WO2010001960A1 - Procédé de fabrication d'une protéine recombinante à l'aide de la bactérie brevibacillus obtenue par génie génétique - Google Patents

Procédé de fabrication d'une protéine recombinante à l'aide de la bactérie brevibacillus obtenue par génie génétique Download PDF

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WO2010001960A1
WO2010001960A1 PCT/JP2009/062121 JP2009062121W WO2010001960A1 WO 2010001960 A1 WO2010001960 A1 WO 2010001960A1 JP 2009062121 W JP2009062121 W JP 2009062121W WO 2010001960 A1 WO2010001960 A1 WO 2010001960A1
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protein
recombinant protein
brevibacillus
producing
fructose
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修 小田原
輝明 武居
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株式会社カネカ
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    • 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
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for producing a recombinant protein using a recombinant Brevibacillus bacterium.
  • Non-patent Document 1 Production of recombinant proteins using Brevibacillus bacteria as a host is used for the production of various heterologous proteins.
  • Various culture conditions have been studied to improve the secretory production of recombinant protein per cell and to increase the density of cells during culture.
  • glucose has been used as the main carbon source of the medium.
  • Sucrose, glycerin and the like are usually used.
  • fructose is used as a suitable carbon source for improving the secretory production amount and increasing the density of the cells, although it is the same monosaccharide as glucose.
  • Non-patent Document 2 This is considered to be caused by the fact that fructose is more rapidly colored in the presence of amino acids than glucose (Non-patent Document 2), so that the culture supernatant is easily colored.
  • the colored substance derived from the fructose binds to the expressed recombinant protein, and the protein to which the colored substance is bound is treated as an impurity derived from the target substance in the purification process. Need to be removed. The generation of these impurities is presumed to prevent the use of fructose as the main carbon source because it results in a significant reduction in the yield of the target product and is difficult to remove due to its approximate properties.
  • Antibody drugs are mainly produced using cultured CHO cells as glycoproteins of about 150 kDa.
  • affinity chromatography having antibody binding ability is generally used, and the most commonly used protein such as protein A, protein G and protein L is immobilized on an appropriate resin. Chromatography with a carrier. Among the proteins used as ligands for these purification carriers, protein A is particularly frequently used.
  • an affinity carrier having a protein as a ligand is required to have high quality as a material for producing pharmaceuticals.
  • Protein ligands themselves are also required to have the same level of quality as protein pharmaceuticals, and it is not possible to inexpensively supply affinity carriers using these as raw materials.
  • These affinity carriers account for a large proportion of the antibody drug production cost, which is a major impediment to reducing the cost of antibody drug products. Therefore, a method for procuring these ligand proteins with high quality at low cost has been desired.
  • Patent Document 1 In order to establish a technique for stably producing a large amount of a partial sequence of protein A so far, the present inventors have used a Brevibacillus bacterium as a host to efficiently produce a large amount of the partial sequence of protein A into a culture solution. Has been found to be secreted and stably accumulated, and can be easily separated and recovered with high purity (Patent Document 1).
  • feline proinsulin is one such bioactive protein. Purified feline proinsulin has not been put on the market so far, and the reliability of proinsulin measurement at the time of diagnosis of feline diabetes has been low, which has prevented early detection of feline diabetes.
  • other types of insulin are used as therapeutic agents for diabetes, and it has been desired to develop an industrial production method of cat proinsulin as a raw material for cat insulin.
  • Non-patent Document 3 a technique for expressing feline proinsulin in Escherichia coli has been constructed, and inclusion bodies are formed in Escherichia coli during the culture. Forming inclusion bodies during culture increases the load on the purification process, resulting in higher product costs. Therefore, realization of an economical production method of feline proinsulin in a secretory expression system is strongly desired.
  • the secretory production of the recombinant protein per cell is improved and the density of the cell at the time of cultivation is increased compared to the conventional techniques. It is an object of the present invention to provide new culture conditions for Brevibacillus bacteria that can be achieved and can be prepared at a lower cost and can be scaled up to an industrial scale.
  • the present inventors surprisingly cultivate using, as a carbon source, fructose that has been avoided so far due to the ease of browning.
  • the secretory production amount of the recombinant protein per microbial cell was improved, and the microbial cell during the cultivation was found to have a higher density, thereby completing the present invention.
  • the present invention provides a method for producing a recombinant protein, which comprises culturing using fructose as a carbon source when producing a recombinant protein using a recombinant Brevibacillus bacterium. It is.
