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WO2019156242A1 - Méthode de production de protéine recombinante - Google Patents

Méthode de production de protéine recombinante Download PDF

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Publication number
WO2019156242A1
WO2019156242A1 PCT/JP2019/004736 JP2019004736W WO2019156242A1 WO 2019156242 A1 WO2019156242 A1 WO 2019156242A1 JP 2019004736 W JP2019004736 W JP 2019004736W WO 2019156242 A1 WO2019156242 A1 WO 2019156242A1
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Prior art keywords
recombinant protein
solvent
soluble fraction
protein
reaction mixture
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Japanese (ja)
Inventor
直樹 松坂
利昭 大澤
雅治 横田
千晶 西岡
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Kojima Industries Corp
Spiber Inc
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Kojima Industries Corp
Spiber Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for producing a recombinant protein.
  • Methods for isolating insoluble target proteins include a method using a metal hydroxide such as sodium hydroxide from a suspension of recombinant cells (Patent Document 1), and an organic acid such as formic acid or propionic acid.
  • Patent Document 2 A method (Patent Document 2) and the like using the method have been reported.
  • Patent Document 2 A method (Patent Document 2) and the like using the method have been reported.
  • Patent Document 2 when an insoluble target protein is isolated by these methods, it is difficult to remove impurities present together with the target protein, so that the purity of the isolated target protein is not high. There was a problem.
  • the method using an organic acid has a problem that the protein that is not resistant to the acid is easily decomposed, so that the protein that can be isolated is limited.
  • Impurities present with the target protein can be removed, and as a method without using an organic acid, the target protein is dissolved in an aprotic polar solvent such as dimethyl sulfoxide, and the impurities are removed using a solvent such as water.
  • a purification method Patent Document 3 has been reported. According to this method, the target protein can be purified up to about 70%, but there is a problem that the target protein is limited to a hydrophilic recombinant protein having a hydropathic index (HI) of 0 or less. there were.
  • an object of the present invention is to provide a method for easily producing a recombinant protein using a recombinant cell expressing the target recombinant protein in an insoluble form.
  • the present inventors processed using the first aprotic polar solvent under conditions that the target recombinant protein dissolves, and treated the resulting treatment solution with the second aprotic polar solvent. It is found that the target recombinant protein can be easily purified by aggregating contaminants and obtaining the purified recombinant protein as a soluble fraction by treating with a second solvent containing It came to be completed.
  • the present invention relates to the following inventions, for example.
  • a method for producing the recombinant protein using a recombinant cell expressing the target recombinant protein in an insoluble form in the cell The recombinant cell is treated with a first solvent containing a first aprotic polar solvent with or without inorganic salt added under conditions that allow the recombinant protein to dissolve, and the resulting reaction mixture is treated with a second non-reactive mixture.
  • a method for producing a recombinant protein characterized in that after treatment with a second solvent containing a protic polar solvent and / or a chelating agent, the insoluble fraction is removed to obtain the recombinant protein as a soluble fraction.
  • Method. [3] A method for producing a recombinant protein, comprising the steps of (A), (B) and (C) below. (A) Recombinant cells expressing a target recombinant protein in an insoluble form in a first solvent containing a first aprotic polar solvent with or without an inorganic salt added thereto.
  • the second solvent includes the second aprotic polar solvent, the dipole moment of the first aprotic polar solvent is 3.0 D or more, and the second aprotic polar solvent The method for producing a recombinant protein according to any one of [1] to [5], wherein the dipole moment is 2.5 D or more and is smaller than the dipole moment of the first aprotic polar solvent.
  • the inorganic salt is at least one selected from the group consisting of alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates and thiocyanates.
  • a method for producing the described recombinant protein [8] The method for producing a recombinant protein according to any one of [1] to [7], wherein the recombinant protein is a structural protein. [9] The method for producing a recombinant protein according to [8], wherein the structural protein is derived from spider silk. [10] The manufacturing method of artificial polypeptide fiber including the process of (A), (B), (C) and (E) below.
  • A Recombinant cells expressing a target recombinant protein in an insoluble form in a first solvent containing a first aprotic polar solvent with or without an inorganic salt added thereto. Treating under conditions where the protein dissolves to obtain a first reaction mixture comprising a first soluble fraction and a first insoluble fraction; (B) treating the first reaction mixture with a second solvent containing a second aprotic polar solvent and / or a chelating agent, and comprising a second soluble fraction and a second insoluble fraction; Obtaining a reaction mixture of 2.
  • a polypeptide film by removing the solvent of the soluble fraction of [12] A method for purifying a recombinant protein comprising the steps of (A), (B) and (C) below.
  • the production method of the present invention from a recombinant cell expressing a target recombinant protein as an insoluble substance in a cell, although it is a simple method that does not include complicated steps and has a small number of steps, A highly pure recombinant protein can be easily produced. Moreover, since the recombinant protein obtained by using the production method and purification method of the present invention has high purity, it can be used as it is for the production of, for example, spinning and film formation without going through a further purification step.
  • the method for producing and purifying the recombinant protein of the present invention does not require an acid addition step. Therefore, the target recombinant protein to be produced and purified is not limited to a protein resistant to acids. Furthermore, the production method and purification method of the recombinant protein of the present invention do not require a purification step of replacing the solvent of the soluble fraction containing the recombinant protein with an aqueous solvent such as water. Therefore, the target recombinant protein to be manufactured and purified is not limited to a hydrophilic recombinant protein having a hydropathic index of 0 or less, can be efficiently filtered, and the disposal of the filtration residue is easy It becomes.
  • FIG. 1 is a photograph showing the results of analysis by polyacrylamide gel electrophoresis (SDS-PAGE) on the purification of a target protein examined in Test Example 1.
  • FIG. 2 is a photograph showing the results of SDS-PAGE analysis on the purification of the target protein (PRT918) examined in Test Example 1.
  • FIG. 3 is a photograph of a coagulation solution examined in Test Example 4 when a yarn is formed using the purified protein solution as a dope for spinning.
  • FIG. 4 is a photograph of a yarn formed by using the purified protein solution as a dope for spinning examined in Test Example 4.
  • FIG. 5 is a photograph showing the results of SDS-PAGE analysis on the purification of the target protein (PRT918) examined in Test Example 5.
  • a method for producing a recombinant protein is a method for producing a recombinant protein using a recombinant cell in which a target recombinant protein is expressed as an insoluble substance in a cell.
  • the cells are treated with a first solvent containing a first aprotic polar solvent with or without addition of an inorganic salt under conditions such that the recombinant protein dissolves, and the resulting reaction mixture is treated with a second aprotic polar solvent.
  • the insoluble fraction is removed, and the recombinant protein is obtained as a soluble fraction.
  • the obtained soluble fraction may be further treated with a poor solvent for the recombinant protein to aggregate the recombinant protein and obtain the recombinant protein as an aggregate.
  • the method for producing a recombinant protein includes the following steps (A), (B), and (C).
  • A Recombinant cells expressing a target recombinant protein in an insoluble form in a first solvent containing a first aprotic polar solvent with or without an inorganic salt added thereto. Treating under conditions where the protein dissolves to obtain a first reaction mixture comprising a first soluble fraction and a first insoluble fraction;
  • C removing the second insoluble fraction from the second reaction mixture to obtain the recombinant protein as the second soluble fraction
  • the method for producing a recombinant protein may further include the following step (D).
  • step (D) A process of treating the second soluble fraction obtained in (C) with a poor solvent for the recombinant protein, aggregating the recombinant protein, and obtaining the recombinant protein as an aggregate
  • the target recombinant protein can be easily obtained with high purity. Therefore, as one embodiment of the present invention, there is provided a method for purifying a target recombinant protein comprising the steps (A), (B) and (C).
  • target recombinant protein examples include any protein that is preferably produced on an industrial scale.
  • proteins usable for medical purposes proteins usable for medical purposes, structural proteins, and the like.
  • proteins that can be used for industrial or medical use include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membranes or transport proteins, antigens used for vaccination, vaccines, antigen binding proteins, immune stimulating proteins, Mention may be made of allergens, full-length antibodies or antibody fragments or derivatives.
