WO2006004067A1 - ブレビバチルス属細菌を用いたプロテインa様蛋白質の生産方法 - Google Patents
ブレビバチルス属細菌を用いたプロテインa様蛋白質の生産方法 Download PDFInfo
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- WO2006004067A1 WO2006004067A1 PCT/JP2005/012252 JP2005012252W WO2006004067A1 WO 2006004067 A1 WO2006004067 A1 WO 2006004067A1 JP 2005012252 W JP2005012252 W JP 2005012252W WO 2006004067 A1 WO2006004067 A1 WO 2006004067A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- 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 protein A-like protein having an immunoglobulin binding ability using a bacterium belonging to the genus Brevibacillus. More specifically, the present invention is directed to causing a Brevibacillus bacterium to secrete and express a large amount of a protein A-like protein by genetic recombination technology, and subjecting the expressed protein A-like protein to degradation by a protease or the like. It is related to separating and recovering the protein A-like protein with high purity, and effectively using the separated and recovered protein A-like protein for applications such as column fat for antibody purification.
- Antibody (also referred to as immunoglobulin or Ig) proteins have been used as pharmaceuticals for a long time because they have a function of capturing and eliminating harmful antigens in the living body.
- Ig immunoglobulin
- Protein A is one of the cell wall proteins produced by the Gram-positive bacterium Staphylococcus aureus, and is apparently having a molecular weight of about 42,000. Its structure consists of seven functional domains (ammo terminal force also signal sequence S, immunoglobulin binding domain E, immunoglobulin binding domain D, immunoglobulin binding domain A, immunoglobulin binding domain B, immunoglobulin binding domain C).
- Staphylococcus' auureus bacterial cell wall binding domain X Staphylococcus' auureus bacterial cell wall binding domain X (see Non-Patent Documents 1, 2, and 3).
- Each of the five immunoglobulin-binding domains of protein A domains E, D, A, B, and C
- can bind at the Fc portion in the immunoglobulin molecule see Non-Patent Document 3).
- the relative affinity of this protein A for the immunoglobulin-binding domain is determined by pH, Staphylococcus aureus strain (Cheung, A. et al. Infec. Immun. 1987. 55: 843-847), and class (I g G, IgM, IgA , IgD, IgE) and subclasses (IgGl, IgG2, IgG3, IgG4 , IgAl, IgA2) is known to depend on many factors such as, in particular human in class Imunogu Roblin IgGl , IgG2, IgG4 and mouse IgG2a, IgG2b, IgG3 Fc part shows strong binding.
- Protein A with the above properties can be used for various diagnostics, pharmaceuticals, and basic research because it can bind to immunoglobulin without compromising its antigen-binding ability, affinity, and properties as an immunoglobulin. It is widely used as a ligand for purification carriers for immunoglobulins, especially IgG.
- a serum inhibitory factor (consisting of a specific antigen, antibody, antiglobulin, and immune complex) that inhibits the tumor cytotoxic effect of sensitized peripheral blood lymphocytes is adsorbed to protein A to cause tumors.
- protein A also has the function of activating polyclonal antibody synthesis, so it is expected to be used in various biotechnology fields as well as the original purified spore ligand. Yes.
- Bacillus subtilis which is a Gram-positive bacterium, like Staphylococcus aureus
- Bacillus subtilis which is a Gram-positive bacterium, like Staphylococcus aureus
- it is secreted into the medium by adding a signal sequence of Bacillus subtilis secreted protein to the N terminus of protein A.
- E. coli production systems approximately 47-10 Omg / L
- the protein A produced by Bacillus subtilis is degraded by the extracellular protease inherent in Bacillus subtilis. Therefore, it has not been achieved yet to suppress the degradation of protein A, which has been attempted to use several types of extracellular protease-deficient strains of Bacillus subtilis (Non-patent Document 5) as a host.
- Patent Document 1 Japanese Patent Application No. 07-187019
- Patent Document 2 US Patent No. US5151350
- Patent Document 3 European Patent No. EP0107509
- Patent Document 4 US Patent No. US4617266
- Patent Document 5 European Patent No. EP0124374
- Non-patent literature l Lofdahl, S et al. Proc. Natl. Acad. Sci. USA. 1983. 80: 697-701.
- Non-Patent Document 2 Shuttleworth, H. L et al. Gene. 1987. 58: 283-295.
- Non-Patent Document 3 Uhlen, M. et al. J. Bio. Chem. 1984. 259: 1695-1702.
- Non-Patent Document 4 Nilsson, B et al. Protein Eng. 1987. 1: 107-113.
- Non-Patent Document 5 Fahnestock, S. R et al. Appl. Envron. Microbiol. 1987. 53: 379-384.