  • the secretory production amount of the recombinant protein per cell is improved, and the cell density during culture is increased, It is possible to achieve the productivity of recombinant protein more than 3 times the conventional.
  • FIG. 7 shows a DNA sequence encoding a promoter sequence, Shine-Dalgarno sequence, signal peptide and protein A (SPA ′) of a partial sequence expression vector (Spa′-pNK3260) of protein A (SPA ′) according to Example 2 of the present invention.
  • FIG. 7 shows a DNA sequence encoding a promoter sequence, Shine-Dalgarno sequence, signal peptide and protein A (SPA ′) of a partial sequence expression vector (Spa′-pNK3260) of protein A (SPA ′) according to Example 2 of the present invention.
  • the present invention achieves high secretion expression and / or high density of cells during culture by using fructose as a carbon source when producing a recombinant protein using a recombinant Brevibacillus bacterium. Is.
  • the present invention will be described in detail below.
  • a recombinant Brevibacillus bacterium refers to a bacterium obtained by inserting a DNA encoding a recombinant protein into an expression vector and transforming the expression vector into a host Brevibacillus bacterium.
  • the DNA encoding the recombinant protein may be any DNA as long as the amino acid sequence obtained by translating the base sequence of the DNA constitutes the target recombinant protein.
  • a DNA sequence can be obtained by using a commonly known method such as a polymerase chain reaction (hereinafter abbreviated as PCR) method. Further, it can be synthesized by a known chemical synthesis method (NucleicNacids Res., 1984, 12: 4359), and can also be obtained from a DNA library.
  • the DNA sequence may have a codon substituted with a degenerate codon, and need not be identical to the original DNA sequence as long as it encodes the same amino acid when translated in the genus Brevibacillus .
  • the expression vector includes a DNA sequence encoding the recombinant protein or a partial sequence thereof, and a promoter capable of functioning in Brevibacillus bacteria operably linked to the sequence.
  • the promoter is not limited as long as it can function in bacteria of the genus Brevibacillus, but Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus
  • a promoter derived from a bacterium such as (Corynebacterium) and operable in a bacterium belonging to the genus Brevibacillus is preferred.
  • Brevibacillus bacterial cell wall proteins middle wall protein (MWP) and outer wall protein (OWP) (Udaka, S. et al. Method Enzymol., 1993, 217: 23-33), or Brevibacillus choshinensis HPD31
  • MBP middle wall protein
  • OBP outer wall protein
  • HWP Brevibacillus choshinensis HPD31
  • a promoter of a gene encoding the cell wall protein HWP J. Bacteriol., 1990, 172: 1312-1320 is more preferable.
  • the expression vector preferably further includes a Shine-Dalgarno sequence (SD sequence) and a signal sequence that can function in Brevibacillus bacteria downstream of the promoter.
  • SD sequence Shine-Dalgarno sequence
  • the expression vector may optionally include a marker sequence.
  • the SD sequence following the above promoter is derived from bacteria such as Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus SD sequence operable in Brevibacillus genus bacteria
  • the SD sequence present in the upstream of the gene encoding MWP, OWP or HWP is more preferable.
  • the DNA encoding the secretory signal peptide following the SD sequence is not particularly limited as long as it encodes the following secretory signal peptide, and encodes the same amino acid when translated in Brevibacillus brevis. As long as it is, it does not have to be the same as the original base sequence.
  • the secretory signal peptide is derived from bacteria such as Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus, and the like in the Brevibacillus genus bacteria
  • the secretory signal peptide that can be operated is preferable, and the secretory signal peptide of MWP, OWP, or HWP is more preferable.
  • DNA encoding the above promoter, SD sequence, and secretory signal peptide can be obtained from, for example, Brevibacillus bacteria.
  • Brevibacillus brevis 47 strain (FERM BP-1223), Brevibacillus brevis 47K strain (FERM BP-2308), Brevibacillus brevis 47-5 strain (FERM BP-1664), Brevibacillus choshinensis HPD31
  • FERM BP-1087 a chromosomal DNA of a strain
  • FERM BP-1087 Brevibacillus choshinensis HPD31-S strain
  • FERM BP-4573 Brevibacillus choshinensis HPD31-OK strain
  • any of the above promoters, any of the above SD sequences, any DNA encoding any of the above signal peptides, and DNA encoding the recombinant protein can operate in Brevibacillus bacteria. It is preferable that it is connected to.
  • the vector is preferably a plasmid vector.
