WO2022191223A1 - Procédé d'extraction d'une protéine cible à partir de cellules procaryotes - Google Patents

Procédé d'extraction d'une protéine cible à partir de cellules procaryotes Download PDF

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WO2022191223A1
WO2022191223A1 PCT/JP2022/010198 JP2022010198W WO2022191223A1 WO 2022191223 A1 WO2022191223 A1 WO 2022191223A1 JP 2022010198 W JP2022010198 W JP 2022010198W WO 2022191223 A1 WO2022191223 A1 WO 2022191223A1
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target protein
acid
prokaryotic cells
culture
extracting
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PCT/JP2022/010198
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English (en)
Japanese (ja)
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将弘 荒武
裕幸 渡邉
輝明 武居
紀幸 伊藤
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株式会社カネカ
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Priority to JP2023505596A priority Critical patent/JPWO2022191223A1/ja
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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • This specification discloses a method for extracting a target protein from prokaryotic cells that express the target protein.
  • the cytoplasm of E. coli is a reducing environment, so disulfide bonds involved in protein folding are difficult to form, resulting in the formation of inclusion bodies and insolubilization. , the problem of obtaining an inactive protein may arise.
  • a method of secreting the expressed protein from the cytoplasm to the outside of the inner membrane and accumulating it in the periplasm is known.
  • the periplasm is the space between the inner and outer membranes of E. coli cells and is a chemically oxidizing environment that facilitates the formation of disulfide bonds and the resulting functionally correct folding of proteins (Non-Patent Document 1). .
  • a protein expressed in the cytoplasm of an E. coli host cell has a signal sequence that facilitates transport from the cytoplasm to the periplasm, the protein can be secreted into the periplasm.
  • Patent Documents 1 to 4 below are examples of documents that disclose methods for extracting heterologous proteins from recombinant microbial cells that express heterologous proteins.
  • Patent Document 1 discloses a method for extracting a soluble protein from a microbial population, comprising a microbial population that expresses the soluble protein and an effective amount for extracting the soluble protein from the microbial population, from 25% (v/v) to A method is described comprising contacting with a solution containing 40% (v/v) carboxylic acid. Patent Literature 1 describes bringing the solution into contact with wet cells obtained by collecting cultured cells.
  • Patent Document 2 describes a method for isolating periplasmic polypeptides from prokaryotic cells, in which prokaryotic cells are treated in a solution containing predetermined concentrations of Tris-HCl and EDTA at a pH value of about 7 to about 10, and about 15%. Methods are described that include incubating at about 25° C. for minutes to about 6 hours.
  • Patent Document 3 discloses a periplasmic protein extraction method comprising suspending a cell pellet obtained from a culture of a prokaryotic microorganism expressing a periplasmic protein in an arginine-containing buffer and recovering the protein in the supernatant. Have been described.
  • Patent Document 4 describes a periplasmic protein recovery method characterized by adding a macromolecular flocculant to a suspension of the Gram-negative bacteria when recovering periplasmic proteins from Gram-negative bacteria by the osmotic shock method. ing.
  • Patent Documents 2 to 4 As a method for extracting a target protein in the periplasm from prokaryotic cells containing the target protein in the periplasm, the methods described in Patent Documents 2 to 4 have been conventionally studied. However, many contaminants derived from the cytoplasm of the host prokaryotic cell were extracted together with the target protein, making subsequent purification of the target protein difficult.
  • Patent Document 1 which discloses a method for extracting soluble proteins from a microbial population, states that soluble proteins are proteins that are soluble in the cytoplasm or in the periplasm. No results are given. According to Patent Document 1, the present inventor contacted prokaryotic cells containing a target protein in the periplasm with a solution containing 25% (v/v) to 40% (v/v) carboxylic acid (acetic acid). , and found that the extracted target protein was contaminated with many contaminants, and that the target protein recovery rate was low.
  • a culture solution containing prokaryotic cells expressing a target protein is mixed with a carboxylic acid at a final concentration of 0.5% (v/v) or more and 15% (v/v) or less, and the mixture is a step of preparing, and A method for extracting a target protein from a prokaryotic cell, comprising extracting the target protein from the prokaryotic cell in a mixture.
  • the prokaryotic cell expressably retains a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of a fusion polypeptide between a signal peptide that promotes secretion into the periplasm and a target protein.
  • prokaryotic cell is a Gram-negative bacterial cell.
  • prokaryotic cell is an E. coli cell.