  • Specific examples of the structural protein include spider silk, silkworm silk, keratin, collagen, elastin and resilin, and proteins derived therefrom.
  • a protein derived from spider silk or silkworm silk that is a fibroin-like protein
  • a protein comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m
  • (A) The n motif represents an amino acid sequence composed of 4 to 20 amino acid residues
  • (A) the number of alanine residues relative to the total number of amino acid residues in the n motif is 80% or more.
  • M represents an integer of 8 to 300.
  • a plurality of (A) n motifs may be the same amino acid sequence or different amino acid sequences. May be the same or different amino acid sequences.).
  • proteins containing the amino acid sequences shown in SEQ ID NO: 1 (PRT918) to SEQ ID NO: 5.
  • the hydropathic index of PRT918 (SEQ ID NO: 1) is 0.44
  • the hydropathic index of PRT468 (SEQ ID NO: 5) is -0.59.
  • the value of the hydropathic index is a value calculated according to the method described in International Publication No. 2014/103846.
  • a protein derived from collagen for example, a protein containing a domain sequence represented by Formula 2: [REP2] o (where, in Formula 2, o represents an integer of 5 to 300.
  • REP2 is derived from Gly-XY.
  • X and Y represent any amino acid residue other than Gly, and a plurality of REP2s may be the same amino acid sequence or different amino acid sequences.
  • a protein Collagen-type4-Kai
  • SEQ ID NO: 6 can be mentioned.
  • the amino acid sequence shown in SEQ ID NO: 6 corresponds to the repeat part and motif of the partial sequence of human collagen type 4 (NCBl Genebank accession number: CAA563335.1, GI: 3702452) obtained from the NCBl database.
  • the amino acid sequence shown in SEQ ID NO: 10 (His tag sequence 76 and hinge sequence) is added to the N-terminus of the amino acid sequence from the 301st residue to the 540th residue.
  • the hydropathic index of Collagen-type4-Kai is -0.75.
  • a protein derived from resilin for example, a protein containing a domain sequence represented by Formula 3: [REP3] p (where, in Formula 3, p represents an integer of 4 to 300.
  • REP3 is Ser-JJ-).
  • Plural REP3s may have the same or different amino acid sequences. ). Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 7.
  • the amino acid sequence shown in SEQ ID NO: 7 is the amino acid sequence of resilin (NCBl Genebank accession number NP_6111157.1, Gl: 24654243), wherein Thr at 87th residue is replaced with Ser, and 95th residue
  • An amino acid sequence represented by SEQ ID NO: 10 (His tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence from the 19th to the 321st residues of the substituted sequence in which Asn is replaced with Asp. .
  • the hydropathy index of Resilin-Kai (SEQ ID NO: 7) is -1.22.
  • elastin-derived proteins include proteins having an amino acid sequence such as NCB1 Genebank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specific examples include a protein comprising the amino acid sequence represented by SEQ ID NO: 8.
  • the amino acid sequence represented by SEQ ID NO: 8 is the amino acid sequence represented by SEQ ID NO: 10 at the N-terminus of the amino acid sequence of residues 121 to 390 of the amino acid sequence of Genebank accession number AAC98395 of NCBl (His tag). Sequence and hinge sequence).
  • the hydropathic index of elastin short (SEQ ID NO: 8) is 0.42.
  • keratin-derived proteins examples include Capra hircus type I keratin. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 9 (amino acid sequence of Genebank accession number ACY30466 of NCBl) can be mentioned. The hydropathic index of type I keratin 26 (SEQ ID NO: 9) is -0.53.
  • a recombinant cell is a recombinant cell in which the recombinant protein is expressed in the cell as an insoluble substance.
  • Recombinant cells can be obtained using a general method using genetic engineering techniques.
  • a recombinant cell can be transformed into a host (host cell) with an expression vector having a nucleic acid sequence encoding the protein of interest and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be obtained.
  • Regulatory sequences are sequences that control the expression of recombinant proteins in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, 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, viral vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like.
  • prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • prokaryotes include bacteria such as E. coli, Bacillus subtilis, Pseudomonas, Corynebacterium, and Lactococcus, and more preferably, E. coli cells.
  • An expression vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a target protein can be transcribed is preferably used. .
  • the expression vector according to the present invention is capable of autonomous replication in a prokaryotic cell, and at the same time, a promoter, a ribosome binding sequence, a nucleic acid sequence and a transcription termination sequence according to the present invention. It is preferable that the vector contains A gene sequence that controls the promoter may be included.
  • an inducible promoter that functions in a host cell and can induce expression of a target protein may be used.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure or pH value.
  • prokaryotic hosts such as bacteria include microorganisms belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • microorganisms belonging to the genus Escherichia include, for example, Escherichia coli BL21 (Novagen), Escherichia coli BL21 (DE3) (Life Technologies), Escherichia coli BLR (DE3) (Merck Millipore), Escherichia coli DH1, Escherichia ⁇ Coli GI698, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli K5 (ATCC 23506), Escherichia coli KY3276, Escherichia coli MC1000, Escherichia coli MG1655 (ATCC 47076), Escherichia coli 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia
  • microorganisms belonging to the genus Brevibacillus include, for example, Brevibacillus agri, Brevibacillus bolsterensis, Brevibacillus centroporus Brevibacillus formosas, Brevibacillus invocatus, Brevibacillus latirosporus, Brevibacillus limnophilus, Brevibacillus paravis Brevibacillus reuszeli, Brevibacillus thermolver, Brevibacillus brevis 47 (FERM BP-1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevi Bacillus brevis 47-5Q (JCM8975), Brevibacillus choshinensis HPD31 (FERM BP-1087), Brevibacil Choshinensis HPD31-S (FERM BP-6623), Brevibacillus choshinensis HPD31
  • microorganisms belonging to the genus Serratia include, for example, Serratia lifafaciens ATCC 14460, Serratia entomofila, Serratia ficaria, Serratia cola (Serratia grimesii), Serratia proteamaculans, Serratia odorifera, Serratia primumica, Serratia rubiae, Sera rubia e It is possible.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis and Bacillus amyloliquefaciens.
  • microorganisms belonging to the genus Microbacterium include, for example, Microbacterium / Ammonia Filum ATCC 15354.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam (Corynebacterium glutamicum) ATCC 14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC 14067) ATCC 13826, ATCC 14067, Brevibacterium immariophyllum (Brevibaterium immariophilum) ATCC 14068, Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC 13869) ATCC 13665, ATCC 13869, Brevibacterium roseum ATCC 13825, Brevibacterium saccharolyticum TCC14066, Brevibacterium Chiogenitarisu ATCC19240, Brevibacterium album ATCC15111, mention may be made of Brevibacterium Serinumu ATCC15112 and the like.
  • Examples of microorganisms belonging to the genus Corynebacterium include, for example, Corynebacterium ammoniagenes ATCC6871, ATCC6872, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicolicoside ATCC14067, Lamb (Corynebacterium acetoacidophilum) ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium alkanolyticum ATCC 21511, Corynebacterium carnae ATCC 15991, Corynebacterium glutamicum Arm ATCC13020, ATCC13032, ATCC13060, can be named Corynebacterium Lilium ATCC15990, Corynebacterium Merasekora ATCC 17965, Corynebacterium thermo amino monocytogenes AJ12340 (FERMBP-1539), Corynebacterium Hakyurisu ATCC13868 and the like.
  • microorganisms belonging to the genus Pseudomonas include, for example, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas fluescens, Pseudomonas fluscens, Pseudomonas fluseusas, and (Pseudomonas sp.) D-0110 and the like.
  • Any method can be used for introducing an expression vector into the host cell as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • the protoplast method Japanese Patent Laid-Open No. 63-248394
  • the methods described in Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979), etc. Can be mentioned.
  • Transformation of microorganisms belonging to the genus Brevibacillus can be performed, for example, by the method of Takahashi et al. (J. Bacteriol., 1983, 156: 1130-1134) or the method of Takagi et al. (Agric. Biol. Chem., 1989, 53: 3099). -3100), or the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61: 202-203).