  • plasmid vectors useful for expression of genes of the genus Brevibacillus include, for example, pUB110, which is known as a Bacillus subtilis vector, or pHY500 (JP-A-2-31682), pNY700 (JP-A-4-278091). Gazette), pHY4831 (J. Bacteriol. 1987. 1239-1245), pNU200 (Takazo Takataka, Journal of Japanese Society of Agricultural Chemistry 1987. 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol. -80), pNU211 (J.
  • the above plasmid vector can be prepared by those skilled in the art based on literature information.
  • an expression vector containing a promoter that functions in a bacterium of the genus Brevibacillus, an SD sequence, and a DNA encoding the target protein, or a gene fragment containing each of these nucleotide sequences is directly incorporated into a chromosome and expressed (specifically (Kaihei 9-135893) may also be used.
  • a known method already used in Bacillus subtilis or yeast can be transferred to Brevibacillus bacteria.
  • Brevibacillus bacteria include, but are not limited to, Brevibacillus agri, Brevibacillus bolsterensis, Brevibacillus brevis, Brevibacillus centroporus, Brevibacillus choshinensis, Brevibacillus formosas, Brevibacillus invocatus, ⁇ Includes Latinosporus, Brevibacillus limnophilus, Brevibacillus parabrevis, Brevibacillus reuszeli, Brevibacillus thermolver, etc.
  • the bacterium belonging to the genus Brevibacillus is Brevibacillus brevis 47 strain (FERM BP-1223), Brevibacillus brevis 47K strain (FERM BP-2308), Brevibacillus brevis 47-5 strain (FERM BP-1664), Brevibacillus brevis 47-5Q strain (JCM8975), Brevibacillus choshinensis HPD31 strain (FERM BP-1087), Brevibacillus choshinensis HPD31-S strain (FERM BP-6623), Brevibacillus choshinensis HPD31-OK Strain (FERM ⁇ ⁇ BP-4573) and Brevibacillus choshinensis SP3 strain (Takara).
  • FERM BP-1223 Brevibacillus brevis 47K strain
  • FERM BP-1664 Brevibacillus brevis 47-5Q strain
  • JCM8975 Brevibacillus choshinensis
  • Brevibacillus brevis 47 strain Brevibacillus brevis 47-5Q strain
  • Brevibacillus choshinensis HPD31 strain Brevibacillus choshinensis HPD31-S strain are suitable.
  • Brevibacillus brevis 47-5Q (JCM8975) can be obtained from the independent administrative corporation Biochemicals Research Institute, Bioresource Center (JCM) (2-1 Hirosawa, Wako, Saitama 351-0198).
  • a mutant strain such as a protease-deficient strain or a high-expression strain of the aforementioned Brevibacillus bacterium may be used.
  • Brevibacillus choshinensis HPD31-derived protease mutant Brevibacillus choshinensis HPD31-OK (FERM BP-4573)
  • Brevibacillus choshinensis HPD31-derived spore-forming ability Brevibacillus choshinensis SP3 strain (manufactured by Takara), which is a protease mutant
  • Transformation of a host cell of Brevibacillus genus bacteria used in the present invention can be performed by the known 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).
  • heterologous protein When a heterologous protein is highly expressed in microorganisms including Brevibacillus bacteria, it often forms an inactive protein without being correctly folded, especially when a protein with many disulfide bonds is highly expressed. Insoluble in many cases.
  • expressing the target protein it is known that the insolubilization of the target protein and the decrease in the secretion efficiency can be suppressed by acting chaperone protein, disulfide bond isomerase and / or proline isomerase, etc. .
  • a widely attempted method is a method in which a protein having disulfide redox activity such as PDI (protein disulfide isomerase) and / or DsbA is allowed to act (JP-A 63-294796, JP-A-5-336986). is there.
  • PDI protein disulfide isomerase
  • DsbA DsbA
  • a method for producing a protein having a correct disulfide bond by introducing a gene encoding a protein having disulfide redox activity into a host organism and simultaneously expressing the target protein and a protein having disulfide redox activity is also known.
  • Japanese Patent Laid-Open No. 2000-83670 Japanese Patent Laid-Open No. 2001-514490, etc.
  • the expression of the protein is performed in order to reduce the burden on the host cell due to excessive protein synthesis and to facilitate protein secretion.
  • DsbA of E. coli (Cell, 1991, 67: 582-589, EMBO. J., which is involved in protein disulfide bonds and is considered to be an analog of protein disulfide isomerase when the protein is expressed in Brevibacillus bacteria. 1992, 11: 57-62.)