  • carboxylic acid is one or more selected from formic acid, acetic acid, propionic acid, malonic acid, citric acid and lactic acid.
  • target protein is a minibody.
  • a method of extracting a target protein from a prokaryotic cell comprising: wherein the method of (7) may have the additional features of any of (2)-(6).
  • This specification includes the disclosure of Japanese Patent Application No. 2021-038314, which is the basis of priority of this application.
  • the prokaryotic cell may be any prokaryotic cell that serves as a host for expressing the target protein, and is preferably a bacterium, particularly preferably a periplasmic bacterial cell such as a Gram-negative bacterium.
  • the periplasm refers to the space between the inner membrane (cytoplasmic membrane) surrounding the cytoplasm of prokaryotic cells and the outer membrane.
  • Preferred examples of Gram-negative bacterial cells having a periplasm include cells of bacteria belonging to the genus Escherichia, particularly preferably Escherichia coli cells.
  • a target protein refers to a protein to be produced by a prokaryotic cell.
  • Prokaryotic cells expressably retain nucleic acids comprising nucleotide sequences encoding target protein amino acid sequences.
  • Prokaryotic cells may express the target protein as a fusion polypeptide in which another polypeptide is linked to one or both of its N-terminal and C-terminal sides.
  • polypeptides include, but are not limited to, signal peptides, tag peptides, and the like.
  • Specific examples of signal peptides include signal peptides that promote secretion into the periplasm, which will be described later.
  • tag peptides include tag peptides (histidine tags) composed of a plurality of (eg, 6 to 10) histidine residues and FLAG tag peptides.
  • a prokaryotic cell preferably retains an expressible nucleic acid containing a nucleotide sequence encoding an amino acid sequence of a fusion polypeptide between a signal peptide that promotes secretion into the periplasm and a target protein.
  • the fusion polypeptide expressed in the cytoplasm is secreted into the periplasm through the inner membrane and can accumulate in the periplasm as a target protein detached from the signal peptide.
  • Non-Patent Document 1 As a signal peptide that promotes secretion into the periplasm, for example, the signal peptide described in Non-Patent Document 1 is known, and specific examples include the pelB signal peptide (SEQ ID NO: 5) and the heat-stable enterotoxin II (STII) signal peptide. (SEQ ID NO: 6), outer membrane protein A (OmpA) signal peptide (SEQ ID NO: 7), maltose outer membrane porin (LamB) signal peptide (SEQ ID NO: 8).
  • pelB signal peptide SEQ ID NO: 5
  • STII heat-stable enterotoxin II
  • the target protein is not particularly limited and can be selected according to the purpose.
  • the target protein is preferably a heterologous protein.
  • antibodies or low-molecular-weight antibodies, particularly low-molecular-weight antibodies, are preferred target proteins.
  • Antibody is a general name that focuses on the function of immunoglobulins. All immunoglobulins basically have the same molecular structure, and have a basic structure of a "Y"-shaped four-chain structure (two light and two heavy polypeptide chains).
  • the region corresponding to the lower half vertical bar of the antibody "Y” is called the Fc region, and the upper half “V” is called the Fab region.
  • the Fc region has an effector function that elicits a reaction after an antibody binds to an antigen, and the Fab region has a function of binding to an antigen.
  • the Fab region and Fc region of the heavy chain are connected by a hinge region, and the proteolytic enzyme papain contained in papaya degrades this hinge region to cleave it into two Fab regions (fragments) and one Fc region.
  • a portion (domain) near the tip of the "Y” in the Fab region is called a variable region (V region) because various changes are observed in the amino acid sequence so that it can bind to various antigens.
  • the light chain variable region is called the VL region
  • the heavy chain variable region is called the VH region
  • the Fab region and the Fc region other than the V region are regions that undergo relatively little change and are called constant regions (C regions).
  • the constant region of the light chain is called the CL region
  • the constant region of the heavy chain is called the CH region.
  • the CH region is further divided into three regions CH1 to CH3.
  • the Fab region of the heavy chain consists of the VH region and CH1, and the Fc region of the heavy chain consists of CH2 and CH3.
  • the hinge portion is located between CH1 and CH2.
  • heavy-chain antibodies which are antibodies composed only of heavy chains without light chains.
  • Camelid-derived heavy-chain antibodies are distinguished from normal IgG antibodies (IgG1), which have light chains, and are termed IgG2, IgG3.
  • fish-derived heavy chain antibodies are called IgNAR (new antigen receptor).