  • vectors for introducing a nucleic acid encoding a target protein include, for example, pBTrp2, pBTac1, pBTac2 (all available from Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia) PSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem.
  • pUC18 When using Escherichia coli as a host, pUC18, pBluescript II, pSupex, pET22b, pCold, etc. can be mentioned as suitable vectors.
  • vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, pHY500 (JP-A-2-31682), pNY700 (JP-A-4-278091), pHY4831 (J Bacteriol., 1987, 1239-1245), pNU200 (Shigezo Tsujitaka, Journal of Japanese Society of Agricultural Chemistry 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211 (J.
  • Promoters using prokaryotes as hosts are not limited as long as they function in host cells.
  • promoters derived from Escherichia coli or phages such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, T7 promoter and the like can be mentioned.
  • artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series (Ptrp ⁇ 2), a tac promoter, a lacT7 promoter, and a let I promoter can be used.
  • a transcription termination sequence is not necessarily required for the expression of the nucleic acid, but a transcription termination sequence is preferably arranged immediately below the structural gene.
  • Examples of eukaryotic hosts include yeast, filamentous fungi (molds, etc.) and insect cells.
  • yeast examples include, for example, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces, the genus Trichosporon, and the genus Cwaniomyces. And yeast belonging to the genus Yarrowia and Hansenula.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Schizosaccharomyces pombe Schizosaccharomyces pombe
  • Kluyveromyces lactis Kluyveromyces lactis
  • Kluyveromyces marxianus Kluyveromyces marxianus
  • Trichosporon pullulans Trichosporon pullulans
  • Shiwaniomaisesu - Albius (Schwaniomyces alluvius)
  • Swaniomyces occidentalis Candida utilis, Pichia pastoris ( Ichia pastoris, Pichia angusta, Pichia methanolica, Pichia polymorpha, Pichia stipitropi, H. pichia Etc.
  • Expression vectors when yeast is used as the host cell are usually an origin of replication (if amplification in the host is required) and a selectable marker for propagation of the vector in E. coli, a promoter for recombinant protein expression in yeast, and It is preferred to include a terminator as well as a selection marker for yeast.
  • the expression vector is a non-integrating vector
  • ARS autonomously replicating sequence
  • vectors in the case of using yeast as a host include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3O1, pHIL-S1, pPIC9K, pPICZ ⁇ , PGAZZp, ICPZB Etc.
  • promoters using yeast as a host are not limited as long as they can be expressed in yeast.
  • a glycolytic gene promoter such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MF ⁇ 1 promoter, CUP 1 promoter, pGAP promoter, Examples thereof include pGCW14 promoter, AOX1 promoter, MOX promoter and the like.
  • any method for introducing an expression vector into yeast any method can be used as long as it is a method for introducing DNA into yeast.
  • electroporation Methods Enzymol., 194, 182 (1990)
  • spheroplast Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)
  • lithium acetate method J. Bacteriol., 153, 163 (1983)
  • Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like electroporation (Methods Enzymol., 194, 182 (1990)
  • spheroplast Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)
  • lithium acetate method J. Bacteriol., 153, 163 (1983)
  • filamentous fungi examples include, for example, the genus Acremonium, the genus Aspergillus, the genus Ustilago, the genus Trichoderma, the genus Neurospora, the genus Fusarium, and Fusarium, Penicillium genus, Myceliophtora genus, Botrytis genus, Magnaporthe genus, Mucor genus, Metalithium genus, Monascus genus, Monascus genus And bacteria belonging to the genus Rhizomucor.
  • filamentous fungi include Acremonium alabamense, Acremonium cellulolyticus, Aspergillus algae or aspergillus sp. Aspergillus oryzae, Aspergillus salmon (Aspergillus sake), Aspergillus sojae (Aspergillus sojae), Aspergillus tubigensis (Aspergillus tubigenisgersgers) us niger, Aspergillus nidulans, Aspergillus parasiticus, Aspergillus fisium, Aspergillus fiscus, Aspergillus fiscus Aspergillus flavus, Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus japonicus (Aspergillus japonicus), Trichoderma bi De (Trichoderma viride), Trichoderma Hajianumu (Trichoderma harzianum), Trichoderma reesei (Tricho
  • promoters when using filamentous fungi as a host may be any of genes related to glycolysis, genes related to constitutive expression, enzyme genes related to hydrolysis, and specifically, amyB, glaA, agdA, glaB, TEF1, xynF1 tannasegene, No. 8AN, gpdA, pgkA, enoA, melO, sodM, catA, catB and the like.
  • the expression vector can be introduced into filamentous fungi using a conventionally known method.
  • a conventionally known method for example, the method of Cohen et al. (Calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)], protoplast method [Mol. Gen. Genet. 168: 111 (1979)], competent method [J. Mol. Biol. 56: 209 (1971)], electroporation method and the like.
  • Insect cells include, for example, lepidopteran insect cells. More specifically, insect cells derived from Spodoptera frugiperda such as Sf9 and Sf21, as well as nettle horsetail such as High 5 (Trichoplusia). ) Derived insect cells.
  • vectors when using insect cells as hosts include baculoviruses such as Autographa californica nuclear polyhedrosis virus, which are viruses that infect night stealing insects (Baculovirus virus). , A Laboratory Manual, WH Freeman and Company, New York (1992).
  • baculoviruses such as Autographa californica nuclear polyhedrosis virus, which are viruses that infect night stealing insects (Baculovirus virus). , A Laboratory Manual, WH Freeman and Company, New York (1992).
  • the polypeptide can be expressed by the method described in Freeman and Company, New York (1992), Bio / Technology, 6, 47 (1988), and the like. That is, after a recombinant gene transfer vector and a baculovirus are co-introduced into insect cells to obtain a recombinant virus (expression vector) in the insect cell culture supernatant, the recombinant virus is further infected with insect cells, and the polypeptide Can be expressed.
  • gene transfer vectors used in the method include pVL1392, pVL1393, and pBlueBacIII (both manufactured by Invitrogen).
  • Examples of a method for co-introducing a recombinant gene introduction vector and a baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 227075), the lipofection method (Proc. Natl. Acad. Sci). USA, 84, 7413 (1987)).
  • the recombinant vector further contains a selection marker gene for selecting a transformant.
  • a selection marker gene for selecting a transformant for example, in E. coli, resistance genes for various drugs such as tetracycline, ampicillin, and kanamycin can be used as selection marker genes. Recessive selectable markers that can complement genetic mutations involved in auxotrophy can also be used.
  • yeast a gene resistant to geneticin can be used as a selectable marker gene, and selectable markers such as LEU2, URA3, TRP1, and HIS3, which are complementary to gene mutations involved in auxotrophy, can also be used.
  • selectable markers such as LEU2, URA3, TRP1, and HIS3, which are complementary to gene mutations involved in auxotrophy, can also be used.
  • filamentous fungi as a selectable marker gene, niaD (Biosci. Biotechnol.
  • aureobasidin resistance gene Mol Gen Genet, 261, 290-296, (1999)
  • Benomyl Marker gene selected from the group consisting of sex genes (Proc Natl Acad Sci USA, 83, 4869-4873, (1986)) and hygromycin resistance genes (Gene, 57, 21-26, (1987)), leucine-requiring complementation A gene etc. can be mentioned.
  • a wild-type gene that complements the auxotrophy can be used as a selectable marker gene.
  • Selection of a host transformed with the above expression vector can be performed by plaque hybridization, colony hybridization, or the like using a probe that selectively binds to the nucleic acid.
  • a probe that selectively binds to the nucleic acid.
  • the probe those obtained by modifying a partial DNA fragment amplified by the PCR method with a radioisotope or digoxigenin based on the sequence information of the nucleic acid can be used.
  • the recombinant protein can be produced by culturing a host (recombinant cell) transformed with the above expression vector in a culture medium.
  • the method for culturing the recombinant cell in a culture medium can be performed according to a method usually used for culturing a host.
  • the culture medium contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host, so that the host can be cultured efficiently.
  • a medium that can be used any of a natural medium and a synthetic medium may be used.
  • Any carbon source may be used as long as it can be assimilated by the host.
  • Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolysate, and organic acids such as acetic acid and propionic acid.
  • alcohols such as ethanol and propanol can be used.
  • nitrogen source examples include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts contained in the medium include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. it can.
  • Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and agitation culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during the culture.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter
  • indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter.
  • An acid or the like may be added to the medium.
  • Insect cell culture media include commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400, ExCell405 (all from JRH Biosciences), Grace ' s Insect Medium (Nature, 195, 788 (1962)) can be used.
  • Insect cell culture can be performed, for example, under a culture medium pH of 6 to 7, a culture temperature of 25 to 30 ° C., and a culture time of 1 to 5 days. Moreover, you may add antibiotics, such as a gentamicin, to a culture medium as needed during culture
  • antibiotics such as a gentamicin
  • the transformed plant cell When the host is a plant cell, the transformed plant cell (recombinant cell) may be cultured as it is, or it can be differentiated into a plant organ and cultured.
  • a medium for culturing the plant cells a commonly used Murashige and Skoog (MS) medium, White medium, or a medium in which a plant hormone such as auxin or cytokinin is added to these mediums or the like is used. be able to.
  • the cultured animal cell (recombinant cell) can be cultured under conditions such as a culture medium pH of 5 to 9, a culture temperature of 20 to 40 ° C., and a culture time of 3 to 60 days. can do.
  • antibiotics such as kanamycin and a hygromycin, to a culture medium as needed during culture
  • the target protein can be expressed in recombinant cells as an insoluble substance by the above method.
  • Step (A) the recombinant cell expressing the target recombinant protein in an insoluble form is treated with a first solvent containing a first aprotic polar solvent with or without addition of an inorganic salt. It is a step of obtaining a first reaction mixture containing a first soluble fraction and a first insoluble fraction by treatment under conditions where the recombinant protein dissolves.
  • the first solvent containing the first aprotic polar solvent with or without the addition of an inorganic salt may be added to a recombinant cell such as a living cell or a dried cell, and includes those recombinant cells. You may add to suspension etc.
  • a recombinant cell such as a living cell or a dried cell, and includes those recombinant cells. You may add to suspension etc.
  • the recombinant protein is treated under conditions that allow it to be dissolved, the recombinant protein is dissolved and contained in the first soluble fraction, and impurities that do not dissolve in the first solvent are contained in the first insoluble fraction. become.
  • Contaminant means something other than the target recombinant protein derived from the host cell.
  • the contaminants are not particularly limited, and examples thereof include proteins other than the target recombinant protein, lipids, nucleic acids and the like derived from host cells.
  • a part of the impurities for example, a part of the protein derived from the host cell, may be contained in the first soluble fraction by dissolving in the first solvent.
  • a part of the impurities may be dissolved in the first solvent.
  • the recombinant cell used in step (A) may be an intact cell or a cell after a treatment such as a destruction treatment.
  • the method of the present invention can also be applied to remove residual impurities using recombinant cells that have already undergone a simple purification treatment.
  • first aprotic polar solvent examples include dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), 1,3-dimethyl-2-imidazolidone (DMI), N, N-dimethylformamide ( DMF), N, N-dimethylacetamide (DMA), acetonitrile, acetone, propylene carbonate, hexamethylphosphoramide, N-ethylpyrrolidone, nitrobenzene, furfural, ⁇ -butyrolactone, ethylene sulfite, sulfolane, succinonitrile, ethylene Although carbonate etc. can be mentioned, it is not limited to these.
  • aprotic polar solvents those having a dipole moment of 3.0 D or more are preferable, for example, at least one solvent selected from the group consisting of DMSO, NMP, DMI, DMF, DMA and acetonitrile is preferable. More preferred is at least one solvent selected from the group consisting of NMP, NMF, DMF and DMA.
  • the purity of the first aprotic polar solvent is not particularly limited and is not necessarily high purity in this step, but it is preferably 80% or more.
  • one kind of aprotic polar solvent may be used alone, or two or more kinds of aprotic polar solvents may be appropriately mixed and used.
  • the first aprotic polar solvent-containing solvent may be the first aprotic polar solvent itself, and a solvent other than the first aprotic polar solvent (other solvent) may be used. May contain. Examples of other solvents include methanol, ethanol, and acetone.
  • the content of the first aprotic polar solvent may be, for example, 60 to 100% by volume based on the total amount of the first solvent.
  • the amount of the solvent containing the first aprotic polar solvent (first solvent) added to the recombinant cell may be an amount that can dissolve the target recombinant protein.
  • the ratio of the volume (mL) of the first solvent to the dry weight (g) (volume (mL) / dry weight (g)) may be 5 to 100 times, 10 to 50 times, 20 It may be up to 35 times.
  • the dry weight of the recombinant cell means the weight of the dried bacterial cell when the host is a bacterium, for example.
  • inorganic salts that can be added to the first solvent include alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, thiocyanates, perchlorates, and the like.
  • the inorganic salt may be at least one selected from the group consisting of alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates and thiocyanates.
  • alkali metal halide examples include potassium bromide, sodium bromide, lithium bromide, potassium chloride, sodium chloride, lithium chloride, sodium fluoride, potassium fluoride, cesium fluoride, potassium iodide, sodium iodide, A lithium iodide etc. can be mentioned.
  • alkaline earth metal halide examples include calcium chloride, magnesium chloride, magnesium bromide, calcium bromide, magnesium iodide, calcium iodide and the like.
  • alkaline earth metal nitrates examples include calcium nitrate, magnesium nitrate, strontium nitrate, and barium nitrate.
  • thiocyanate examples include sodium thiocyanate, ammonium thiocyanate, (guanidinium thiocyanate), and the like.
  • perchlorates examples include ammonium perchlorate, potassium perchlorate, calcium perchlorate, silver perchlorate, sodium perchlorate, and magnesium perchlorate.
  • These inorganic salts may be used alone or in combination of two or more.
  • Suitable inorganic salts include alkali metal halides and alkaline earth metal halides, and specific examples of suitable inorganic salts include lithium chloride and calcium chloride.
  • the optimum amount of the inorganic salt may be determined according to the first solvent to be used.
  • an inorganic salt can be added so that it may become more than 0 mol / L and 1.0 mol / L or less with respect to a 1st solvent, for example.
  • the addition amount of the inorganic salt may be 0.7 mol / L or less, 0.6 mol / L or less, or 0.5 mol / L or less, 0.25 mol / L or less, 0.05 mol / L or more, 0 It may be 1 Mmol / L, or 0.2 mol / L or more.
  • the addition amount of the inorganic salt is preferably 0 to 0.7 mol / L, more preferably 0 to 0.25 mol / L with respect to the first solvent. Preferably, it may be 0 to 0.15 mol / L.
  • the addition amount of the inorganic salt is preferably 0 to 0.7 mol / L with respect to the first solvent. Is more preferably 0.25 mol / L, and may be 0 to 0.15 mol / L.
  • the first solvent includes a first aprotic solvent, and may optionally include an inorganic salt.
  • the inorganic salt may be dissolved in the first aprotic solvent, and then the recombinant cell expressing the target recombinant protein in the cell as an insoluble substance may be treated.
  • the solvent may be added simultaneously with or after the solvent is added to the recombinant cells.
  • the amount of the first solvent added to the recombinant cells may be any amount that can dissolve the target recombinant protein.
  • the volume of the first solvent relative to the dry weight (g) of the recombinant cells may be 10 to 100 times, 12 to 50 times, or 20 to 35 times.
  • the dry weight of the recombinant cell means the weight of the dried bacterial cell when the host is a bacterium, for example.
  • Conditions under which the recombinant protein dissolves are the concentration of the inorganic salt added to the first solvent and the target recombinant protein However, it is preferable that the temperature condition is satisfied. Although the recombinant protein dissolves, it is preferable to warm to a temperature at which impurities are difficult to dissolve or not dissolve and maintain for a predetermined time.
  • the temperature for dissolution may be determined according to the concentration of the inorganic salt added to the first solvent and the target recombinant protein, and examples include temperatures of 30 to 100 ° C. and 40 to 60 ° C. .