  • chaperone proteins such as DnaK, DnaJ, GrpE (JP-A-9-180558) can be expressed simultaneously.
  • enzyme PDI Japanese Patent Application No. 2001-567367
  • disulfide oxidoreductase Japanese Patent Application Laid-Open No.
  • the medium used for culturing the recombinant Brevibacillus bacterium is not particularly limited as long as it contains fructose as long as it can produce recombinant protein with high efficiency and high yield.
  • a known carbon source or nitrogen source such as glucose, sucrose, glycerol, polypeptone, meat extract, yeast extract, casamino acid, and amino acid can be used.
  • inorganic salts such as potassium salt, sodium salt, phosphate, magnesium salt, manganese salt, zinc salt and iron salt may be added as necessary.
  • anti-foaming effects such as soybean oil, lard oil, surfactant, etc., or change the permeability of the cell membrane material, and increase in the production of recombinant protein per cell is expected.
  • a compound to be prepared may be added.
  • the use of a surfactant is preferable because the effect of the present invention may be enhanced.
  • the surfactant is not particularly limited as long as it does not adversely affect the growth of recombinant Brevibacillus bacteria and / or recombinant protein production, and is preferably a polyoxyalkylene glycol surfactant.
  • auxotrophic host cells When using auxotrophic host cells, nutrients required for growth may be added. If necessary, antibiotics such as penicillin, erythromycin, chloramphenicol and neomycin may be added.
  • protease inhibitors may be added at an appropriate concentration in order to suppress degradation of the target protein by the host-derived protease existing outside the cell body, and to lower the molecular weight.
  • protease inhibitors include phenylmethane sulfonylPfluoride (PMSF), Benzamidine, 4- (2-aminoethyl) -benzenesulfonyl fluoride (AEBSF), Antipain, Chymostatin, Leupeptin, Pepstatin A, Phosphoramidon, Aprotinin, Ethylenediaminetetra acetic acid (EDTA)
  • PMSF phenylmethane sulfonylPfluoride
  • AEBSF 4- (2-aminoethyl) -benzenesulfonyl fluoride
  • Antipain Chymostatin, Leupeptin, Pepstatin A, Phosphoramidon, Aprotinin, Ethylenediamine
  • the initial concentration is 1%. Above or 9% or less is preferable, more preferably 1 to 9% of the initial. More preferably, fructose is added in a timely manner so that the fructose concentration is 9% or less, particularly 4% or less during the culture.
  • Fructose addition methods include divided or continuous addition. Examples of such a method include, but are not limited to, a method of adding fructose after 6 hours from the start of culture.
  • Antibody-binding protein is not a protein that is recognized as an antigen by an antibody, but is a protein that can bind to a portion other than the antigen recognition site of an antibody (for example, an Fc portion).
  • the structure is not particularly limited as long as it is a protein that can bind to a site different from the antigen recognition site of the antibody. Examples of such proteins include protein A, protein G, and protein L.
  • Protein A is a kind of cell wall protein produced by the Gram-positive bacterium Staphylococcus aureus and is a protein having a molecular weight of about 42,000. Its structure consists of seven functional domains (signal sequence S from the amino terminus, immunoglobulin binding domain E, immunoglobulin binding domain D, immunoglobulin binding domain A, immunoglobulin binding domain B, immunoglobulin binding domain C, Staphylococcus aureus) Bacterial cell wall binding domain X) (Proc. Natl. Acad. Sci. USA, 1983, 80: 697-701, Gene, 1987, 58: 283-295, J. Bio. Chem., 1984, 259 : 1695-1702).
  • the relative affinity of this protein A for the immunoglobulin binding domain includes pH, Staphylococcus aureus strain (Infec. Immun., 1987, 55: 843-847), and immunoglobulin classes (IgG, IgM, IgA, IgD, IgE) and subclasses (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) are known to depend on many factors, particularly in the immunoglobulin class, human IgG1, human IgG2, human IgG4 and mouse IgG2a, It shows strong binding to the Fc part of mouse IgG2b and mouse IgG3.
  • immunoglobulin classes IgG, IgM, IgA, IgD, IgE
  • subclasses IgG1, IgG2, IgG3, IgG4, IgA1, IgA2
  • human IgG1, human IgG2, human IgG4 and mouse IgG2a It shows strong binding to the Fc part
  • Protein G is one of cell wall proteins produced by Group C and G Streptococcus bacteria, and has a molecular weight of about 59,000.
