WO2013065772A1 - タンパク質の分泌生産法 - Google Patents
タンパク質の分泌生産法 Download PDFInfo
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- WO2013065772A1 WO2013065772A1 PCT/JP2012/078285 JP2012078285W WO2013065772A1 WO 2013065772 A1 WO2013065772 A1 WO 2013065772A1 JP 2012078285 W JP2012078285 W JP 2012078285W WO 2013065772 A1 WO2013065772 A1 WO 2013065772A1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/241—Tumor Necrosis Factors
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Definitions
- the present invention relates to a coryneform bacterium for efficiently secreting and producing a heterologous protein and a method for secreting and producing a heterologous protein.
- the heterologous protein secreted and produced in the present invention is a multimer protein.
- Non-patent Document 1 Bacillus bacteria
- Non-patent Document 2 methanol-assimilating yeast Pichia pastoris
- Aspergillus filamentous fungi Non-Patent Documents 3 and 4
- Patent Document 1 Non-patent document 5
- secretion of proteases such as subtilisin
- non-patent document 6 protein secretion using signal peptides of cell surface proteins PS1 and PS2 (also referred to as CspB) of coryneform bacteria
- CspB protein secretion using signal peptides of cell surface proteins PS1 and PS2
- PS2 Secretion of fibronectin binding protein using signal peptide of (CspB)
- Non-patent Document 7 secretion of protransglutaminase using signal peptide of cell surface protein PS2 (CspB) or SlpA (also called CspA) of coryneform bacteria
- Patent document 3 protein secretion using a mutant secretion apparatus
- Secretion of pro-transglutaminase by mutants (Patent Document 5), secretion of the protein utilizing the Tat-dependent signal peptide (Patent Document 6) has been reported and the like.
- Metallopeptidase is a protease and requires various metal ions such as zinc ion and calcium ion for activation, and has an activity of degrading various proteins.
- metallopeptidases belonging to the M23 / M37 family (also referred to as M23 / M37 metallopeptidases) are metalloendopeptidases that require zinc ions.
- the Cgl0858 gene of C. glutamicum is known from the sequence information to be a gene encoding a protein containing a region homologous to the motif of M23 / M37 metallopeptidase. However, the function of the protein encoded by the Cgl0858 gene in C. glutamicum is unknown.
- Penicillin-binding proteins are proteins that bind to ⁇ -lactam antibiotics, and are a generic term for proteins whose enzyme functions are inhibited by binding to ⁇ -lactam antibiotics. In general, PBPs are membrane-bound proteins and are thought to be essential for cell wall synthesis in eubacteria. PBPs are classified into high molecular weight type (HMW-PBPs) and low molecular weight type (LMW-PBPs) according to their molecular weight.
- HMW-PBPs high molecular weight type
- LMW-PBPs low molecular weight type
- HMW-PBPs have a high transpeptidase active domain that has a transpeptidase activity that crosslinks to the peptidoglycan part that constitutes the cell wall and a transglycosylase active domain that has a transglycosylase activity domain that forms a polysaccharide chain from a disaccharide.
- the molecular weight PBPs are classified into class A (class A HMW-PBPs) and high molecular weight PBPs class B ⁇ ⁇ (class B HMW-PBPs) having only the transpeptidase active domain.
- C.sglutamicum PBPs The knowledge about C.sglutamicum PBPs is detailed in Non-Patent Documents 8 and 9 etc.
- C. glutamicum at least nine PBPs homologs have been found so far. Five of them are HMW-PBPs, and the breakdown is two classes A HMW-PBPs (PBP1a, PBP1b) and three classes B HMW-PBPs (FtsI, PBP2a, PBP2b). It is known that class A HMW-PBPs of C. glutamicum is a factor involved in cell elongation, and classB HMW-PBPs is a factor involved in the formation of peptidoglycan in the septum during cell division.