  • the upper limit value of the temperature for dissolving may be 100 ° C., 90 ° C., 80 ° C. or 70 ° C.
  • the lower limit value of the temperature for dissolving may be 30 ° C., 40 ° C., or 50 ° C. .
  • the time for the dissolution is not particularly limited as long as the target recombinant protein is sufficiently dissolved and the impurities are less dissolved. However, considering industrial production, it is 10 to 120 minutes. It is preferably 10 to 60 minutes, more preferably 10 to 30 minutes.
  • the target recombinant protein is a structural protein such as spider silk, silkworm silk, keratin, collagen, elastin, and resilin
  • the following conditions can be exemplified.
  • the amount of the first solvent added to the recombinant cells expressing the structural protein is the ratio of the volume (mL) of the first solvent to the dry weight (g) of the recombinant cells (volume (mL) / dry weight).
  • (G) is preferably 5 to 100 times, more preferably 10 to 50 times, and even more preferably 20 to 35.
  • the inorganic salt added to the first solvent is selected from the group consisting of lithium chloride and calcium chloride.
  • One or more inorganic salts are preferable, and lithium chloride is more preferable, and the concentration of the inorganic salt in the first solvent is preferably 0 to 1.0 mol / L with respect to the first solvent. 0.6 mol / L is more preferable, and 0 to 0.5 mol / L is more preferable.
  • the target recombinant protein is a protein comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m as shown in SEQ ID NOs: 1 to 5 (wherein in Formula 1)
  • the n motif represents an amino acid sequence composed of 4 to 20 amino acid residues
  • (A) the number of alanine residues relative to the total number of amino acid residues in the n motif is 80% or more.
  • m represents an integer of 8 to 300
  • a plurality of (A) n motifs may be the same or different amino acid sequences.
  • the REP to be may be the same amino acid sequence or different amino acid sequences.), For example, the following conditions can be mentioned.
  • the amount of the first solvent added to the recombinant cells expressing the target recombinant protein is the ratio of the volume (mL) of the first solvent to the dry weight (g) of the recombinant cells (volume (mL). / Dry weight (g)) is preferably 5 to 100 times, more preferably 10 to 50 times, and even more preferably 20 to 35 times.
  • the inorganic salt added to the first solvent one or more inorganic salts selected from the group consisting of lithium chloride and calcium chloride are preferable, and lithium chloride is more preferable.
  • the concentration of the inorganic salt in the first solvent is preferably 0 to 1.0 mol / L, more preferably 0 to 0.6 mol / L, and more preferably 0 to 0.5 mol / L with respect to the first solvent. Further preferred.
  • the target recombinant protein can be dissolved by treatment at a temperature of 30 to 100 ° C. and 40 to 60 ° C. using the first aprotic polar solvent.
  • the upper limit value of the temperature for dissolving may be 100 ° C., 90 ° C., 80 ° C. or 70 ° C.
  • the lower limit value of the temperature for dissolving may be 30 ° C., 40 ° C., or 50 ° C.
  • the dissolution time is, for example, preferably 10 to 120 minutes, more preferably 10 to 60 minutes, and further preferably 10 to 30 minutes.
  • the first reaction mixture obtained by step (A) contains a first soluble fraction and a first insoluble fraction.
  • the first soluble fraction contains the target recombinant protein, and may contain impurities derived from host cells in some cases.
  • the first insoluble fraction contains contaminants derived from host cells.
  • the first reaction mixture is donated to step (B) without fractionation. Further, if necessary, the fraction may be fractionated by, for example, filtration to remove the first insoluble fraction, and then provided to step (B).
  • step B the first reaction mixture obtained in step (A) is treated with a second solvent containing a second aprotic polar solvent and / or a chelating agent, and the second soluble fraction and the second A second reaction mixture containing the insoluble fraction.
  • step (B) after the step (A), the obtained first reaction mixture is treated with a second solvent, and the resulting insoluble fraction (second insoluble fraction) is removed. By removing this insoluble fraction, the recombinant protein is obtained as a soluble fraction (second soluble fraction).
  • a method for removing the insoluble fraction include general methods such as centrifugation, filter filtration using a drum filter, a press filter, and the like. In the case of filter filtration, a soluble fraction containing the target recombinant protein can be recovered more efficiently by using a filter aid such as celite and diatomaceous earth and a precoat agent in combination.
  • impurities can be removed by separating a soluble fraction and an insoluble fraction by a simple method without adding an aqueous solvent such as water. That is, in this method, since it is not necessary to add an aqueous solvent, the used first aprotic polar solvent can be purified and reused in the step (A), which is economical. .
  • the second solvent includes a second aprotic polar solvent and / or a chelating agent.
  • the second aprotic polar solvent is a different type of solvent from the first aprotic polar solvent, and is a solvent (poor solvent) that makes it difficult to dissolve impurities dissolved in the first solvent. Is preferred.
  • Examples of the second aprotic polar solvent include ketones and nitrites described later, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and 1,3-dimethyl-2-imidazolidone (DMI).
  • DMSO dimethyl sulfoxide
  • NMP N-methyl-2-pyrrolidone
  • DMI 1,3-dimethyl-2-imidazolidone
  • N, N-dimethylacetamide (DMA) propylene carbonate, hexamethylphosphoramide, N-ethylpyrrolidone, nitrobenzene, furfural, ⁇ -butyrolactone, ethylene sulfite, sulfolane, and Although ethylene carbonate can be mentioned, it is not limited to these.
  • ketones include acetone, methyl ethyl ketone, methyl butyl ketone, and methyl isobutyl ketone.
  • the ketone is preferably at least one selected from the group consisting of acetone and methyl ethyl ketone, more preferably acetone.
  • Nitriles may be saturated or unsaturated, but are preferably saturated. Nitriles may have 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, and more preferably 2 to 4 carbon atoms. Specific examples of nitriles include acetonitrile, propionitrile, succinonitrile, butyronitrile, and isobutyronitrile.
  • the second solvent may contain a solvent other than the second aprotic polar solvent in addition to the second aprotic polar solvent.
  • the content of the second aprotic polar solvent in the second solvent may be, for example, 85 to 100% by volume, or 90 to 100% by volume with respect to the total amount of the second solvent.
  • the dipole moment of the second aprotic polar solvent may be 2.5D or more and may be smaller than the dipole moment of the first aprotic polar solvent.
  • Examples of the aprotic polar solvent having a dipole moment of 2.5D or more include acetone, acetonitrile, and the like.
  • the second solvent may contain only the second aprotic polar solvent, and may further contain other solvents depending on the type of the target recombinant protein.
  • the second solvent is preferably composed only of the second aprotic polar solvent, and is preferably acetone.
  • the chelating agent may be a polyamine from the viewpoint that the recombinant protein can be more easily produced.
  • a polyamine is a polymer having a plurality of amino groups.
  • the polyamine may be linear or branched.
  • the polyamine may contain a primary amino group, a secondary amino group, and a tertiary amino group.
  • the polyamine may have, for example, a structural unit (ethyleneimine skeleton) represented by the formula I: —C 2 H 4 —NH—. That is, the polyamine may be polyethyleneimine.
  • the weight average molecular weight of the polyamine may be, for example, 10,000 or more, 30000 or more, or 50000 or more.
  • the weight average molecular weight of the polyamine may be, for example, 100,000 or less and may be 80,000 or less.
  • the weight average molecular weight is measured from a gel permeation chromatograph (GPC) using a standard polystyrene calibration curve.
  • the content of the chelating agent in the second solvent may be, for example, 0.001 to 10% by mass and 0.005 to 5% by mass with respect to the total amount of the second solvent.
  • the second solvent may contain a chelating agent and water, may contain only a chelating agent and water, or may contain a chelating agent, water, and another solvent.
  • the treatment in the step (B) is performed, for example, by adding a second solvent to the first reaction mixture obtained in the step (A) and bringing the first reaction mixture and the second solvent into contact with each other.
  • a second solvent to the first reaction mixture obtained in the step (A) and bringing the first reaction mixture and the second solvent into contact with each other.