  • the structure consists of five functional domains (from the amino terminus, signal sequence SS, albumin binding domain by repetition of sequences A and B, immunoglobulin binding domain by repetition of sequences C and D, cell wall transmembrane domain W, and transmembrane domain. M).
  • This immunoglobulin G binding domain of protein G exhibits extensive binding to the Fc portion of mammalian IgG compared to that of protein A (J. Immunol., 1984, 133: 969-974, J. Biol. Chem. , 1991, 266: 399-405).
  • Protein L is a kind of protein produced by Peptostreptococcus magnus and has a molecular weight of about 79,000.
  • the structure consists of 6 functional domains (from the amino terminus, signal sequence SS, amino terminal domain A, immunoglobulin binding domain B, 5 repeats, unknown function domain 2 repeats, transmembrane domain W, transmembrane domain M) It is composed of
  • the immunoglobulin binding domain of this protein L shows binding to the kappa light chain of immunoglobulin.
  • “Partial sequence” of protein A, protein G, and protein L is a protein that is composed of an arbitrary part of amino acid sequences constituting protein A, protein G, and protein L and has antibody binding activity.
  • the amino acid sequence shown after the 31st Ala of SEQ ID NO: 9 obtained by removing the above-mentioned signal sequence S and cell wall binding domain X from protein A corresponds to the“ partial sequence ”of protein A.
  • a peptide having an antibody binding activity obtained by removing one or a plurality of immunoglobulin binding domains is also included in the “partial sequence” as long as it has an antibody binding activity.
  • “Functional variants and their linking sequences” of protein A, protein G and protein L are amino acids in the state in which at least one of the immunoglobulin binding domains of protein A, protein G and protein L retains antibody binding activity. This refers to antibody-binding proteins that have been subjected to substitution, insertion, and deletion treatments, and have changed sensitivities to physicochemical environmental factors such as drugs, enzymes, heat, and pH, and constructs that are homozygous or heterozygically linked. The combination and the number of connections are not limited.
  • Physiologically active protein is a protein used as a pharmaceutically active ingredient, and specifically includes peptide hormones, cytokines, growth factors, hematopoietic factors, enzymes, and precursors thereof.
  • Peptide hormones are peptides that are produced and secreted by endocrine cells in animal tissues according to information inside and outside the body, and are transported to the target cells by the bloodstream to regulate the activity of the target cells as foreign signals.
  • peptide hormones or precursors thereof include insulin, proinsulin, or preproinsulin, particularly feline insulin, feline proinsulin, or feline preproinsulin.
  • Insulin is a peptide hormone that is synthesized as proinsulin which is a biosynthetic precursor in the rough endoplasmic reticulum of pancreatic B cells, is converted into insulin, is stored in B granules, and is released into the blood in response to secretory stimulation. is there.
  • Cat feinsulin is a biosynthetic precursor of cat insulin.
  • Example 1 Construction of Brevibacillus expression vector pNK3260
  • the MWP P5 promoter contained in pNH326 (J. Bacteriol., 1995, 177: 745-749) was converted to the MWP P2 promoter to obtain the Brevibacillus expression vector pNK3260. It was constructed as follows. First, using pNH326 as a template, PCR was performed using two oligonucleotide primers Primer-1 and Primer-2 having the nucleotide sequences shown in SEQ ID NOs: 3 and 4, and a portion of pNH326 excluding the MWP P5 promoter was amplified. The ends were digested with restriction enzymes EcoRI and HindIII.
  • a double-stranded DNA fragment containing the MWP P2 promoter having the base sequence shown in SEQ ID NO: 5 was prepared according to a conventional method, and its ends were digested with restriction enzymes MunI and HindIII. These two DNA fragments were ligated using T4 DNA ligase to construct pNK3260.
  • Staphylococcus aureus cowan I strain (JCM2179) Staphylococcus aureus cowan I strain (JCM2179) was prepared from T2 liquid medium (polypeptone 1%, Yeast extract 0.2%, glucose 1%, fish meat extract 0.5%, pH 7.0) and cultured with shaking at 37 ° C. overnight. The cells were collected from the obtained culture broth by centrifugation, and washed twice with 10 mM Tris-HCl buffer (pH 8.0). The bacterial cells were suspended in the same buffer, lysed with 1% SDS, heated at 60 ° C.
  • Staphylococcus aureus cowan I strain (JCM2179) can be obtained from RIKEN BioResource Center, Microbial Materials Development Office (JCM) (2-1 Hirosawa, Wako, Saitama 351-0198) .