- the present invention develops a novel technique for improving the ability of a coryneform bacterium to produce a multimeric protein, and provides a coryneform bacterium that secretes and produces a multimeric protein, and a method for producing a secreted multimeric protein using the bacterium. Is an issue.
- the present inventors have increased expression of a gene encoding a metallopeptidase in a method for producing a heterologous protein using a coryneform bacterium as an expression host.
- a coryneform bacterium as an expression host.
- the present invention is as follows.
- a coryneform bacterium that has the ability to secrete and produce a multimeric protein and is modified so that expression of a gene encoding a metallopeptidase is increased.
- the coryneform bacterium in which the expression of the gene is increased by increasing the copy number of the gene or modifying the expression regulatory sequence of the gene.
- the coryneform bacterium, wherein the metallopeptidase is a protein comprising a region homologous to a motif possessed by M23 / M37 metallopeptidase or M23 / M37 metallopeptidase.
- the coryneform bacterium wherein the metallopeptidase is a protein shown in the following (A) or (B).
- A a protein having the amino acid sequence shown in SEQ ID NO: 4
- B The amino acid sequence shown in SEQ ID NO: 4 has an amino acid sequence containing 1 to 10 amino acid substitutions, deletions, insertions or additions, and a large amount when expression is increased in coryneform bacteria
- the coryneform bacterium further modified so that the activity of the penicillin-binding protein is reduced.
- coryneform bacterium wherein the activity of cell surface protein is reduced.
- the coryneform bacterium wherein the coryneform bacterium belongs to the genus Corynebacterium or Brevibacterium.
- the coryneform bacterium, wherein the coryneform bacterium is Corynebacterium glutamicum.
- the coryneform bacterium has a gene construct for secretory expression of a multimeric protein, The gene construct is connected to a promoter sequence that functions in a coryneform bacterium, a nucleic acid sequence that encodes a signal peptide that functions in a coryneform bacterium connected downstream of the promoter sequence, and a nucleic acid sequence that encodes the signal peptide.
- the coryneform bacterium comprising a nucleic acid sequence encoding the produced multimeric protein.
- the coryneform bacterium, wherein the multimeric protein is an antibody-related molecule.
- the coryneform bacterium wherein the antibody-related molecule is one or more proteins selected from Fab, F (ab ′) 2 and Fc fusion protein.
- the coryneform bacterium, wherein the multimeric protein is vascular endothelial growth factor-A (VEGF-A).
- a method for producing a multimeric protein comprising culturing the coryneform bacterium and recovering the secreted multimeric protein.
- FIG. 1 shows the alignment of the amino acid sequences of the protein encoded by Cgl0858 of C.Cglutamicum ATCC13032 and the protein encoded by the Cgl0858 homolog of C. glutamicum ATCC13869.
- FIG. 2 is a photograph showing the results of non-reducing SDS-PAGE when the H chain region and L chain region of the trastuzumab Fab fragment were co-expressed in the C. glutamicum YDK010 strain and its metallopeptidase expression-enhanced strain.
- FIG. 3 is a photograph showing the results of Western blotting when expressing the F (ab ′) 2 fragment of trastuzumab in the C.
- FIG. 4 shows the C. glutamicum YDK010 strain and its metallopeptidase expression-enhanced strain, and the C. glutamicum YDK010 ⁇ PBP1a strain metallopeptidase expression-enhanced strain when the H chain region and L chain region of the trastuzumab Fab fragment were coexpressed. It is a photograph which shows the result of non-reducing SDS-PAGE.
- FIG. 5 is a photograph showing the results of non-reducing SDS-PAGE when vascular endothelial growth factor-A (VEGF-A) was expressed in the C.
- VEGF-A vascular endothelial growth factor-A
- FIG. 6 is a photograph showing the results of non-reducing SDS-PAGE when the adalimumab Fab (H & L) fragment was expressed in the C. glutamicum YDK010 ⁇ PBP1a strain and its enhanced metallopeptidase expression.