  • the first reaction mixture may be brought into contact with a mixture of two or more solvents in advance, and the two or more solvents are added separately. By doing so, it may be brought into contact with the first reaction mixture.
  • the target recombinant protein is a hydrophobic protein
  • filterability and ease of disposal of filtration residue are further improved.
  • the above hydrophobic protein refers to a protein having a hydropathic index (HI) of more than 0.
  • the amount of the solvent to be added is preferably set in a timely manner according to the target recombinant protein, and is set to a concentration at which the target recombinant protein does not precipitate. Any concentration may be used as long as the target recombinant protein does not precipitate, and the addition amount may be set appropriately according to the state of aggregation of the protein and the presence of contamination.
  • the volume ratio of the addition amount of the second solvent to the addition amount of the first solvent may be 0.2 to 0.5.
  • the ratio of the addition amount (mL) of the first solvent to the addition amount (g) of the second solvent may be 1 to 8, 1 to 5, 1 to 4, 2 May be from 3.5 to 2.5, may be from 2.5 to 3.5, and may be from 2.5 to 3.0.
  • the ratio (A 1 : A 2 ) between the addition amount (A 1 ) of the first solvent and the addition amount (A 2 ) of the second solvent is 4 (vol / vol): 1 (vol / vol) 3 (vol / vol): 1 (vol / vol), 2 (vol / vol): 1 (vol / vol), 1 (vol / vol): 1 ( vol / vol).
  • the ratio between the added amount of DMSO and the added amount of acetone is, for example, preferably 4 (vol / vol): 1 (vol / vol), more preferably 3 (vol / vol): 1 (vol / vol), and still more preferably 2 (vol / vol): 1 (vol / vol), and 1 (vol / vol): 1 (vol / vol) may be used.
  • the standing time for treating the first reaction mixture with the second solvent is not particularly limited as long as impurities are sufficiently precipitated and the target recombinant protein is less precipitated. In consideration of production, it is preferably 10 to 120 minutes, more preferably 10 to 75 minutes, further preferably 10 to 60 minutes, and may be 10 to 30 minutes.
  • the second reaction mixture obtained by step (B) includes a second soluble fraction and a second insoluble fraction.
  • the second soluble fraction contains the desired recombinant protein and does not contain host cell contaminants or is less than the amount contained in the first reaction mixture. Yes.
  • the second insoluble fraction contains contaminants derived from the host cells.
  • the second reaction mixture preferably has improved filterability than the first reaction mixture, and is easily fractionated by filtration.
  • Step (C) is a step of removing the second insoluble fraction from the second reaction mixture obtained in step (B) to obtain the recombinant protein as a second soluble fraction.
  • Examples of a method for removing the second insoluble fraction include general methods such as centrifugation, filter filtration using a drum filter, a press filter, and the like. .
  • filter filtration the second soluble fraction containing the target recombinant protein can be recovered more efficiently by using a filter aid such as celite and diatomaceous earth and a precoat agent in combination.
  • the recombinant protein obtained as the second soluble fraction in the step (C) has a high purity of the recombinant protein, and can be used as it is for the production of, for example, spinning and film formation without further purification steps. Can do.
  • the method including the steps (A), (B), and (C), it is possible to separate a soluble fraction and an insoluble fraction by a simple method without adding an aqueous solvent such as water. Can be removed. That is, in this method, since it is not necessary to add an aqueous solvent, the used first aprotic polar solvent can be purified and reused in the step (A), which is economical. Thus, filtration can be performed efficiently, and disposal of the filtration residue is facilitated. Furthermore, this method does not require a step of adding an acid. Therefore, the recombinant protein to be purified is not limited to a protein resistant to acids.
  • Step (D) is a step of treating the second soluble fraction obtained in (C) with a poor solvent for the recombinant protein to aggregate the recombinant protein and obtain the recombinant protein as an aggregate.
  • the poor solvent for the recombinant protein is preferably a solvent that makes the target recombinant protein difficult to dissolve in the second solvent.
  • the poor solvent for the recombinant protein include aprotic polar solvents, protic polar solvents, esters, ethers, and aromatic hydrocarbons. Specific examples of these solvents are as described above.
  • the amount of the poor solvent added to the recombinant protein may be appropriately determined according to the recombinant protein so that the recombinant protein precipitates.
  • the poor solvent of the recombinant protein is usually added in the same amount as the soluble fraction.
  • the addition amount may be appropriately adjusted according to the state of aggregation of the recombinant protein and the presence of contamination.
  • Examples of a method for recovering the aggregated target recombinant protein as an aggregate include general methods such as centrifugation, filter filtration using a drum filter, a press filter, and the like.
  • the target recombinant protein can be more efficiently recovered as an aggregate by using a filter aid such as celite and diatomaceous earth and a precoat agent in combination.
  • the target recombinant protein can be purified without adding an aqueous solvent such as water. Therefore, after recovering the recombinant protein as an aggregate, it is easier and economical to purify and reuse the second solvent used and the poor solvent of the target recombinant protein.
  • the target recombinant protein is a structural protein produced for the purpose of producing a polypeptide fiber, for example, a protein derived from spider silk, silkworm silk, keratin, etc.
  • the second soluble protein obtained in step (C) The fraction can be used as such for the production of polypeptide fibers.
  • using as it is means that no further purification step is required. This indicates that the purity of the recombinant protein contained in the second soluble fraction obtained in step (C) is sufficiently high to be used for spinning.
  • an artificial polypeptide fiber can be produced by wet spinning.
  • the method for producing an artificial polypeptide fiber includes the following steps (A), (B), (C) and (E): (A) Recombinant protein expressed in cells with the target recombinant protein insoluble in a first solvent containing a first aprotic polar solvent with or without addition of an inorganic salt A first reaction mixture comprising a first soluble fraction and a first insoluble fraction, under a condition in which is dissolved.
  • Step (E) is a step of obtaining an undrawn yarn by applying the second soluble fraction as a filamentous liquid to the coagulation liquid and coagulating and collecting the recombinant protein in a filamentous form.
  • the recombinant protein coagulates.
  • the second soluble fraction obtained in step (C) is applied to the coagulation solution.
  • the recombinant protein coagulates.
  • the second soluble fraction as a thread-like liquid to the coagulation liquid
  • the recombinant protein coagulates in the form of a thread and a thread (unstretched thread) can be formed.
  • the undrawn yarn can be formed in accordance with, for example, a method described in Japanese Patent No. 5585932.
  • the second soluble fraction obtained in the step (C) does not require a further purification step, and the step (E) is used as a so-called dope solution.
  • the viscosity suitable for spinning is generally 10 to 50,000 cP (centipoise), and the viscosity can be measured using, for example, a trade name “EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd. If the viscosity of the second soluble fraction obtained in step (C) is not within the range of 10 to 50,000 cP (centipoise), the viscosity of the second soluble fraction is adjusted to the viscosity that can be spun. Also good.
  • the aprotic polar solvent illustrated as a suitable solvent in description of a process (A) can be used for adjustment of a viscosity.
  • the aprotic polar solvent may contain a suitable inorganic salt exemplified in the description of the step (A).
  • the concentration of the inorganic salt added to the aprotic polar solvent when adjusting the viscosity may be higher than that for the purpose of purification.
  • an inorganic salt may be added so as to be 0.5 to 2.0 M with respect to the dope solution.
  • the second soluble fraction can actually be used as it is for spinning in the step (E) as a dope solution can be confirmed, for example, by the following simple method. That is, an appropriate amount of the second soluble fraction is put into a syringe having a needle having an inner diameter of 0.1 mm, and the tip of the needle is put into methanol (corresponding to a coagulation solution) in a 300 mL beaker and pushed out. When the recombinant protein coagulates as a thread in methanol and can be removed from the methanol as a thread with tweezers, the soluble fraction can be used for spinning as a dope solution. When the yarn is not formed, it can be used as a dope solution by reviewing the viscosity and inorganic salt concentration of the soluble fraction and finely adjusting them.
  • the polypeptide solution (dope solution) prepared in (1) above is heated to 100 ° C or higher. By the heating, dehydration condensation occurs between the polypeptides to polymerize. By using a known dehydration condensation catalyst in combination, the polymerization efficiency can be dramatically increased.
