 300 mM imidazole, pH 8.0) was passed through the column to elute the polypeptide. Further, 3 mL of the second eluate (8 M urea, 100 mM sodium chloride, 25 mM Tris hydrochloride, 500 mM imidazole, pH 8.0) was flown to elute the polypeptide.
 図4は、カラム精製の際の各溶液を変性剤非存在下でSDS-PAGE法により分析した結果を示す写真である。図4中、「through」は溶解液をカラムに負荷した際の通過液、「wash」はカラムを洗浄した際の通過液、「elute1」は第1の溶出液で溶出した際の溶出液、「elute2」は第2の溶出液で溶出した際の溶出液である。図4に示すとおり、各ポリペプチドは二量体に相当する分子量を有するバンドとして検出された。この結果より、各ポリペプチドは、変性剤非存在下では二量体化することが確認でき、自己会合能を有することが確認された。 FIG. 4 is a photograph showing the results of analyzing each solution at the time of column purification by the SDS-PAGE method in the absence of a denaturing agent. In FIG. 4, “through” is a flow-through when the column is loaded with the lysate, “wash” is a flow-through when the column is washed, “elute1” is an eluate when eluted with the first eluate, “Elute2” is the eluate when eluted with the second eluate. As shown in FIG. 4, each polypeptide was detected as a band having a molecular weight corresponding to a dimer. From this result, it was confirmed that each polypeptide dimerizes in the absence of a denaturing agent, and that it has a self-association ability.
(4)変性剤存在下での自己会合能の確認
 上記(3)と同様の条件でカラム精製した配列番号18で示されるアミノ酸配列を有するポリペプチド(His-ADF4-NRC)を使用して、当該ポリペプチドが変性剤(DTT:ジチオトレイトール)存在下で選択的な自己会合能を有するかを確認した。対照として、当該ポリペプチドに代えて、非特許文献(Proteins:Struct.Funct.Genet.,vol.49,pp.255-265,(2002年)、Proteins:Struct.Funct.Bioinformat.,vol.58,pp.271-277,(2005年))に記載の方法に従って調製したSNase(K116C)を使用した。
(4) Confirmation of self-association ability in the presence of a denaturant Using a polypeptide (His-ADF4-NRC) having the amino acid sequence represented by SEQ ID NO: 18 column-purified under the same conditions as in (3) above, It was confirmed whether the polypeptide has a selective self-association ability in the presence of a denaturant (DTT: dithiothreitol). As a control, instead of the polypeptide, a non-patent document (Proteins:Struct. Funct. Genet., vol. 49, pp. 255-265, (2002)), Proteins: Struct. Funct. Bioinformat., vol. 58. , Pp. 271-277, (2005)) and SNase (K116C) prepared according to the method.
 His-ADF4-NRC溶液(溶媒:5Mグアニジンチオシアン酸塩、10mMトリス塩酸塩、pH8)にDTTを5mMとなるように添加した。DTTを添加した溶液を透析チューブ(BioDesignDialysisTubing #D100、8000MWCO,BioDesign Inc.製)に移し、透析液(5Mグアニジンチオシアン酸塩、10mMトリス塩酸塩、pH8)に入れて、12時間透析を行った。DTT添加前、DTT添加直後、透析開始から3時間後、6時間後及び12時間後にHis-ADF4-NRC溶液から試料を採取し、SDS-PAGE法により分析した。SNase(K116C)についても同様に分析を行った。図5は、変性剤添加前、変性剤(DTT)添加後、透析開始から3時間後、6時間後及び12時間後のポリペプチド(His-ADF4-NRC)溶液をSDS-PAGE法により分析した結果を示す写真である。 DTT was added to a His-ADF4-NRC solution (solvent: 5M guanidine thiocyanate, 10 mM Tris hydrochloride, pH 8) so as to be 5 mM. The solution to which DTT was added was transferred to a dialysis tube (BioDesignDiallysisTubing #D100, 8000MWCO, manufactured by BioDesign Inc.), put in a dialysate (5M guanidine thiocyanate, 10 mM tris hydrochloride, pH 8), and dialyzed for 12 hours. Samples were collected from the His-ADF4-NRC solution before the addition of DTT, immediately after the addition of DTT, and 3 hours, 6 hours and 12 hours after the start of dialysis, and analyzed by SDS-PAGE. The same analysis was performed for SNase (K116C). In FIG. 5, polypeptide (His-ADF4-NRC) solutions before denaturant addition, denaturant (DTT) addition, and 3 hours, 6 hours, and 12 hours after the start of dialysis were analyzed by SDS-PAGE. It is a photograph which shows a result.