  • PCR is performed using these two oligonucleotide primers Primer-3 and Primer-4, and a signal sequence from protein A (S domain)
  • S domain a DNA fragment (about 0.9 kbp) encoding a portion excluding the cell wall binding domain (X domain) (hereinafter referred to as SPA ′) was amplified.
  • the obtained DNA fragment was digested with restriction enzymes NcoI and BamHI, and then separated and recovered from an agarose gel.
  • the Brevibacillus expression vector pNK3260 constructed in Reference Example 1 was similarly digested with restriction enzymes NcoI and BamHI, purified and recovered, and dephosphorylated by alkaline phosphatase treatment.
  • FIG. 2 shows a DNA encoding a promoter, SD sequence, signal peptide and protein A (SPA ') contained in Spa'-pNK3260.
  • the base sequence shown in SEQ ID NO: 8 shows the promoter, SD sequence, signal peptide and DNA encoding protein A (SPA ′) contained in Spa′-pNK3260, and SEQ ID NO: 9 shows the signal peptide and protein A (SPA ′).
  • SEQ ID NO: 8 shows the promoter, SD sequence, signal peptide and DNA encoding protein A (SPA ′) contained in Spa′-pNK3260
  • SEQ ID NO: 9 shows the signal peptide and protein A (SPA ′).
  • MWP-P2 is the P2 promoter region of Brevibacillus brevis cell wall protein MWP
  • SDM is the SD sequence of Brevibacillus brevis cell wall protein MWP
  • SP ′ is the Brevibacillus brevis cell wall.
  • spam ′ is a DNA sequence encoding SPA ′
  • Nm is a neomycin resistance gene coding region
  • Rep / pUB110 is a replica of vector pNK3260 Means the starting point.
  • P2-35 and “P2-10” mean the ⁇ 35 region and the ⁇ 10 region of the P2 promoter of Brevibacillus brevis cell wall protein MWP, respectively.
  • This Spa'-pNK3260 was used to transform Brevibacillus choshinensis HPD31-OK strain (FERM BP-4573) by a known method.
  • Comparative Example 1 Production of recombinant protein using glucose as a carbon source 1
  • the transformant obtained in Example 2 was treated with 3YC medium (polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO 4 .7H 2 O 0.01%, CaCl 2 .7H 2 O 0. 0.01%, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O 0.0001% pH 7.0) with a glucose concentration of 1, 3, 9% 500 ppm of Adecanol LG109 (manufactured by Adeka Co., Ltd.) was added to the prepared medium and cultured under aerobic conditions at 30 ° C.
  • Adecanol LG109 manufactured by Adeka Co., Ltd.
  • the culture solution is collected, the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein SPA ′ in the culture supernatant is analyzed by high performance liquid chromatography. The concentration was analyzed. As a result, the glucose concentration was 0.3 g / L at 1%, 0.9 g / L at 3%, and 0.7 g / L at 9%.
  • the culture solution was collected 48 hours after the start of the culture and analyzed for turbidity at 660 nm using a spectrophotometer.
  • the glucose concentration was 17 at 3%, 24 at 9%, and 16 at 9%.
  • Example 3 Production of recombinant protein using fructose as a carbon source 1
  • the transformant obtained in Example 2 was cultured in the same manner as in Comparative Example 1 except that the carbon source of the medium was changed from glucose to fructose 1, 3, and 9%.
  • the culture solution was collected and the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein SPA ′ in the culture supernatant was analyzed by high performance liquid chromatography. The concentration was analyzed.
  • the fructose concentration was 1%, 0.6 g / L, 3%, 1.1 g / L, 9%, 0.9 g / L.
  • the results are shown in Table 1.
  • the culture solution was collected 48 hours after the start of culture and analyzed for turbidity at 660 nm using a spectrophotometer.
  • the fructose concentration was 17% at 1%, 29 at 9%, 19 at 9%, and 19 at any concentration, which was equal to or higher than the addition of glucose.
  • the results are shown in Table 1.
  • Example 2 Production of recombinant protein using glucose as a carbon source 2
  • the transformant obtained in Example 2 was treated with 3YC medium (polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO 4 .7H 2 O 0.01%, CaCl 2 .7H 2 O 0. .01%, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O 0.0001% pH 7.0) 500 ppm of fats and oils) was added and cultured under aerobic conditions at 30 ° C.