- FIG. 7 is a photograph showing the results of non-reducing SDS-PAGE when expressing the Fab (H & L) fragment of trastuzumab in C.amicglutamicum ATCC13869 strain (wild strain) and its metallopeptidase expression-enhanced strain.
- Coryneform bacterium of the present invention is a coryneform bacterium having an ability to secrete and produce a multimer protein, and has been modified so that expression of a gene encoding a metallopeptidase is increased.
- Coryneform bacteria hereinafter also referred to as “bacteria of the present invention” or “coryneform bacteria of the present invention” are provided.
- secreting means that the protein is transferred outside the bacterial cell (outside the cell).
- a protein is “secreted” when all the molecules of the protein are finally completely free in the medium, as well as when all the molecules of the protein are present on the surface of the cell. This includes the case where some molecules of the protein are present in the medium and the remaining molecules are present on the surface of the cells.
- the ability to secrete and produce multimeric protein means that when the bacterium of the present invention is cultured in a medium, the multimeric protein is secreted in the medium or on the surface of the cell, The ability to accumulate to the extent that it can be recovered from the surface of the body.
- the accumulation amount is, for example, preferably 10 ⁇ g / L or more, more preferably 1 ⁇ mg / L or more, particularly preferably 100 ⁇ mg / L, and even more preferably 1 ⁇ g / L or more as the accumulation amount in the medium. Good.
- the accumulated amount is, for example, the accumulated amount on the surface of the bacterial cell, when the multimeric protein on the surface of the bacterial cell is collected and suspended in the same amount of liquid as the medium, the concentration of the multimeric protein in the suspension is The amount may be preferably 10 ⁇ g / L or more, more preferably 1 ⁇ mg / L or more, and particularly preferably 100 ⁇ mg / L or more.
- the “protein” secreted and produced in the present invention is a concept including an embodiment called a peptide or polypeptide.
- heterologous protein refers to a protein that is exogenous to coryneform bacteria that express and secrete the protein.
- the heterologous protein may be, for example, a protein derived from a microorganism, a protein derived from a plant, a protein derived from an animal, a protein derived from a virus, or an artificial protein.
- the protein may be a protein whose amino acid sequence is designed.
- a multimeric protein refers to a protein that can exist as a multimer composed of two or more subunits.
- each subunit may be linked by a covalent bond such as a disulfide bond, or may be linked by a non-covalent bond such as a hydrogen bond or a hydrophobic interaction, or by a combination thereof. May be.
- Multimers preferably contain one or more intermolecular disulfide bonds.
- the multimer may be a homomultimer composed of a single type of subunit or a heteromultimer composed of two or more types of subunits.
- the multimeric protein is a heteromultimer, at least one subunit among the subunits constituting the multimer may be a heterologous protein. That is, all the subunits may be derived from different species, or only some of the subunits may be derived from different species.
- the multimeric protein may be a protein that is naturally secreted or a protein that is non-secreted in nature, but is preferably a protein that is naturally secreted. Specific examples of the “multimeric protein” will be described later.
- the multimeric protein secreted and produced in the present invention may be only one type, or two or more types.
- the multimeric protein is a heteromultimer, all subunits constituting the heteromultimer are secreted and produced.
- coryneform bacteria are aerobic Gram-positive bacilli, and examples of coryneform bacteria include Corynebacterium bacteria, Brevibacterium bacteria, and Microbacterium bacteria.
- Coryneform bacteria were previously classified as genus Brevibacterium, but now also include bacteria integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)).
- Corynebacterium bacteria were previously classified as Corynebacterium ammoniagenes, but some have been reclassified as Corynebacterium stationis by 16S rRNA sequence analysis. Included (Int. J. Syst. Evol. Microbiol., 60,879874-879 (2010)).
- coryneform bacteria is that, compared to filamentous fungi, yeast, Bacillus bacteria, etc., which are conventionally used for secretory production of heterologous proteins, there are very few proteins that are secreted outside the cells. Simplification and omission of the purification process can be expected, and it grows well on simple media containing sugar, ammonia, inorganic salts, etc., and excels in medium cost, culture method, and culture productivity And so on.