  • the dope solution subjected to the polymerization reaction can be diluted by adding ethanol (ethyl alcohol), methanol (methyl alcohol), water, or the like, if necessary, and used for producing wet spinning.
  • the soluble fraction (dope) after heating is applied to the coagulation liquid as a thread-like fluid.
  • the coagulation liquid used for wet spinning may be any solution as long as it can be desolvated.
  • As the coagulation liquid for removing the solvent and forming fibers it is preferable to use a lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol, 2-propanol or acetone. Water may be appropriately added to the coagulation liquid.
  • the temperature of the coagulation liquid is preferably 5 to 30 ° C. from the viewpoint of spinning stability.
  • the method for applying the second soluble fraction (dope solution) as a thread-like fluid is not particularly limited, and examples thereof include a method of extruding from a spinneret into a coagulating liquid in a desolvation tank. An undrawn yarn is obtained by the solidification of the recombinant protein.
  • the extrusion speed in the case of extruding the second soluble fraction (dope solution) into the coagulating liquid can be appropriately set according to the diameter of the die, the viscosity of the soluble fraction, and the like.
  • the extrusion speed is preferably 0.2 to 6.0 mL / h per hole, more preferably 1.4 to 4.0 mL / h per hole. preferable.
  • the length of the solvent removal tank (coagulation liquid tank) into which the coagulation liquid is put is not particularly limited, but the length may be, for example, 200 to 500 mm.
  • the take-up speed of the undrawn yarn formed by coagulation of the recombinant protein may be 1 to 14 m / min, for example, and the residence time may be 0.01 to 0.15 min, for example.
  • the take-up speed of the undrawn yarn is preferably 1 to 3 m / min from the viewpoint of solvent removal efficiency.
  • the unstretched yarn formed by coagulation of the recombinant protein may be further stretched (pre-stretched) in the coagulation liquid, but the coagulation liquid is kept at a low temperature from the viewpoint of suppressing evaporation of the lower alcohol used in the coagulation liquid. It is preferable to take it off from the coagulation liquid in an undrawn yarn state.
  • the method for producing an artificial polypeptide fiber according to the present embodiment can also include a step of further stretching the unstretched yarn obtained in the step (E).
  • the stretching may be single-stage stretching or multi-stage stretching of two or more stages. When drawn in multiple stages, the molecules can be oriented in multiple stages and the total draw ratio can be increased, which is suitable for producing fibers with high toughness.
  • the target recombinant protein is a structural protein produced for the purpose of producing a polypeptide film, for example, a protein derived from spider silk, silkworm silk, keratin, etc.
  • the second soluble protein obtained in step (C) The fraction can be used as such for the production of polypeptide films.
  • using as it is means that no further purification step is required. This indicates that the purity of the recombinant protein contained in the soluble fraction obtained in step (C) is sufficiently high for use in forming a polypeptide film.
  • a polypeptide film can be produced by performing a film formation step after steps (A) to (C).
  • the method for producing a polypeptide film includes the following steps (A) to (C) and (F): (A) Recombinant protein expressed in cells with the target recombinant protein insoluble in a first solvent containing a first aprotic polar solvent with or without addition of an inorganic salt A first reaction mixture containing a first soluble fraction and a first insoluble fraction by treatment under a condition in which (B) treating the first reaction mixture with a second solvent containing a second aprotic polar solvent and / or a chelating agent to provide a second soluble fraction and a second insoluble fraction; Obtaining a reaction mixture of (C) removing the second insoluble fraction from the second reaction mixture to obtain the recombinant protein as the second soluble fraction; (F) On the flat plate resistant to the solvent of the second soluble fraction obtained in (C), a coating film is formed using the second soluble fraction, and the second soluble fraction is formed from the coating film.
  • A Recombinant protein expressed in cells with the target
  • step (F) a coating film is formed on the flat plate resistant to the solvent of the second soluble fraction using the second soluble fraction, and the solvent of the second soluble fraction is removed from the coating film. In this step, a polypeptide film is obtained.
  • the flat plate forming the coating film a flat plate resistant to the solvent of the second soluble fraction, such as a glass plate, is used.
  • the thickness of the coating film is not particularly limited, but may be, for example, 1 to 1000 ⁇ m.
  • Examples of a method for forming a polypeptide film include a casting method.
  • a cast film is formed by casting the second soluble fraction on a flat plate to a thickness of several microns or more using a jig such as a doctor coat or knife coater, Thereafter, the polypeptide film can be obtained by removing the solvent by drying under reduced pressure or immersion in a solvent removal tank.
  • SEQ ID NO: 1 SEQ ID NO: 1 (PRT918), SEQ ID NO: 2 (PRT410), SEQ ID NO: 3 (PRT587), SEQ ID NO: 4 (PRT799), or SEQ ID NO: 5
  • PRT928 SEQ ID NO: 1
  • SEQ ID NO: 2 SEQ ID NO: 4
  • SEQ ID NO: 5 A nucleic acid encoding fibroin having a sequence derived from spider silk having the amino acid sequence shown by PRT468) was synthesized. The nucleic acid was added with an NdeI site at the 5 ′ end and an EcoRI site downstream of the stop codon. The hydropathy index and molecular weight (kDa) of each protein are as shown in Table 2.
  • the nucleic acids encoding these five types of target proteins were each cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • Escherichia coli BLR (DE3) was transformed with pET-22b (+) expression vectors recombined with the four types of nucleic acids, respectively, to obtain transformed Escherichia coli (recombinant cells) expressing the target protein. .
  • the transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium (Table 3) containing ampicillin so that the OD 600 was 0.005.
  • the culture temperature was kept at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours) to obtain a seed culture.
  • the seed culture solution was added to a jar fermenter to which 500 mL of production medium (Table 4) was added so that the OD 600 was 0.05.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • a feed solution (glucose 455 g / 1 L, yeast extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the culture solution temperature 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 cultured for 20 hours. Thereafter, 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target protein.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • SDS-PAGE is performed using cells prepared from the culture solution before and after IPTG addition, and the appearance of the target protein size band depending on the addition of IPTG, the target protein is expressed as an insoluble substance. Confirmed that. The collected cells were dried to obtain dry cells in which the target protein was expressed as an insoluble material.
  • the ratio of the addition amount of DMSO and the addition amount of acetone is 5 (vol / vol): 1 (vol / vol), 5 (vol / vol): 2 (vol / vol. vol) or 5 (vol / vol): 3 (vol / vol).
  • the extract (soluble fraction) was added to an equal volume of ethanol (a poor solvent for the desired recombinant protein) to precipitate the hydrophobic protein (PRT918).
  • the fibroin precipitated was recovered by centrifugation (conditions: 20 ° C., 500 ⁇ g, 30 min.). The precipitated fibroin was washed 3 times with the same amount of RO water as the extract. After washing, the sample was lyophilized. After completion of lyophilization, the purified powder was recovered. The obtained powder was analyzed by SDS-PAGE. The results are shown in FIG.
  • the ratio of the addition amount of DMSO and the addition amount of acetone is 5 (vol / vol): 1 (vol / vol), 5 (vol / vol): 2 (vol / vol. vol) or 5 (vol / vol): 3 (vol / vol).
  • the extract (soluble fraction) was added to an equal amount of ethanol (a poor solvent for the desired recombinant protein) to precipitate the hydrophobic protein (PRT918).
  • the fibroin precipitated by centrifugation (conditions: 20 ° C., 500 ⁇ g, 30 min.) was collected.
  • the precipitated fibroin was washed 3 times with the same amount of RO water as the extract. After washing, the sample was lyophilized. After completion of lyophilization, the purified powder was recovered. The obtained powder was analyzed by SDS-PAGE. The results are shown in Table 5 and FIG.
  • the ratio of the DMSO addition amount and the acetone addition amount was 2 (vol / vol): 1 (vol / vol).
  • stirring was stopped and the mixture was allowed to stand at room temperature for 60 minutes (including stirring time).
  • a filter aid (equal to the amount of bacterial cells) was added with stirring.