  • minibody is an antibody fragment lacking a portion of a full-length antibody (whole antibody, such as whole IgG), and is not particularly limited as long as it has the ability to bind to an antigen. .
  • minibodies preferably do not have CH2 and CH3 domains.
  • minibodies preferably contain either or both of a heavy chain variable region (VH) and a light chain variable region (VL).
  • VH or VL amino acid sequence can contain additions, deletions and/or substitutions.
  • VH or VL, or a portion of both can be deleted as long as they bind to the antigen.
  • the low-molecular-weight antibody is not particularly limited and can be appropriately selected according to the purpose.
  • -NAR single chain antibody
  • Fab single chain antibody
  • Fab' single chain antibody
  • F(ab') 2 single chain antibody
  • single chain antibody: scFv single chain antibody
  • dibody tribody
  • minibody minibody
  • VHH camelid-derived heavy chain antibody variable regions
  • scFv single chain antibodies
  • the low-molecular-weight antibody may be obtained by binding two or more antibody fragments, and the low-molecular-weight antibody obtained by binding two or more antibody fragments may be obtained by binding antibody variable regions with a linker such as a peptide linker.
  • a linker such as a peptide linker.
  • a prokaryotic cell may retain a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of the target protein (or the fusion protein) in an expression vector or as part of genomic DNA. may be
  • a plasmid vector or the like containing the nucleic acid can be used as an expression vector.
  • Expression vectors are preferably capable of autonomous replication in prokaryotic cells.
  • the expression vector preferably contains a DNA containing a nucleotide sequence encoding the amino acid sequence of the target protein (or the fusion protein) and a promoter operably linked to a position capable of transcribing the DNA.
  • the expression vector is preferably capable of autonomous replication in prokaryotic cells, and is composed of a promoter, a ribosome binding sequence, a base sequence encoding the amino acid sequence of the target protein (or the fusion protein), and a transcription termination sequence. It is a recombinant DNA containing the base sequence of
  • Suitable plasmid vectors include pET-28b (available from Merck), pQEK1, pCA24N (DNA RESEARCH, 12, 191-299 (2005)), pACYC177, pACYC184 (available from Nippon Gene), pQE30, pQE60.
  • pQE70, pQE80 and pQE9 available from QIAGEN
  • pTipQC1 available from QIAGEN or Hokkaido System Science
  • pTipRT2 available from Hokkaido System Science
  • pBS vector Phagescript vector, Bluescript vector, pNH8A, pNH16A, pNH18A and pNH46A (available from Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540 and pRIT5 (available from Addgene); pRSF (available from MERCK); and pAC (available from Nippon Gene).
  • pUCN18 can be prepared by modifying pUC18 (available from Takara Bio Inc.)), pSTV28 (available from Takara Bio Inc.), pUCNT (International Publication No. 94/03613), and the like.
  • an inducible promoter may be used as the promoter contained in the expression vector.
  • the promoter may be operably linked to an operator sequence to form an inducible promoter.
  • inducible promoters include isopropyl- ⁇ -thiogalactopyranoside (IPTG)-inducible promoter, light-inducible promoter that induces gene expression under light irradiation, araBAD promoter (arabinose-inducible), rhaBAD promoter (rhamnose-inducible). ), tet promoter (drug-inducible), penP promoter (drug-inducible), cspA promoter (temperature-inducible promoter that responds to low temperature), promoters containing tetO or lacO operators as operator sequences, etc.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • araBAD promoter aromaticnose-inducible
  • rhaBAD promoter rhamnose-inducible
  • tet promoter drug-inducible
  • penP promoter drug-inducible
  • IPTG-inducible A promoter can be exemplified, and IPTG-inducible A promoter, araBAD promoter, rhaBAD promoter, tet promoter, penP promoter, cspA promoter, or a promoter containing the tetO or lacO operator as operator sequence is preferred.
  • the copy number of the expression vector in the cell is preferably 2 or more, more preferably 3 or more, more preferably 5 or more, more preferably 10 or more, More preferably 15 or more, more preferably 20 or more.
  • the method of introducing the expression vector into prokaryotic cells is not particularly limited. It can be carried out.
  • Homologous recombination can be used in a mode in which a prokaryotic cell retains, as part of its genomic DNA, a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of the target protein (or the fusion protein).
  • Prokaryotic cells that express the target protein can be cultured in an appropriate medium.
  • the medium may be either a synthetic medium or a natural medium, as long as it contains nutrients such as carbon sources, nitrogen sources, inorganic salts, vitamins, yeast extract, etc. necessary for growth of prokaryotic cells and expression of target proteins.