  • 3YC medium polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO 4 .7H 2 O 0.01%, CaCl 2 .7H 2 O 0. .01%, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O 0.0001% pH 7.0
  • the culture solution was collected and the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein SPA ′ in the culture supernatant was analyzed by high performance liquid chromatography. The concentration was analyzed. As a result, it was 1.3 g / L.
  • the culture solution was collected 48 hours after the start of the culture and analyzed for turbidity at 660 nm using a spectrophotometer. As a result, it was 23.
  • Example 4 Production of recombinant protein using fructose as a carbon source 2
  • the transformant obtained in Example 2 was treated with 3YC2 medium (peptone 1%, yeast extract 0.5%, fructose 2%, phosphate 0.3%, MgSO 4 .7H 2 O 0.01%, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O 0.0001% pH 7.0) 750 ppm of Disperse CC-118 was added, The cells were cultured under aerobic conditions while controlling the pH from 7.0 to 7.8. At 30 hours of culture, 2% fructose was added.
  • 3YC2 medium peptone 1%, yeast extract 0.5%, fructose 2%, phosphate 0.3%, MgSO 4 .7H 2 O 0.01%, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O
  • the culture solution is collected and the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the concentration of the recombinant protein SPA ′ in the culture supernatant is measured by high performance liquid chromatography.
  • concentration of the recombinant protein SPA ′ in the culture supernatant is measured by high performance liquid chromatography.
  • Example 5 Production of recombinant protein using fructose as a carbon source 3
  • the transformant obtained in Example 2 was treated with 3YC3 medium (1% peptone, 0.5% yeast extract, 4% fructose, 0.3% phosphate, 0.01% MgSO 4 .7H 2 O, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O 0.0001% pH 7.0, fructose 4% from 6 to 48 hours after the start of culture 750 ppm of Disperse CC-118 was added to the continuous addition, and the cells were cultured under aerobic conditions of 30 ° C. while controlling the pH from 7.0 to 7.8.
  • 3YC3 medium 1% peptone, 0.5% yeast extract, 4% fructose, 0.3% phosphate, 0.01% MgSO 4 .7H 2 O, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%,
  • the culture solution is collected, the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the concentration of the recombinant protein SPA ′ in the culture supernatant is measured by high performance liquid chromatography.
  • the concentration of the recombinant protein SPA ′ in the culture supernatant is measured by high performance liquid chromatography.
  • it was 5.0 g / L, and by carrying out the culture by a novel technique, it was about 4 times as compared with 1.3 g / L in the conventional culture method using glucose shown in Comparative Example 2. It was found that the secretion amount of the recombinant protein increased.
  • Table 2 The results are shown in Table 2.
  • the culture solution was collected 72 hours after the start of the culture, and the turbidity at 660 nm was analyzed using a spectrophotometer. As a result, it was 45.
  • the cell density at the end of the culture was greatly doubled compared with 23 in the conventional culture method using glucose shown in Comparative Example 2 and about twice. It turned out to increase. The results are shown in Table 2.
  • Example 6 Preparation of Transformant Expressing 5 Conjugates of Functional Variant of C Domain of Protein BR> ⁇ Protein of Concatenated Protein by Modifying 29th Gly of C Domain of Protein A to Ala Back translation was performed from an amino acid sequence (SEQ ID NO: 10, hereinafter referred to as C-G29A), and a DNA sequence encoding the protein was designed.
  • the codon usage of the protein is close to the codon usage of HWP (J. Bacteriol., 172, p. 1312-1320, 1990), a cell surface protein that is expressed in large amounts in the Brevibacillus choshinensis HPD31 strain.
  • the codons were distributed so that the sequence identity of the base sequences encoding each of the five domains was low.
  • a restriction enzyme recognition site for PstI on the 5 ′ side and XbaI on the 3 ′ side of the sequence encoding the 5 linking domain was prepared.
  • the sequence of the prepared DNA fragment is shown in SEQ ID NO: 11.
  • the prepared DNA fragment was digested with PstI and XbaI (both manufactured by Takara), and fractionated and purified by agarose gel electrophoresis.
  • pNCMO2 manufactured by Takara
  • pNCMO2 manufactured by Takara
  • pNCMO2 manufactured by Takara
  • Example 3 Production of Recombinant Protein Using Glucose as a Carbon Source 3
  • the transformant obtained in Example 6 was treated with 3YC medium (polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO 4 .7H 2 O 0.01%, CaCl 2 .7H 2 O 0. .01%, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O 0.0001% pH 7.0) and aerobic conditions at 30 ° C. Cultured under.