 図5に示すとおり、配列番号18で示されるアミノ酸配列を有するポリペプチド(His-ADF4-NRC)は、変性剤添加直後は単量体となっているものの、透析開始から3時間後には、ほとんどが二量体化していた。一方、SNase(K116C)透析開始から12時間後であっても二量体化していないタンパク質が存在することが確認された。 As shown in FIG. 5, the polypeptide having the amino acid sequence represented by SEQ ID NO: 18 (His-ADF4-NRC) was a monomer immediately after the addition of the denaturant, but almost 3 hours after the initiation of dialysis. Was dimerized. On the other hand, it was confirmed that even after 12 hours from the start of dialysis of SNase (K116C), there was a protein that did not dimerize.
 変性剤存在下では立体構造を保つことができず、タンパク質同士の相互作用はランダムな熱ゆらぎによって制御されるため、タンパク質間相互作用の効率は非常に低くなる。しかし、一般的なタンパク質(SNase(K116C))が二量体していない透析開始から3時間後(すなわち、変性剤の影響が残っていると考えられる。)には、配列番号18で示されるアミノ酸配列を有するポリペプチド(His-ADF4-NRC)は、非常に高い効率で二量体化が起きることが確認された。すなわち、配列番号18で示されるアミノ酸配列を有するポリペプチド(His-ADF4-NRC)は、変性剤存在下であっても選択的な自己会合能を有しているといえる。また、配列番号18で示されるアミノ酸配列を有するポリペプチド(His-ADF4-NRC)は、選択的な相互作用を介して短時間でジスルフィド結合の形成が可能である。なお、SNase(K116C)の一部が二量体化しているのは、ブラウン運動により分子が衝突することで二量体化しているものと考えられる。 ③ In the presence of a denaturant, the three-dimensional structure cannot be maintained, and the interaction between proteins is controlled by random thermal fluctuations, so the efficiency of protein-protein interaction is extremely low. However, a general protein (SNase (K116C)) is not dimerized, and 3 hours after the start of dialysis (that is, the effect of the denaturant is considered to remain), it is shown by SEQ ID NO: 18. It was confirmed that a polypeptide having an amino acid sequence (His-ADF4-NRC) undergoes dimerization with extremely high efficiency. That is, it can be said that the polypeptide having the amino acid sequence represented by SEQ ID NO: 18 (His-ADF4-NRC) has a selective self-association ability even in the presence of a denaturing agent. In addition, the polypeptide having the amino acid sequence represented by SEQ ID NO: 18 (His-ADF4-NRC) can form a disulfide bond in a short time through selective interaction. Note that it is considered that a part of SNase (K116C) is dimerized by the collision of molecules due to Brownian motion.
[試験例3:自己会合能を有するポリペプチドの設計及び評価]
(1)自己会合能を有するポリペプチドの設計
 既知の大瓶状腺スピドロイン(MaSp)のC末端のアミノ酸配列(配列番号27~191で示されるアミノ酸配列)に基づいて、プロファイル隠れマルコフモデル(HMM)を作成した。次いで、作成したHMMを入力値としてhmmemitプログラムを実行することにより、E値が10-5よりも小さいアミノ酸配列を生成した(10000配列)。生成したアミノ酸配列に対して、連続する5アミノ酸残基ごとに凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値を算出した。算出した平均値の最小値が-4.5以下になるアミノ酸配列として、配列番号10、12、14及び16で示されるアミノ酸配列を選出した。
[Test Example 3: Design and evaluation of polypeptide having self-association ability]
(1) Design of polypeptide capable of self-association Based on the C-terminal amino acid sequence of known large ampullate spidroin (MaSp) (amino acid sequences represented by SEQ ID NOs: 27 to 191), profile hidden Markov model (HMM) It was created. Then, the HMMmit program was executed using the created HMM as an input value to generate an amino acid sequence having an E value smaller than 10 −5 (10000 sequences). With respect to the generated amino acid sequence, the average value of the aggregation free energy g(k) per 1 amino acid residue was calculated for every 5 consecutive amino acid residues. The amino acid sequences represented by SEQ ID NOs: 10, 12, 14 and 16 were selected as the amino acid sequences having the calculated minimum value of −4.5 or less.