  • the carbon source may be any carbon source that can be assimilated by the prokaryotic cells, including carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid. can be done.
  • nitrogen sources include ammonia, ammonium salts such as ammonium chloride and ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone.
  • inorganic salts include trisodium phosphate, sodium monohydrogen phosphate, magnesium sulfate, iron (II) sulfate, manganese (II) chloride, sodium chloride, and potassium carbonate.
  • vitamins examples include biotin and thiamine.
  • substances required for the growth of the prokaryotic cells for example, required amino acids in the case of amino acid-requiring strains) can be added.
  • a medium containing glucose, yeast extract, trisodium phosphate, sodium monohydrogen phosphate, ammonium chloride, magnesium sulfate, iron (II) sulfate, and manganese (II) chloride is preferably used for culturing the prokaryotic cells.
  • the pH of the medium is preferably adjusted to 6-8.
  • the culture conditions for culturing the prokaryotic cells are not particularly limited, but preferable examples include shaking culture and agitation culture. In addition, it is preferable to culture while aerating with air.
  • the culture temperature is 20-50°C, preferably 25-40°C, more preferably 25-35°C.
  • the culture time is 3 hours to 5 days, preferably 5 hours to 4 days.
  • a method disclosed herein for extracting a target protein from a prokaryotic cell expressing the target protein comprises: A step of mixing a carboxylic acid at a final concentration of 0.5% (v/v) or more and 15% (v/v) or less with a culture medium containing prokaryotic cells expressing the target protein to prepare a mixed solution. ,as well as, The method is characterized by including a step of extracting the target protein from the prokaryotic cell in the mixture.
  • contamination of the target protein to be extracted can be suppressed, and the recovery rate of the target protein can be increased.
  • This method is particularly useful for extracting target proteins present in the periplasm.
  • the mixed concentration of carboxylic acid is less than 0.5% (v / v), and if it exceeds 15% (v / v), the recovery rate of the target protein is reduced, or the target protein extracted There is a tendency that the amount of contaminants mixed in is increased.
  • the mixed concentration of carboxylic acid is more preferably 3% (v/v) or more and 10% (v/v) or less.
  • the mixed concentration of carboxylic acid in the culture medium containing prokaryotic cells expressing the target protein is the ratio of the volume of the mixed carboxylic acid to the total volume of the culture medium after mixing the total amount of carboxylic acid (i.e., final concentration).
  • the volume of carboxylic acid refers to the volume of the carboxylic acid that is liquid at normal temperature and pressure when using a carboxylic acid such as formic acid, acetic acid, propionic acid, and lactic acid that is liquid at normal temperature and pressure.
  • a carboxylic acid that is solid at normal temperature and normal pressure, such as citric acid it refers to the volume of a saturated aqueous solution of the carboxylic acid at 25°C.
  • the mixed concentration of the carboxylic acids in the culture solution containing the prokaryotic cells expressing the target protein refers to the total mixed concentration of each of the multiple carboxylic acids.
  • the mixed concentration of the carboxylic acid in the culture medium containing prokaryotic cells expressing the target protein is preferably 0.5% (v/v) or more, more preferably 1% (v/v) or more, more preferably 3%. (v/v) or more, particularly preferably 5% (v/v) or more.
  • the mixed concentration of the carboxylic acid is within this range, contamination of the target protein with contaminants can be suppressed particularly effectively, and the recovery rate of the target protein can be further increased.
  • the mixed concentration of carboxylic acid is particularly preferably 1% (v/v) to 15% (v/v), more preferably 3% (v/v) to 15% (v/v), more preferably 5%. (v/v) to 15% (v/v), more preferably 5% (v/v) to 10% (v/v).
  • carboxylic acid is not particularly limited.
  • a carboxylic acid may be a hydroxy acid.
  • preferred carboxylic acids include one or more selected from formic acid, acetic acid, propionic acid, malonic acid, citric acid and lactic acid. More preferably, it is one or more selected from formic acid, acetic acid and propionic acid, and more preferably one or more carboxylic acid selected from formic acid and acetic acid.
  • a “culture medium containing prokaryotic cells expressing a target protein” means a prokaryotic It is a suspension in which biological cells are suspended.
  • a culture solution is preferably a culture solution obtained by culturing prokaryotic cells that express the target protein in the medium described above.
  • the target protein expressed in prokaryotic cells is extracted into the liquid fraction by mixing a predetermined amount of carboxylic acid in the culture solution before separating the cultured prokaryotic cells from the medium. can do.