- Non-Patent Document 6 Cruido, M et al., J. Basic Microbiology. 1991. 31: 337-345.
- Non-Patent Document 7 Sjostrom, J, -E et al. J. bacteriol. 1975. 123: 905-915.
- Non-Patent Document 8 Bjorck, Shira. 1984. J. Immunol. 133, 969-974.
- Non-Patent Document 9 Kastern, W. et al. J Biol Chem. 1992. 267: 12820-12825
- Non-Patent Document 10 Udaka, S. et al. Method Enzymol. 1993. 217: 23-33.
- An object of the present invention is to provide a protein A production technique that is more efficient than production methods using Escherichia coli and Bacillus subtilis.
- the present invention by using a Brevibacillus genus bacterium as a host, protein A, which greatly exceeds the production amount reported when a microorganism such as Escherichia coli or Bacillus subtilis is used as the host, is added to the culture solution. Can be secreted and purified to high purity without impairing the immunoglobulin binding function. Therefore, the present invention eliminates the low productivity of Protein A and the complexity of the purification process, which contributes to the high price to date.
- the present invention includes one or more of the following objects.
- the present invention provides a DNA sequence comprising a DNA sequence encoding a protein A-like protein or a partial sequence thereof, and a promoter operable in Brevibacillus bacteria operably linked to the sequence. .
- the present invention provides an expression vector comprising the DNA sequence.
- the present invention provides a Brevibacillus bacterium transformant comprising the expression vector.
- the present invention includes culturing the transformant and recovering a protein A-like protein secreted and produced by the transformant or a protein having a partial sequence thereof.
- a method for producing a protein or a protein having a partial sequence thereof is provided.
- the present invention provides a method for producing a protein A-like protein or a protein having a partial sequence thereof by the production method, and immobilizing the protein-like protein or a protein having a partial sequence thereof on an appropriate substrate. There is provided a method for producing an immunoglobulin adsorbent.
- FIG. 1 is a view showing the base sequence and amino acid sequence of protein A of Staphylococcus 'aureus' Cowan I strain (numbers indicate the number of amino acid residues).
- FIG. 2 is a diagram showing the gene sequence and amino acid sequence of protein A of the Staphylococcus aureus strain (numbers indicate the number of amino acid residues).
- FIG. 3 is a view showing a protein A (SPA) expression vector (Spa-pNH301).
- SPA protein A
- FIG. 4 shows the nucleotide sequence and amino acid sequence from the promoter sequence of the protein A (SPA) expression vector (Spa-pNH301) to the region encoding protein A (SPA).
- SPA protein A
- FIG. 5 is a diagram showing the results of SDS-PAGE analysis of puffin A (SPA) produced by Brevibacillus choshinensis HPD31-OK strain.
- SPA puffin A
- FIG. 6 is a view showing the results of an antibody binding test of puffin A (SPA) produced by Brevibacillus choshinensis HPD31-OK strain.
- SPA puffin A
- FIG. 7 is a diagram showing a protein A (SPA ′) expression vector (Spa′—pNK3260).
- FIG. 8 shows a promoter sequence of protein A (SPA,) expression vector (Spa, pNK3260), a Shine-Dalgarno sequence, a signal peptide and protein A (SPA ').
- SPA protein A
- FIG. 9 shows the results of SDS-PAGE analysis of the behavior and accumulation of peptin A (SPA,) produced by Brevibacillus choshinensis HPD31—OK strain in the culture medium.
- SPA peptin A
- the present inventors secrete and express active protein A in a large amount of culture medium by using Brevibacillus genus bacteria that are Gram-positive bacteria. It has been found that the problem of low productivity in E. coli and Bacillus subtilis and the problem of degradation of expressed protein A in Bacillus subtilis are effectively improved. That is, by using Brevibacillus bacteria, the expression level obtained with Bacillus subtilis can be easily secured and further accumulated in the medium.
- the present invention will be described in detail based on embodiments.
- protein A is one of the cell wall proteins produced by the Gram-positive bacterium Staphylococcus aureus.
- protein A is derived from the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2).
- the staphylococcus cauleus' Cowan I strain QCM2179) (Non-patent document 2), the amino acid sequence shown in Fig. 2 (SEQ ID NO: 4) (Non-patent document 6, Finck- Barbancon, V. et al. FEMS Microbiol. Lett. 1992.