(2)自己会合能を有するポリペプチドの製造
 配列番号30で示されるアミノ酸配列を有するポリペプチド(His-ADF3-9467)を設計した。配列番号30で示されるアミノ酸配列は、配列番号15で示されるアミノ酸配列のN末端に配列番号17で示すアミノ酸配列(タグ配列)を付加したものである。また、配列番号15で示されるアミノ酸配列は、配列番号16で示されるアミノ酸配列のN末端に付加配列を付加したものである。
(2) Production of polypeptide having self-association ability A polypeptide (His-ADF3-9467) having the amino acid sequence represented by SEQ ID NO: 30 was designed. The amino acid sequence represented by SEQ ID NO: 30 is obtained by adding the amino acid sequence (tag sequence) represented by SEQ ID NO: 17 to the N-terminus of the amino acid sequence represented by SEQ ID NO: 15. The amino acid sequence shown by SEQ ID NO:15 is the amino acid sequence shown by SEQ ID NO:16 with an additional sequence added to the N-terminus.
 配列番号30で示されるアミノ酸配列を有するポリペプチドは、当該ポリペプチドをコードする核酸を使用した他は、試験例2と同様の手順で製造及び可溶化し、試験例2と同様の手順で変性剤存在下での自己会合能の確認を行った。変性剤存在下での自己会合能の確認では、DTT添加直後、透析開始から1.5時間後、3時間後、6時間後及び8時間後にポリペプチド(His-ADF3-9467)溶液から試料を採取し、SDS-PAGE法により分析した。結果を図6に示す。 The polypeptide having the amino acid sequence represented by SEQ ID NO: 30 was produced and solubilized by the same procedure as in Test Example 2 except that the nucleic acid encoding the polypeptide was used, and denatured by the same procedure as in Test Example 2. The self-association ability in the presence of the agent was confirmed. To confirm the self-association ability in the presence of a denaturant, a sample was prepared from the polypeptide (His-ADF3-9467) solution immediately after the addition of DTT, 1.5 hours, 3 hours, 6 hours, and 8 hours after the start of dialysis. Collected and analyzed by SDS-PAGE. Results are shown in FIG.
 図6は、変性剤(DTT)添加直後(0時間)、透析開始から1.5時間後、3時間後、6時間後及び8時間後のポリペプチド(His-ADF3-9467)溶液をSDS-PAGE法により分析した結果を示す写真である。図6に示すとおり、配列番号30で示されるアミノ酸配列を有するポリペプチド(His-ADF3-9467)は、変性剤添加直後は単量体となっているものの、透析開始から1.5時間後には二量体が形成されており、変性剤存在下であっても選択的な自己会合能を有することが確認された。 FIG. 6 shows SDS-polypeptide (His-ADF3-9467) solutions immediately after the addition of denaturant (DTT) (0 hour), 1.5 hours, 3 hours, 6 hours, and 8 hours after the start of dialysis. It is a photograph which shows the result analyzed by the PAGE method. As shown in FIG. 6, the polypeptide having the amino acid sequence represented by SEQ ID NO: 30 (His-ADF3-9467) is a monomer immediately after addition of the denaturant, but 1.5 hours after the initiation of dialysis. It was confirmed that a dimer was formed and that it had selective self-association ability even in the presence of a denaturant.

Claims (15)

  1.  下記式(1)で算出される凝集自由エネルギーg(k)の1アミノ酸残基あたりの平均値が-4.0以下であるアミノ酸配列を有する、ポリペプチド。
    Figure JPOXMLDOC01-appb-M000001
    [式(1)中、ε i,j(L)及びε i,j(L)は、アミノ酸残基のペアに対して算出されたエネルギーである。δi≦k<i+Lは、kがi~i+L-1に属するときは1であり、それ以外は0である。δj≦k<j+Lは、kがj~j+L-1に属するときは1であり、それ以外は0である。i及びjは、始点を1としたアミノ酸配列中の残基番号に対応する。λは、2.0である。]
    A polypeptide having an amino acid sequence in which the average value of the aggregation free energy g(k) calculated by the following formula (1) per amino acid residue is −4.0 or less.
    Figure JPOXMLDOC01-appb-M000001
    [In the formula (1), ε p i,j (L) and ε a i,j (L) are energies calculated for a pair of amino acid residues. δ i ≦k<i+L is 1 when k belongs to i to i+L−1, and is 0 otherwise. δ j≦k<j+L is 1 when k belongs to j to j+L−1, and is 0 otherwise. i and j correspond to the residue numbers in the amino acid sequence with the start point as 1. λ is 2.0. ]
  2.  配列番号3~16のいずれかで示されるアミノ酸配列と60%以上の配列同一性を有するアミノ酸配列を含む、ポリペプチド。 A polypeptide comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 3 to 16.