  • the sample solution was put into a filter precoated with a filter aid and filtered.
  • the filterability was evaluated as “ ⁇ ” when filtration was impossible and “+” when filtration was possible.
  • Table 6 When DMSO containing 0.25MCaCl 2 (first protic polar solvent) was used, filterability was improved and residue treatment was easier.
  • the extract (soluble fraction) was added to an equal amount of ethanol (a poor solvent for the desired recombinant protein) to precipitate the hydrophobic protein (PRT918).
  • the fibroin precipitated by centrifugation (conditions: 20 ° C., 500 ⁇ g, 30 min.) was collected.
  • the precipitated fibroin was washed 3 times with the same amount of RO water as the extract. After washing, the sample was lyophilized. After completion of lyophilization, the purified powder was recovered.
  • the required amount of dry cells of E. coli expressing PRT918 was weighed into an Eppendorf tube, 500 ⁇ L of DMSO containing 0.5 M calcium chloride was added, and the mixture was heated at a temperature of 60 ° C. for 30 minutes to dissolve PRT918. After standing to cool to room temperature, 500 ⁇ L of RO water was added and stirred. PRT918 could not be centrifuged. If sedimentation by centrifugation does not occur under these conditions, it has been confirmed that filtration is not possible.
  • acetone second aprotic polar solvent
  • the ratio of the DMSO addition amount and the acetone addition amount was 2 (vol / vol): 1 (vol / vol).
  • stirring was stopped and the mixture was allowed to stand at room temperature for 60 minutes (including stirring time).
  • a filter aid (equal to the amount of bacterial cells) was added with stirring. After stirring for 5 minutes, the sample solution was put into a filter precoated with a filter aid and filtered.
  • the extract (soluble fraction) was added to an equal amount of ethanol (a poor solvent for the target recombinant protein) to precipitate each fibroin.
  • the fibroin precipitated was recovered by centrifugation (conditions: 20 ° C., 500 ⁇ g, 30 min.). The precipitated fibroin was washed 3 times with the same amount of RO water as the extract. After washing, the sample was lyophilized. After completion of lyophilization, the purified powder was recovered.
  • Table 7 shows the filterability relationship in comparison between the conventional method and this method, and the evaluation results of the filterability of each protein by this method.
  • the filterability was evaluated as “ ⁇ ” when filtration was impossible and “+” when filtration was possible.
  • the purified protein solution obtained by the present purification method has a higher purity of the target protein as it can be used as a dope solution for spinning without further purification steps. That is, it was found that the spinning dope solution can be prepared with very few steps as compared with the conventional preparation step of spinning dope solution using recombinant protein (aggregation, washing, drying, re-dissolution, etc.).
  • the polyethyleneimine aqueous solution was prepared by diluting a 30 mass% polyethyleneimine aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd., trade name: 30% polyethyleneimine P-70 solution) containing polyethyleneimine having a weight average molecular weight of 70,000 with water.
  • the mass ratio of water and PEI in the second solvent (the amount of water added (g): the amount of PEI added (g)) was 2.25: 0, 2.248: 0.002, 0.899: 0. 001 or 1.337: 0.014. After 5-10 minutes from the addition of the second solvent, stirring of the reaction mixture was stopped and allowed to stand at room temperature. The time from the addition of the second solvent to the end of standing was 30 minutes.
  • the desired recombinant protein PRT918 was produced under conditions (conditions 1 to 4) in which the type and addition amount of the second solvent were different.
  • the extract (soluble fraction) was added to an equal volume of ethanol (a poor solvent for the desired recombinant protein) to precipitate fibroin (PRT918).
  • the fibroin precipitated was recovered by centrifugation (conditions: 20 ° C., 11000 ⁇ g, 15 minutes). The precipitated fibroin was washed 3 times with the same amount of RO water as the extract. The sample after washing was freeze-dried. After completion of lyophilization, the purified powder was recovered. The obtained powder was analyzed by SDS-PAGE (FIG. 5).
  • the photograph in FIG. 5 is stained with an InVision (trademark) His-tagged in-gel staining reagent (manufactured by Thermo Fisher Scientific) that reacts with the His tag region of PRT918 after electrophoresis. PRT918 is detected as a band between 40-50 kDa molecular weight markers.
  • the analysis results of the recombinant proteins produced under Condition 4, Condition 3, Condition 2, and Condition 1 are shown in order from the right lane. The purity was calculated in Band% by using the ImageLab software (manufactured by BIO-RAD) and the emission intensity of 40 to 50 KDa and lower.
  • the purity was evaluated as “D” when less than 40%, “C” when 40% or more and less than 70%, “B” when 70% or more and less than 80%, and “A” when 80% or more.
  • D when less than 40%
  • C when 40% or more and less than 70%
  • B when 70% or more and less than 80%
  • A when 80% or more.

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Abstract

La présente invention concerne une méthode de production d'une protéine recombinante à l'aide d'une cellule recombinante dans laquelle la protéine recombinante souhaitée est exprimée sous la forme d'un matériau insoluble, la méthode étant caractérisée en ce que la cellule recombinante est traitée avec un premier solvant contenant un premier solvant polaire aprotique avec ou sans ajout d'un sel inorganique dans les conditions où la protéine recombinante peut être dissoute dans le solvant pour produire un mélange réactionnel, puis le mélange réactionnel est traité avec un second solvant contenant un second solvant polaire aprotique, puis une fraction insoluble est retirée du produit résultant pour produire une fraction soluble contenant la protéine recombinante.
PCT/JP2019/004736 2018-02-09 2019-02-08 Méthode de production de protéine recombinante Ceased WO2019156242A1 (fr)

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JP2022545183A (ja) * 2019-08-22 2022-10-26 ボルト スレッズ インコーポレイテッド スパイダーシルクタンパク質の改善した抽出方法

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JP2004315541A (ja) * 1995-06-07 2004-11-11 Chiron Corp タンパク質の可溶化、精製、および再生の方法
CN101748516A (zh) * 2008-12-15 2010-06-23 上海正家牛奶丝科技有限公司 一种蛋白质合成纤维制造过程中的溶剂除杂方法
JP2010183904A (ja) * 2009-01-15 2010-08-26 Preventec Inc 組換え植物で発現して難抽出化した組換えタンパク質の抽出・精製方法
WO2014103799A1 (fr) * 2012-12-26 2014-07-03 スパイバー株式会社 Film de protéine de soie d'araignée et son procédé de production
WO2016163337A1 (fr) * 2015-04-09 2016-10-13 Ｓｐｉｂｅｒ株式会社 Solution de solvant polaire et procédé de production associé
WO2018066558A1 (fr) * 2016-10-03 2018-04-12 Ｓｐｉｂｅｒ株式会社 Procédé de purification de protéine recombinée

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Publication number Priority date Publication date Assignee Title
JP2004315541A (ja) * 1995-06-07 2004-11-11 Chiron Corp タンパク質の可溶化、精製、および再生の方法
CN101748516A (zh) * 2008-12-15 2010-06-23 上海正家牛奶丝科技有限公司 一种蛋白质合成纤维制造过程中的溶剂除杂方法
JP2010183904A (ja) * 2009-01-15 2010-08-26 Preventec Inc 組換え植物で発現して難抽出化した組換えタンパク質の抽出・精製方法
WO2014103799A1 (fr) * 2012-12-26 2014-07-03 スパイバー株式会社 Film de protéine de soie d'araignée et son procédé de production
WO2016163337A1 (fr) * 2015-04-09 2016-10-13 Ｓｐｉｂｅｒ株式会社 Solution de solvant polaire et procédé de production associé
WO2018066558A1 (fr) * 2016-10-03 2018-04-12 Ｓｐｉｂｅｒ株式会社 Procédé de purification de protéine recombinée

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022545183A (ja) * 2019-08-22 2022-10-26 ボルト スレッズ インコーポレイテッド スパイダーシルクタンパク質の改善した抽出方法
JP7750520B2 (ja) 2019-08-22 2025-10-07 ボルト スレッズ インコーポレイテッド スパイダーシルクタンパク質の改善した抽出方法

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