- Non-patent Document 7 derived from Woods46 strain (Non-patent document 3), derived from 8325-4 strain (Non-patent document 3), and This refers to the cloned plasmid DNA, ie, the spa gene product encoded by pSPl, pSP3, etc. (Non-patent Document 7)
- the protein A-like protein referred to in the present invention includes protein A or a protein substantially the same as protein A. Protein A-like proteins also have at least 60% when compared to the amino acid sequence of protein A using a sequence comparison algorithm known to those skilled in the art and aligned for maximum match, A protein having an amino acid sequence having an amino acid residue identity of preferably 80%, more preferably 90-95%, and most preferably at least 99%, and having an immunoglobulin binding activity is included.
- the amino acid sequences having identity are preferably 50 residues or longer, and more Preferably, it is 100 residues or more, more preferably 150 residues or more, and in the most preferred embodiment, it has identity over its full length.
- BLAST algorithm An example of an algorithm that is suitable for determining percent sequence identity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). ing. Software for performing BLAST analyzes is available to the public through the National Center for Biotechnology Information (http: ⁇ www.ncbi.nlm.nih.gov/).
- the protein A-like protein is, for example, an amino acid sequence encoded by a DNA that is neutralized under stringent conditions to DNA having a sequence complementary to the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. It can be a powerful protein.
- hybridization conditions under the stringent conditions are: preferably about 7% sodium dodecyl sulfate (SDS), about 0.5 M NaPO, ImM EDTA at about 50 ° C. Isesi
- FIG. 1 SEQ ID NOs: 1 and 2
- FIG. 2 SEQ ID NOs: 3 and 4
- Protein A-like protein includes, for example, those obtained by rearranging the immunoglobulin binding domains (E, D, A, B, C) constituting protein A in an arbitrary permutation.
- the "protein A-like protein” includes, for example, protein G (Non-patent Document 8) possessed by Streptococcal bacteria of groups C and G, or Peptostreptococcus magnus (Peptostreptococcus magnus). Protein L (Non-patent Document 9) and V, and protein having the same immunoglobulin binding function as Protein A are also included.
- the “partial sequence” of a protein A-like protein refers to a protein that is composed of any part of the amino acid sequence constituting the protein A-like protein and has an immunoglobulin binding activity.
- the amino acid sequence shown after the 24th Ala of SEQ ID NO: 8 obtained by removing a part of the above-mentioned sidinal sequence S and cell wall binding domain X from protein A see “Example 1” SPA ”. See FIGS. 3 and 4, SEQ ID NOs: 7 and 8.) and protein A force 31st of SEQ ID NO: 19 obtained by removing all of signal sequence S and cell wall binding domain X described above.
- Each of the amino acid sequences shown after Ala correspond to “partial sequences” of protein A-like proteins. To do.
- amino acid sequence constituting the immunoglobulin binding domain of the above proteins G and L can also be mentioned as a "partial sequence”.
- the immunoglobulin A binding domain of protein A referred to in this specification is, for example, from 37 amino acid residues to 327 amino acid residues (domain E force C is also shown in FIG. 1), and the 37 amino acid residue in FIG. 355 amino acid residues from the group (domain E force C).
- the DNA sequence encoding the protein A-like protein used in the present invention may be any amino acid sequence capable of translating the DNA sequence and constituting a protein A-like protein.
- a DNA sequence can be obtained by a commonly used known method such as polymer It can be obtained using the 'chain' reaction (hereinafter abbreviated as PCR) method.
- PCR 'chain' reaction
- the DNA sequence must be identical to the original DNA sequence as long as it encodes the same amino acid when translated in the genus Brevibacillus where the codon can be replaced with a degenerate codon. No sex.
- the “expression vector” of the present invention includes a DNA sequence encoding a protein A-like protein or a partial sequence thereof, and a promoter capable of functioning in Brevibacillus bacteria operably linked to the sequence.
- the promoter may be any one that can function in bacteria of the genus Brevibacillus, but is not limited to Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus Middle wall protein (MWP), a promoter derived from bacteria such as (Corynebacterium) that can operate in bacteria belonging to the genus Brevibacillus, outer wall protein (OWP) (non-patented) Reference 10) or a promoter of a gene encoding Brevibacillus choshinensis HPD31 cell wall protein HWP (Ebisu.
- the P5 promoter region “MWP-P5” of the Brevibacillus brevis cell wall protein MWP shown in Example 1 (see FIGS. 3 and 4, SEQ ID NOs: 7 and 8), and the Brevibacillus shown in Example 5 are used.
- Each of the P2 promoter region “MWP-P2” (see FIGS. 7 and 8, SEQ ID NOS: 18 and 19) of the brevis cell wall protein MWP corresponds to a “promoter that can function in Brevibacillus bacteria”.
- the "expression vector” preferably further includes a Shine-Dalgarno sequence and a signal sequence that can function in Brevibacillus bacteria downstream of the promoter.