  • the culture medium is preferably a suspension containing 50 to 200 g/L wet weight of prokaryotic cells.
  • the target protein is extracted from the prokaryotic cells in a mixture formed by mixing the aforementioned amount of carboxylic acid with the culture medium containing the prokaryotic cells expressing the target protein.
  • the extraction time is not particularly limited, it is preferably 0.5 hours or longer, more preferably 1 hour or longer, and preferably 10 hours or shorter, more preferably 8 hours or shorter, and more preferably 5 hours or shorter.
  • the extraction temperature is not particularly limited, it is preferably in the range of 15°C to 30°C, more preferably in the range of 20°C to 30°C.
  • the mixed liquid may be left still, or the mixed liquid may be temporarily or continuously stirred.
  • cells After extraction of the target protein, cells can be removed by suitable solid-liquid separation means such as centrifugation and filtration, and the liquid fraction in which the target protein has been eluted can be recovered.
  • suitable solid-liquid separation means such as centrifugation and filtration
  • the target protein can be further purified and recovered from the liquid fraction.
  • the plasmid used for E. coli transformation is the constructed vector. It was prepared by introducing into E. coli DH5 ⁇ competent cells (manufactured by Takara Bio Inc.) and culturing and amplifying the resulting transformant. A plasmid was prepared from the plasmid-carrying strain using a QIAprep spin miniprep kit (manufactured by QIAGEN).
  • An E. coli strain was prepared that expresses anti-Fc VHH and Caplacizumab (tandem VHH), which are minibodies, as target proteins.
  • the amino acid sequence of anti-Fc VHH is described in SEQ ID NO: 163 of EP2170960B1, and the amino acid sequence of Caplacizumab (tandem VHH) is described in SEQ ID NO: 1 of WO2009/115614A2.
  • the gene encoding anti-Fc VHH with the pelB signal peptide (SEQ ID NO: 5) added upstream and the gene encoding Caplacizumab with the STII signal peptide (SEQ ID NO: 6) added upstream of synthetic DNA was prepared and utilized in vector construction. Both the pelB signal peptide and the STII signal peptide are signal peptides that facilitate transport of expressed proteins to the periplasm.
  • Prime STAR Max DNA Polymerase manufactured by Takara Bio Inc.
  • the reaction conditions were performed according to the method described in the attached manual.
  • Example 1 Target protein expression vector construction
  • a nucleic acid fragment with NdeI restriction enzyme sites added upstream of each synthetic gene and a Bpu1102I restriction enzyme site downstream thereof was used as a template for the pelB signal peptide-added anti-Fc VHH synthetic DNA, primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2). ), and PCR using the STII signal peptide-added Caplacizumab synthetic DNA as a template and primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4), respectively.
  • -28b manufactured by Merck
  • Example 2 Acquisition of transformed E. coli] 0.1 ⁇ L of each of the prepared expression vectors and 1 ⁇ L of E. coli competent cell line BL21 (DE3) were mixed on ice and allowed to stand for 30 minutes. Warmed to 42° C. for 45 seconds and chilled on ice. After cooling, SOC medium (20 g/L Bacto tryptone (Becton Dickinson and Company (BD)), 5 g/L Bacto yeast extract (BD), 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium sulfate, 10 mM chloride 100 ⁇ L of magnesium, 20 mM glucose) was added, and recovery culture was performed at 37° C. for 1 hour.
  • SOC medium (20 g/L Bacto tryptone (Becton Dickinson and Company (BD)
  • BD Bacto yeast extract
  • 10 mM sodium chloride 2.5 mM potassium chloride
  • 10 mM magnesium sulfate 10 mM chloride 100 ⁇ L of magnesium, 20
  • LBK selection agar plate (10 g / L polypeptone (BD), 5 g / L bacto yeast extract (BD), 10 g / L sodium chloride, 50 ⁇ g / L kanamycin, 15 g / L agarose), 37 ° C., 16 hours A strain that grows in static culture was selected, and anti-Fc VHH-expressing E. coli and Caplacizumab-expressing E. coli were obtained.
  • Experiment 3 Culture of transformed E. coli
  • TB medium 24 g / L bacto yeast extract (BD), 12 g / L bacto tryptone (BD), 10 g in a 500 mL Sakaguchi flask.