- coryneform bacteria include the following species. Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum Brevibacterium flavum Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum
- coryneform bacteria include the following strains. Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum ATCC 14020 Brevibacterium flavum ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum ATCC 13869 Brevibacter
- strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.
- Corynebacterium glutamicum AJ12036 isolated as a streptomycin (Sm) -resistant mutant from wild-type Corynebacterium glutamicum ATCC 13869 is more protein-rich than its parent strain (wild-type). It is predicted that there is a mutation in the functional gene related to secretion of the protein, and the secretory production ability of the heterologous protein is extremely high, about 2 to 3 times as the accumulated amount under the optimum culture condition, and is suitable as a host bacterium.
- the above-described coryneform bacterium as a parent strain may be selected as a host by selecting a strain having enhanced protein secretory production ability using a mutation method or a gene recombination method. For example, after treatment with a chemical mutagen such as ultraviolet irradiation or N-methyl-N′-nitrosoguanidine, a strain with enhanced protein secretory production ability can be selected.
- a chemical mutagen such as ultraviolet irradiation or N-methyl-N′-nitrosoguanidine
- a strain modified so as not to produce cell surface protein from such a strain is used as a host, purification of a heterologous protein secreted in the medium or on the surface of the cell body is facilitated, which is particularly preferable.
- modification can be carried out by introducing mutation into the coding region of cell surface protein on the chromosome or its expression regulatory region by mutation or gene recombination.
- C. glutamicum YDK010 strain WO2004 / 029254
- CspB cell line protein PS2
- CspB cell line protein PS2 deficient of C. glutamicum AJ12036
- a coryneform bacterium having the ability to secrete and produce a multimeric protein can be obtained by introducing and retaining a genetic construct for secretory expression of a multimeric protein into the coryneform bacterium as described above. That is, the bacterium of the present invention has a gene construct for secretory expression of multimeric protein.
- the “gene construct for secretory expression of multimeric protein” and its introduction method will be described later.
- the bacterium of the present invention has been modified so that expression of a gene encoding a metallopeptidase is increased.
- the bacterium of the present invention can be obtained by modifying a coryneform bacterium having the ability to secrete and produce a multimeric protein so that expression of a gene encoding a metallopeptidase is increased.
- the bacterium of the present invention can also be obtained by imparting the ability to secrete and produce multimeric proteins after modifying the coryneform bacterium so that the expression of the gene encoding the metallopeptidase is increased.
- the modification for constructing the bacterium of the present invention can be performed in any order.
- the bacterium of the present invention may be obtained from a strain capable of secreting and producing a multimeric protein before being modified so that expression of a gene encoding a metallopeptidase is increased.
- the bacterium of the present invention secretes and produces multimeric protein even if it has a gene construct for secretory expression of multimeric protein before it is modified so that expression of the gene encoding metallopeptidase is increased. It was obtained from a strain that could not be obtained, and may be modified so that expression of the gene encoding the metallopeptidase is increased so that the multimeric protein can be secreted and produced. .
- the metallopeptidase and the gene encoding it will be described below.
- Metallopeptidase is a protease and requires various metal ions such as zinc ion and calcium ion for activation, and has an activity of degrading various proteins. In the present invention, this activity is also referred to as metallopeptidase activity.
- the metallopeptidase whose expression is enhanced in the present invention is not particularly limited, it is preferably M23 / M37 metallopeptidase.
- M23 / M37 metallopeptidase is a metalloendopeptidase that requires zinc ions.
- Specific examples of the M23 / M37 metallopeptidase include a protein encoded by the ale-1 gene of Staphylococcus capitis EPK1 strain.
- the metallopeptidase whose expression is enhanced in the present invention may be a protein containing a region homologous to the motif of the metallopeptidase as described above.