- the expression vector may optionally include a marker sequence.
- the "Schein Dalgarno sequence" following the above promoter sequence includes Escherichia coli, Bacillus subtilis, Brevibacillus, Staphylococcus, Streptococcus, Streptomyces, Corynebacterium Preferred is a Shine-Dalgarno sequence derived from bacteria such as Brevibacillus and capable of operating in Brevibacillus bacteria. More preferred is a protein middle wall protein (MWP), an outer wall protein (OWP) which is the same protein, or a Shine-Dalgarno sequence existing upstream of a gene encoding Brevibacillus choshinensis HPD31 cell wall protein HWP.
- the DNA sequence encoding the secretory signal peptide following the Shine Dalgarno sequence is not particularly limited as long as it is a DNA sequence encoding the following secretory signal peptide.
- secretory signal peptides include Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus, and Brevibacillus genus.
- a secretion signal peptide is more preferred. Further, the amino acid sequence of a conventional secretory signal peptide may be improved.
- signal peptide of middle w all protein Met- Lys- Lys- Val- Val- Asn- Ser- Val- Leu- Ala- er- Ala- Leu- Ala- Leu- Thr- Val — Ala— Pro— Met -Ala- Phe -Ala (SEQ ID NO: 11) Met-Lys-Lys-Are -Are-Val-Val-Asn-Asn-Ser-Val-Leu-Leu-Leu-Leu- Leu— Leu— Ala— Ser -Ala -Leu- Ala -Leu- Thr-Val -Ala -Pro -Met- Ala-Phe-Ala (SEQ ID NO: 12)
- a secretory signal peptide added or eliminated may be used.
- the secretory signal peptide used in the secretory protein of the genus Brevibacillus can be used.
- the signal peptide inherent to the protein A Figs. 1 and 2), that is, Met-Lys-Lys-Lys-Asn — lie— Tyr— Ser— lie— Arg— Lys— Leu— Gly— Val— Gly— lie— Ala-Ser-Val-Thr-Leu-Gly-Thr-Leu-Leu-lie-Ser-Gly-Gly-Val-Thr-Pro-Ala-Ala-Asn-Ala!
- a DNA sequence encoding the above promoter sequence, Shine-Dalgarno sequence, and secretory signal peptide can obtain, for example, Brevibacillus bacteria.
- Brevibacillus brevis 47 JCM6285
- JP-A-60-58074 Brevibacillus brevis 47K
- FERM BP-23008 see Non-Patent Document 10
- Brevibacillus brevis 47-5 FERM BP— 1664
- Brevibacillus choshinensis HPD31 FERM BP—1087) (see Japanese Laid-Open Patent Publication No.
- Brevibacillus choshinensis HPD31—S (FERM BP— 6623), or Brevibacillus thyonusinensis HPD31 — It can be obtained by specifically increasing the chromosomal DNA of OK (FERM BP-4573) (see JP-A-6-296485) as a saddle type by a known PCR method.
- any of the above promoters, any of the above Shine-Dalgarno sequences, any of the above signal peptide sequences, and the protein A-like protein or part of the protein A-like protein is preferred. It is preferred that the DNA sequence encoding the target sequence is operably linked in a Brevibacillus bacterium.
- a plasmid vector is preferable as the 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). No. gazette), pHY4831 (J. Bacteriol. 1987. 1239-1245), pNU200 (Takashige Takashi, Journal of Japanese Society of Agricultural Chemistry 1987. 61: 669-676), pNU100 (Appl. Microbiol. Biotech nol., 1989, 30 : 75-80), pNU211 (J.
- JP 2002-238569 A is a shuttle vector of Escherichia coli and Brevibacillus spp. Being limited to these There.
- an expression vector containing a promoter that functions in bacteria of the genus Brevibacillus, a Shine-Dalgarno sequence, and a DNA sequence encoding the target protein, or a gene fragment containing each of these sequences is directly integrated into the chromosome for expression.
- Japanese Patent Laid-Open No. 9-135693 Japanese Patent Laid-Open No. 9-135693
- Such a method is a known method already used in Bacillus subtilis and yeast.
- the protein A-like protein or a protein consisting of a partial sequence thereof may be produced in either a secreted form or a non-secreted form. However, it is secreted into the culture medium for ease of separation and purification. Is preferred ⁇ .
- DNA encoding a signal peptide that functions in the genus Brevibacillus is added upstream of DNA encoding the polypeptide or It is preferable to connect.
- the present invention also provides a Brevibacillus genus transformant transformed with the above expression vector.
- any Brevibacillus bacterium can be used as a host cell.