  • the culture apparatus used was Marubishi Bioengineering Co., Ltd.'s Bioneer-5L, the aerated air speed was 2.5 L/min, the agitation speed was 400 rpm, the culture temperature was 30°C, and the pH was 6.9 to 7. .1. Also, the pH fluctuation during the culture was controlled within the above range by adding 12.5% aqueous ammonia or 4N sulfuric acid.
  • the pump was started to supply a mixture of 423 g/L glucose and 13 g/L magnesium sulfate heptahydrate.
  • a Peristaltic Biominipump manufactured by ATTO Corporation was used for the supply, and the flow rate of the pump was adjusted so that the dissolved oxygen concentration was maintained at 40% saturation.
  • the start of feeding was 16 hours after the start of culture.
  • the culture temperature was changed from 30°C to 25°C. After the culture temperature is lowered, IPTG (isopropyl- ⁇ -thiogalactopyranoside) is added to the culture solution to a final concentration of 0.8 mM, thereby expressing low-molecular-weight antibodies (anti-Fc VHH or Caplacizumab). induced.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the turbidities of the anti-Fc VHH-expressing E. coli and Caplacizumab E. coli cultures were 65.9 and 66.2, respectively.
  • the yield of wet cells was 110 g and 108 g per 1 L of culture solution.
  • the Escherichia coli culture solution (hereinafter sometimes referred to as "Escherichia coli culture solution") after culturing for 72 hours was sonicated to obtain a low-molecular-weight antibody extract.
  • Escherichia coli culture solution For ultrasonic crushing, an ultrasonic disperser UH-50 manufactured by SMT was used, and the treatment was performed on ice 60 times with intervals of 1 second at maximum output. The obtained solution was centrifuged (15,000 G, 5 minutes) to remove bacterial cell residues, and a low-molecular-weight antibody extract was obtained from the supernatant.
  • a low-molecular-weight antibody band was confirmed at the position of the molecular weight estimated from the amino acid sequence (anti-Fc VHH: 14 kDa, Caplacizumab: 28 kDa).
  • concentration of each low-molecular-weight antibody was calculated using the anti-Fc VHH purified preparation as a control.
  • the concentration of impurities was calculated from the difference obtained by subtracting the area value of the target low-molecular-weight antibody from the area value of the entire lane, using the purified anti-Fc VHH preparation as a control.
  • the impurity concentrations in the culture solutions of anti-Fc VHH-expressing E. coli and Caplacizumab-expressing E. coli were 15.89 g/L and 14.23 g/L, respectively.
  • the recovery rate and degree of impurity reduction in the extraction method of the target protein (anti-Fc VHH or Caplacizumab) described in Comparative Examples 1 to 5 and Example 1 below were calculated.
  • Target protein extraction was carried out by a heating method, as described in A. P. J. Middelberg Biotechnology Advances 13 (1995) 491-551.
  • the E. coli culture was heated for 1 hour in a water bath set at 55°C.
  • the E. coli culture solution after heating was cooled on ice, and the supernatant was recovered by centrifugation (15,000 G, 5 minutes).
  • Example 1 Extraction of target protein from Escherichia coli culture solution with carboxylic acid> Carboxylic acids (formic acid, acetic acid, propionic acid, malonic acid, citric acid, lactic acid) were added to each final concentration (v/v) to the E. coli culture solution, and allowed to stand at room temperature for 1 hour. The supernatant was recovered by centrifugation (15,000 G, 5 minutes). However, since malonic acid and citric acid are solids at normal temperature and normal pressure, the concentration was calculated with a saturated solution as 100%.
  • Carboxylic acids formic acid, acetic acid, propionic acid, malonic acid, citric acid, lactic acid

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Abstract

Le problème à résoudre par la présente invention est d'empêcher la contamination d'une protéine cible par des substances étrangères et d'augmenter le taux de récupération de la protéine cible, dans un procédé d'extraction d'une protéine cible à partir de cellules procaryotes exprimant la protéine cible.　La présente invention concerne un procédé d'extraction d'une protéine cible à partir de cellules procaryotes qui est caractérisé en ce qu'il comprend : une étape de mélange d'une solution de culture contenant des cellules procaryotes qui expriment la protéine cible avec un acide carboxylique à une concentration finale de 0,5 à 15 % (v/v) inclus pour préparer un liquide mélangé ; et une étape d'extraction de la protéine cible à partir des cellules procaryotes dans le liquide mélangé.
PCT/JP2022/010198 2021-03-10 2022-03-09 Procédé d'extraction d'une protéine cible à partir de cellules procaryotes WO2022191223A1 (fr)

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