  • 3YC medium polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO 4 .7H 2 O 0.01%, CaCl 2 .7H 2 O 0. .01%, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O 0.0001% pH 7.0
  • the culture solution was collected, the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein C-G29A in the culture supernatant was analyzed by high performance liquid chromatography. The concentration of was analyzed. As a result, it was 1.4 g / L.
  • the culture solution was collected 48 hours after the start of the culture and analyzed for turbidity at 660 nm using a spectrophotometer. As a result, it was 26.
  • Example 7 Production of recombinant protein using fructose as a carbon source 4
  • the transformant obtained in Example 6 was cultured in the same manner as in Comparative Example 2 except that the carbon source of the medium was changed from glucose to fructose 3%. After 48 hours from the start of the culture, the culture solution was collected, the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein C-G29A in the culture supernatant was analyzed by high performance liquid chromatography. The concentration of was analyzed. As a result, when it was cultured with glucose shown in Comparative Example 3, it was 1.4 g / L, whereas it was 2.2 g / L. The results are shown in Table 3.
  • Example 8 Preparation of transformant expressing feline proinsulin fusion protein From the amino acid sequence of feline proinsulin shown in (Non-patent Document 3) and E and D domains of protein A, feline proinsulin fusion protein The amino acid sequence was designed (SEQ ID NO: 12). In consideration of codon usage, a feline proinsulin fusion protein gene having the base sequence shown in SEQ ID NO: 13 was prepared. The prepared DNA fragment was digested with NcoI and EcoRI (both manufactured by Takara), and fractionated and purified by agarose gel electrophoresis.
  • pNH326 which is a plasmid vector for Brevibacillus spp., was digested with NcoI and EcoRI and purified and recovered. After mixing both, it was ligated using Ligation High (manufactured by TOYOBO) to construct a plasmid vector pNH326EDCIP. Using the plasmid vector obtained by the above operation, a transformant of Brevibacillus choshinensis HPD31-OK was prepared.
  • Comparative Example 4 Production of recombinant protein using glucose as a carbon source 4
  • the transformant obtained in Example 8 was treated with 3YC medium (polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO 4 .7H 2 O 0.01%, CaCl 2 .7H 2 O 0. .01%, MnSO 4 .4H 2 O 0.001%, FeSO 4 .7H 2 O 0.001%, ZnSO 4 .7H 2 O 0.0001% pH 7.0)
  • the cells were cultured under aerobic conditions at 30 ° C.
  • the culture solution was collected, the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the concentration of the feline proinsulin fusion protein in the culture supernatant by SDS-PAGE was analyzed.
  • Example 9 Production of recombinant protein using fructose as a carbon source 5
  • the transformant obtained in Example 8 was cultured in the same manner as in Comparative Example 3 except that the carbon source of the medium was changed from glucose to fructose 3%. After 48 hours from the start of the culture, the culture solution was collected, the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the concentration of the feline proinsulin fusion protein in the culture supernatant by SDS-PAGE was analyzed by ChemiDoc XRS system (Bio-Rad). As a result, about twice the production amount when cultured with glucose shown in Comparative Example 4 was confirmed. The results are shown in Table 4.

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Abstract

Dans la production d'une protéine recombinante à l'aide d'une bactérie Brevibacillus obtenue par génie génétique, la culture de la bactérie est effectuée à l'aide de fructose comme source de carbone. Ceci est une nouvelle condition pour cultiver la bactérie Brevibacillus, qui permet l'amélioration de la quantité de la protéine recombinante sécrétée/produite par cellule et l'augmentation de la densité de cellules pendant la culture et permet également la préparation de la protéine recombinante à un coût inférieur par comparaison avec ceux obtenus par des techniques classiques, et peut être portée à l'échelle industrielle.
PCT/JP2009/062121 2008-07-03 2009-07-02 Procédé de fabrication d'une protéine recombinante à l'aide de la bactérie brevibacillus obtenue par génie génétique WO2010001960A1 (fr)

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WO2015190457A1 (fr) * 2014-06-09 2015-12-17 株式会社カネカ Procédé de production de protéines recombinées à l'aide de brevibacillus recombiné

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WO2015190458A1 (fr) * 2014-06-09 2015-12-17 株式会社カネカ Procédé de production d'une protéine recombinante à l'aide de bactéries recombinantes du genre brevibacillus
WO2015190457A1 (fr) * 2014-06-09 2015-12-17 株式会社カネカ Procédé de production de protéines recombinées à l'aide de brevibacillus recombiné
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