- Brevibacillus spp. But not limited to Brevibacillus agn, B. Dorstelensis, B. brevis, B. centrosporu s, B. choshinensis, B. formosus, B. invocatus, B. laterosporus, B. limnophilus, B. p arabrevis Including B. reuszeri and B. thermoruber.
- the bacterium belonging to the genus Brevibacillus is Brevibacillus brevis 47 (JCM6285), Brevibacillus brevis 47K (FE RM BP-2308), Brevibacillus brevis 47-5Q (JCM8970), Brevibacillus choshinensis HPD31 strain (FERM BP 1087) and Brevibacillus thyotusinensis HPD31—OK strain (FERM BP-4573) are also selected from the group of forces.
- Brevibacillus brevis 47 strain Brevibacillus brevis 47-5Q strain
- Brevibacillus choshinensis HPD31 strain Brevibacillus choshinensis HPD31-S strain are suitable.
- a mutant strain such as a protease-deficient strain or a high-expressing strain of the aforementioned Brevibacillus bacterium may be used. More specifically, Brevibacillus thiocinensis HPD31-OK (Japanese Patent Laid-Open No. 6-296485), a protease mutant derived from Brevibacillus choshinensis HPD31, and Breviba obtained as a human salivary amylase high-producing strain. Bacillus brevis 47K (Konishi, H. et al. Appl Microbio 1. Biotechnol. 1990. 34: 297-302) can be used. Alternatively, a mutant of any strain contained in the Brevibacillus genus bacteria group may be used.
- Brevibacillus brevis 47K (FERM BP-2308), Brevi Bacillus brevis 47-5 (FERM BP-1664), Brevibacillus choshinensis HPD31 (FERM BP-1087), Brevibacillus choshinensis HPD31- S (FE RM BP-6623), and Brevibacillus choshinensis HPD31— OK (FERM BP— 4573) is registered with the Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) (IPO D; Tsukuba Sakai Higashi 1-chome 305-8566 Ibaraki) Have been deposited.
- Patent Organism Depositary National Institute of Advanced Industrial Science and Technology
- Brevibacillus brevis 47 JCM6285
- Brevibacillus brevis 47-5Q strain JCM8970
- JCM BioResource Center
- 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. Often insolubilized inside and outside the cell.
- the target protein when the target protein is expressed, the decrease in the secretion efficiency of the target protein insoluble protein can be suppressed by the action of chaperone protein, disulfide-binding heterozygous enzyme and Z or proline isomeric enzyme. It is known. Widely tried
- V is a method in which a protein having disulfide acid reducing activity such as PDI (protein disulfide isomerase) and / or DsbA is allowed to act (JP-A 63-294796, JP-A-5-336986) ).
- PDI protein disulfide isomerase
- a gene encoding a protein having disulfide redox activity is introduced into a host organism, and the target protein and a protein having disulfide redox activity are expressed simultaneously to produce a protein having a positive U-disulfide bond.
- Japanese Patent Laid-Open No. 2000-83670, Special Table 2001-514490, etc. Japanese Patent Laid-Open No. 2000-83670, Special Table 2001-514490, etc.
- transformation of host cells of the genus Brevibacillus used in the present invention can be carried out by the known method of Takahahi et al. (Takahashi. W et al. J. Bacteriol. 1983. 156: 1130-1134), Takaahi et al. By the method (Takagi. H. et al. 1989. Agric. Biol. Chem, 53: 3099-3100) or Okamoto et al. (Okamoto. A. et al. 1997. Biosci. Biotechnol. Biochem. 61: 202-203) Can be implemented.
- the medium used for culturing the obtained transformant is not particularly limited as long as it can produce a protein A-like protein or a protein comprising a partial sequence thereof with high efficiency and high yield.
- carbon sources and nitrogen sources such as glucose, sucrose, glycerol, polypeptone, meat extract, yeast extract and casamino acid can be used.
- inorganic salts such as potassium salt, sodium salt, phosphate salt, magnesium salt, manganese salt, zinc salt and iron salt are added as necessary.
- auxotrophic host cells nutrient substances required for growth may be added. If necessary, antibiotics such as penicillin, erythromycin, chloram fecol, neomycin may be added.
- protease inhibitors namely Phenylmethane sulfonyl fluoride (PMSF), Benzamidine, 4- (2 -aminoethyl) -benzenesulfonyl fluoride (AEBSF), Anti pain, hymostatin, Leupeptin, Pepstatin A, Phosphoramidon, Aprotinin, Ethylenedi It is also possible to add aminetetra acetic acid (EDTA) and Z or other commercially available protease inhibitors at appropriate concentrations.