  3.  N末端及びC末端のいずれか一方又は両方にタグ配列を含む、請求項1又は2に記載のポリペプチド。 The polypeptide according to claim 1 or 2, comprising a tag sequence at either or both of the N-terminus and the C-terminus.
  4.  前記タグ配列が、配列番号17で示されるアミノ酸配列を含む、請求項3に記載のポリペプチド。 The polypeptide according to claim 3, wherein the tag sequence comprises the amino acid sequence shown by SEQ ID NO:17.
  5.  配列番号3~16のいずれかで示されるアミノ酸配列を含む、ポリペプチド。 A polypeptide comprising the amino acid sequence shown in any of SEQ ID NOs: 3 to 16.
  6.  配列番号18~31のいずれかで示されるアミノ酸配列を含む、ポリペプチド。 A polypeptide comprising the amino acid sequence shown in any of SEQ ID NOs: 18 to 31.
  7.  請求項1~6のいずれか一項に記載のポリペプチドをコードする核酸。 A nucleic acid encoding the polypeptide according to any one of claims 1 to 6.
  8.  請求項1~6のいずれか一項に記載のポリペプチドと、タンパク質とが融合されたアミノ酸配列を含む、融合タンパク質。 A fusion protein comprising the amino acid sequence obtained by fusing the polypeptide according to any one of claims 1 to 6 and a protein.
  9.  前記タンパク質のアミノ酸配列と、前記ポリペプチドのアミノ酸配列とを含む、請求項8に記載の融合タンパク質。 The fusion protein according to claim 8, comprising the amino acid sequence of the protein and the amino acid sequence of the polypeptide.
  10.  前記タンパク質のアミノ酸配列中に、前記ポリペプチドのアミノ酸配列を含む、請求項8に記載の融合タンパク質。 The fusion protein according to claim 8, which comprises the amino acid sequence of the polypeptide in the amino acid sequence of the protein.
  11.  前記タンパク質のN末端又はC末端から融合部位までのアミノ酸残基数が、前記タンパク質の総アミノ酸残基数に対して、10%以下である、請求項10に記載の融合タンパク質。 The fusion protein according to claim 10, wherein the number of amino acid residues from the N terminus or C terminus of the protein to the fusion site is 10% or less with respect to the total number of amino acid residues of the protein.
  12.  前記タンパク質が、構造タンパク質である、請求項8~11のいずれか一項に記載の融合タンパク質。 The fusion protein according to any one of claims 8 to 11, wherein the protein is a structural protein.
  13.  前記タンパク質が、クモ糸タンパク質である、請求項8~12のいずれか一項に記載の融合タンパク質。 The fusion protein according to any one of claims 8 to 12, wherein the protein is a spider silk protein.
  14.  請求項8~13のいずれか一項に記載の融合タンパク質をコードする核酸。 A nucleic acid encoding the fusion protein according to any one of claims 8 to 13.
  15.  請求項1~6のいずれか一項に記載のポリペプチドと、タンパク質とを融合させることを含む、前記タンパク質に自己会合能を付与する方法。 A method for imparting a self-association ability to the protein, which comprises fusing the polypeptide according to any one of claims 1 to 6 with a protein.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012165476A1 (en) * 2011-06-01 2012-12-06 スパイバー株式会社 Artificial polypeptide fiber and method for producing same
JP2015518489A (en) * 2012-05-02 2015-07-02 スパイバー テクノロジーズ アーベーSpiber Technologies Ab Structure of a spider silk fusion protein incorporating an immunoglobulin fragment as an affinity ligand
JP2016531845A (en) * 2013-09-17 2016-10-13 ボルト スレッズ インコーポレイテッド Methods and compositions for synthesizing improved silk fibers
WO2018002216A1 (en) * 2016-07-01 2018-01-04 Anna Rising Engineered spider silk proteins and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012165476A1 (en) * 2011-06-01 2012-12-06 スパイバー株式会社 Artificial polypeptide fiber and method for producing same
JP2015518489A (en) * 2012-05-02 2015-07-02 スパイバー テクノロジーズ アーベーSpiber Technologies Ab Structure of a spider silk fusion protein incorporating an immunoglobulin fragment as an affinity ligand
JP2016531845A (en) * 2013-09-17 2016-10-13 ボルト スレッズ インコーポレイテッド Methods and compositions for synthesizing improved silk fibers
WO2018002216A1 (en) * 2016-07-01 2018-01-04 Anna Rising Engineered spider silk proteins and uses thereof

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