- PMSF Phenylmethane sulfonyl fluoride
- AEBSF 4- (2 -aminoethyl) -benzenesulfonyl fluoride
- EDTA aminetetra acetic acid
- Z commercially available protease inhibitors at appropriate concentrations.
- the culture temperature is about 15-42 ° C, preferably about 28-37 ° C, and it is desirable to perform aerobic culture under aeration and stirring conditions. You can cultivate anaerobically ⁇
- a protein A-like protein or a protein having a partial arrangement ability thereof is produced in large amounts outside the cell body, that is, in the culture supernatant. Since it accumulates, the active protein can be collected and purified from the culture supernatant.
- the protein remaining in the microbial cells and on the surface of the bacterium can be extracted by crushing the bacterium by a known method such as a method using ultrasonic wave, French press, alkali or SDS treatment.
- the obtained protein is obtained by known protein purification methods such as salting out using ammonium sulfate sodium sulfate, concentration with ethanol or acetone, gel filtration, ion exchange, hydroxyapatite, It can be effectively purified using various chromatography utilizing the antibody binding activity and Z or chromatography utilizing the affinity of the protein.
- the Brevibacillus genus bacterial transformant transformed with the "expression vector" of the present invention can stably express the protein and secrete and accumulate it in a large amount in the culture supernatant. Specifically, by culturing in an appropriate medium, active protein A, which appears in the molecular weight of about 40,000 to 50,000 in SDS-PAGE, can be secreted and accumulated in a large amount in the culture supernatant.
- active protein A which appears in the molecular weight of about 40,000 to 50,000 in SDS-PAGE, can be secreted and accumulated in a large amount in the culture supernatant.
- at least about 15 Omg / L culture solution preferably about 200 mg / L culture solution, more preferably about 500 mg / L culture solution, most preferably about lOOOmg / L or more. Can achieve the quantity.
- the production volume can vary depending on the culture conditions.
- the “substrate” for immobilizing the protein-like protein or the protein having a partial arrangement ability thereof includes an inorganic substrate such as activated carbon, glass beads, and silica gel, and a crosslinked polyvinyl chloride.
- -Synthetic polymers such as alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, cross-linked polystyrene, or organic base materials such as resin, crystalline cellulose, cross-linked cellulose, cross-linked agarose, cross-linked dextrin, cellulose, poly Butyl alcohol, Ethylene acetate Butyl copolymer saponification product, Polyacrylamide, Polyacrylic acid, Polymethacrylic acid, Polymethyl methacrylate, Polyacrylic acid grafted polyethylene, Polyacrylamide grafted polyethylene, Glass, and these Although composite base materials, such as organic mono-organic and organic mono-inorganic obtained by a combination, are mentioned as a representative example, this invention is not limited only to these.
- the substrate is made of water, nylon 6, nylon 6, 6, nylon 11, polyethylene, poly (salt vinylidene), poly (butyl chloride), poly (vinylinol acetate), polystyrene, styrene divininolebenzene.
- Polymer, styrene divinylbenzene, poly (trifluoroethylene), poly (black trifluoroethylene), poly (ethylene terephthalate), polypropylene, poly (methyl acrylate), polyacrylic ester, poly (Methyl methacrylate), polymethacrylic acid ester, cross-linked polyatalylate, cross-linked polyamide and the like are selected from the group consisting of synthetic polymer compounds.
- any of a spherical shape, a granular shape, a flat membrane shape, a fibrous shape, a hollow fiber shape, and the like can be used effectively.
- the average particle size is preferably about 5 ⁇ m to 1000 ⁇ m, more preferably about 20 to 800 ⁇ m, most preferably about 30 to 600 ⁇ m.
- protein A-like protein or a protein comprising a partial sequence thereof is immobilized on the substrate by covalent or non-covalent bonding, such as affinity, association, antigen-antibody reaction, It can be carried out by hydrogen bonding, condensation or the like.
- covalent or non-covalent bonding such as affinity, association, antigen-antibody reaction
- it can be carried out by hydrogen bonding, condensation or the like.
- molecular modifications such as addition, substitution and deletion of amino acid residues by means well known to those skilled in the art.
- the protein A-like protein can be easily immobilized on the substrate.
- the immunoglobulin-adsorbing carrier obtained by the production method of the present invention can be suitably used as a carrier for purifying immunoglobulin, particularly IgG. It can also be applied to disease treatment, such as removal of IgG with plasma power.
- Staphylococcus aureus strain ATCC6538P was cultured at 37 ° C in the T2 liquid medium (polypeptone 1%, yeast extract 0.2%, glucose 1%, fish extract 0.5%, pH 7.0) did.
- the bacterial cells were collected from the obtained culture broth by centrifugation and washed twice with 10 mM Tris-HCl buffer (pH 8.0). The cells were suspended in the same buffer, lysed with 1% SDS, heated at 60 ° C for 30 minutes, and then extracted with total methods such as phenol extraction and ethanol precipitation.
- the obtained DNA fragment was digested with restriction enzymes Ncol and BamHI, and then separated and recovered from an agarose gel.
- the Brevibacillus expression vector pNH301 (Shiga. Y. et al. Appl. Environ. Mi crobiol. 1992. 58: 525-531.) was also digested with restriction enzymes Ncol and BamHI and purified and recovered. Then, dephosphorylation treatment was performed by alkaline phosphatase treatment. [0057] The above-mentioned DNA fragment encoding SPA treated with restriction enzyme and the above-mentioned expression vector pNH301 were ligated using T4DNA ligase V to construct SPA expression plasmid Spa-pNH301 (Figs. 3, 4; SEQ ID NO: 7, 8). In Fig. 3 and Fig.
- MWP-P5 is Brevibacillus brevis cell wall protein MWP P5 promoter region
- SDM is Brevibacillus brevis cell wall protein MWP SD sequence
- SP is Brevibacillus brevis cell wall
- spa means the DNA sequence encoding “SPA”
- Nm means the neomycin metagene coding region
- RepZpUB110 means the replication origin of vector pNH301.
- FIG. 4 "1 3 5 35” Oyobi "1 3 5 10"
- their respective, Brevibacillus' brevis cell wall protein 35 area of P5 promoter MWP and - 10 means region.
- This Spa-pNH301 was used to transform Brevibacillus brevis 47K or Brevibacillus choshinensis HPD31 ⁇ koji strain by a known method.
- Each of the Brevibacillus choshinensis HPD31—OK strain having only the transformant obtained in Example 1 and the control vector PNH301 was used as a production medium 3 YC (polypeptone S 3%, yeast extract 0.5 %, Glucose 3%, MgSO7 ⁇ ⁇ 0.0
- the culture was performed at 30 ° C under aerobic conditions for 3 days. After removing the cells from the culture by centrifugation (10,000 rpm, 4 ° C, 5 minutes), the cells were subjected to SDS-PAGE under reducing conditions using a 10-20% gradient gel by a conventional method. After electrophoresis, the gel was stained with CBB to detect the SPA band ( Figure 5). As a result of SDS-PAGE analysis, a large amount of SPA was confirmed in the culture supernatant.
- Genomic DNA prepared from Staphylococcus aureuska described in Example 1 is used as a cage, and two oligonucleotides, primers 5 and TTGCTCCCATGGCTTTCGCTGCGCAACACGATGAAGCT-3 '(SEQ ID NO: 5), 5'-CGCGGATCCTTATAGTTCGCGACGACG-3' (sequence) (Number 9)
- a DNA fragment is amplified by PCR using 5'-CGCGGATCCTCAACGTATATAAGTTAAAAT-3 '(SEQ ID NO: 10).
- the obtained DNA fragment encoding protein A is ligated between Ncol of PNH301 and BamHI by the method described in Example 1.
- a transformant is obtained by transforming the obtained plasmid into Brevibacillus brevis 47K or Brevibacillus choshinenensis HPD31-OK strain. After the transformant is cultured by the culture method described in Example 2, the protein A secreted into the culture medium is confirmed.
- Example 1 Measurement of antibody binding ability of protein A produced by Brevibacillus genus To confirm that the SPA produced by the transformant obtained in Example 1 has antibody binding ability, mouse anti-human IgG antibody and Binding studies were performed using alkaline phosphatase-labeled Usagi anti-mouse IgG antibody.
- the MWP P5 promoter contained in pNH326 (lshihara. T et al., 1995. J. Bacteriol, 177: 745-749) was converted to the MWP P2 promoter, and the Brevibacillus expression vector pNK3260 was constructed as follows.
- PCR was performed using pNH326 as a saddle and two oligonucleotide primers 5'-GGAATTCTG ATTTC ACTTTTTGC ATTCTAC A-3 '(SEQ ID NO: 13) and 5'- AGTGCACTCGCAC TTACTGT-3' (SEQ ID NO: 14).
- the portion of pNH326 excluding the P5 promoter of MWP was amplified, and its ends were digested with restriction enzymes EcoRI and Hindlll.
- TTCGGTACCCCGGGTTCGAAATCGATAAGCTTCTGT-3 ′ (SEQ ID NO: 15) was prepared according to a standard method, and its ends were digested with restriction enzymes Muni and Hindlll. These two DNA fragments were ligated using T4DNA ligase to construct pNK3260.
- the Brevibacillus expression vector pNK3260 constructed in Example 4 was similarly digested with restriction enzymes Ncol and BamHI, purified and recovered, and subjected to dephosphorylation treatment with alkaline phosphatase treatment.
- 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 Brevibacillus brevis.
- a modified signal peptide sequence in which the signal peptide sequence of the cell wall protein MWP is partially modified, “spa,” is the DNA sequence encoding SPA
- “Nm” is the neomycin metagene coding region
- rRep / pUBHOj is a replica of the vector pNK3260 Means the starting point.
- P2-35” and “P2-10” mean the 35 and 10 regions of the P2 promoter of Brevibacillus brevis cell wall protein MWP, respectively.
- Example 6 Peristalsis in an adherent of protein A secreted and expressed by Brevibacillus bacteria
- the transformant obtained in Example 5 was added to the production medium 3YC (polypeptone S 3%, yeast exhalation 0.5%, glucose 3%, MgSO ⁇ 7 ⁇ . 0.01%, CaCl ⁇ 7 ⁇ O 0.01 %, M
- lane number 1 is The molecular weight marker
- lane number 2 is Repligen protein A (rPA-50) 0.52 / zg used as a control
- lane number 3 is SPA, expression vector 24 hours after the start of culture.
- the target SPA ' was expressed in a large amount 48 hours after the start of culture (lane number 4), and the concentration of SPA' continued to increase thereafter. Finally, it accumulated in the culture supernatant at a concentration of about 2 g / L. The concentration of SPA 'in the culture supernatant was measured using the ChemiDoc XRS system (Bio-Rad), using the Repligen protein A (rPA-50) 0.52 ⁇ g band migrated in lane number 2 as a control. did.
- Example 7 Confirmation of the N-terminal amino acid west R row of protein A cattle bovine by a trophoblastic body
- the human IgG binding activity of SPA prepared above was evaluated as follows. First, 5 ⁇ g / m SPA, was diluted with PBS buffer (Takara Bio) so that it was L, and 100 L was dispensed into a 96-well immunoplate (NU NC). After reacting at 37 ° C for 1 hour, the plate was washed twice with PBS buffer (250 L), and 250 L of 3% ushi serum albumin ZPBS solution was added and blocked at 4 ° C. Next, add 25 g / ml human IgG (Sigma) solution 100 1 prepared in PBS buffer containing 0.1% BSA and react at 37 ° C for 1.5 hours. Washed with PBS buffer containing% Tween 20.
- productivity exceeding the expression level of recombinant protein A in Escherichia coli and Bacillus subtilis previously reported could be achieved. It shows that productivity problems can be solved.
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JP2006528867A JPWO2006004067A1 (ja) | 2004-07-06 | 2005-07-01 | ブレビバチルス属細菌を用いたプロテインa様蛋白質の生産方法 |
EP05755845A EP1767640A4 (en) | 2004-07-06 | 2005-07-01 | PROCESS FOR PREPARING A PROTEIN-A-like PROTEIN USING A BACTERIUM OF THE GENUS BREVIBACILLUS |
US11/630,334 US8597908B2 (en) | 2004-07-06 | 2005-07-01 | Process for producing protein A-like protein with use of Brevibacillus genus bacterium |
CA002570253A CA2570253A1 (en) | 2004-07-06 | 2005-07-01 | Process for producing protein a-like protein with use of brevibacillus genus bacaterium |
US14/066,231 US8889389B2 (en) | 2004-07-06 | 2013-10-29 | Process for producing protein A-like protein with use of Brevibacillus genus bacterium |
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US14/066,231 Division US8889389B2 (en) | 2004-07-06 | 2013-10-29 | Process for producing protein A-like protein with use of Brevibacillus genus bacterium |
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- 2005-07-01 EP EP10171148.9A patent/EP2251425B1/en not_active Not-in-force
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Also Published As
Publication number | Publication date |
---|---|
EP1767640A4 (en) | 2008-03-26 |
EP2251425B1 (en) | 2016-04-20 |
JP2010246569A (ja) | 2010-11-04 |
EP2251425A1 (en) | 2010-11-17 |
US8889389B2 (en) | 2014-11-18 |
EP1767640A1 (en) | 2007-03-28 |
US20140080179A1 (en) | 2014-03-20 |
US8597908B2 (en) | 2013-12-03 |
JP2014064589A (ja) | 2014-04-17 |
JPWO2006004067A1 (ja) | 2008-04-24 |
US20070243582A1 (en) | 2007-10-18 |
CA2570253A1 (en) | 2006-01-12 |
JP2016063844A (ja) | 2016-04-28 |
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