- the metallopeptidase whose expression is enhanced in the present invention is preferably a protein containing a region homologous to the motif of M23 / M37 metallopeptidase.
- a protein containing a region homologous to the motif of M23 / M37 metallopeptidase for example, a protein encoded by Cgl0858 gene of C. glutamicum ATCC13032 or a Cgl0858 homolog gene of C.glutamicum ATCC13869 Examples include proteins.
- the protein containing a region homologous to the motif of M23 / M37 metallopeptidase include a protein encoded by the nlpD gene of Escherichia coli (E. coli) K12 MG1655 strain.
- the metallopeptidase whose expression is enhanced in the present invention has a property of increasing the secreted production amount of a multimeric protein as compared with an unmodified strain when the expression is increased in a coryneform bacterium.
- the metallopeptidase whose expression is enhanced in the present invention may or may not have a metallopeptidase activity.
- the property of increasing the secretory production of multimeric protein when the expression is increased in coryneform bacteria as compared to that of the non-modified strain means that the non-modified strain such as wild It refers to the property of imparting the ability to secrete and produce a higher amount of multimeric protein than the strain or parent strain.
- Secretion production of a larger amount of a multimeric protein than that of an unmodified strain is not particularly limited as long as the secretory production amount of the multimeric protein is increased as compared with that of the unmodified strain, but for example, in the medium and / or the cell surface
- the amount of accumulated multimeric protein is preferably 10% or more, more preferably 20% or more, particularly preferably 30% or more, and even more preferably 100% or more larger than the non-modified strain. It may be.
- secretory production of a multimeric protein in an amount larger than that of the unmodified strain means that the multimeric protein cannot be detected when the culture supernatant of the non-modified non-modified strain is subjected to SDS-PAGE and stained with CBB.
- the expression of that gene is increased based on the strain belonging to coryneform bacteria
- the amount of multimeric protein secreted and produced when the modified strain is cultured in a medium is quantified, and secreted production is obtained when the unmodified strain (non-modified strain) is cultured in a medium. This can be confirmed by comparison with the amount of multimeric protein produced.
- a coryneform bacterium having a gene construct for secretory expression of a multimeric protein can be used as the unmodified strain herein.
- a strain obtained by introducing a gene construct for secretory expression of multimeric protein into C. glutamicum AJ12036 (FERM BP-734) or C. glutamicum YDK010 strain can be used as the non-modified strain.
- the metallopeptidase has “the property of increasing the secretory production amount of a multimeric protein when the expression is increased in a coryneform bacterium compared to an unmodified strain” specifically, a metallopeptidase It may be active.
- Metallopeptidase activity can be measured by methods well known to those skilled in the art. Specifically, the metallopeptidase activity can be measured by, for example, a metalloprotease assay kit (manufactured by Oxford Biomedical Research).
- the ale-1 gene of Staphylococcus capitis (S. capitis) EPK1 strain is registered in the NCBI database as GenBank accession BAA13069 (VERSION BAA13069.1 GI: 1890068).
- the Cgl0858 gene of C. glutamicum ATCC13032 corresponds to the complementary sequence of sequences 916, 967 to 917,680 in the genome sequence registered as GenBank accession BA000036 (VERSION BA000036.3 GI: 42602314) in the NCBI database.
- the amino acid sequence of the protein encoded by the Cgl0858 gene of C. glutamicum ATCC13032 is shown in SEQ ID NO: 98.
- nucleotide sequence of the Cgl0858 homolog gene of C. glutamicum ATCC13869 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 3 and 4, respectively.
- the nlpD gene of Escherichia coli (E. coli) K12 MG1655 strain corresponds to the complementary sequence of the 2,865,636-2,866,775 position in the genome sequence registered as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990) in the NCBI database To do.
- the nlpD gene of E. coli K12 MG1655 is synonymous with ECK2737 and JW2712.
- the base sequence of the gene encoding metallopeptidase may vary depending on the genus, species, or strain to which the bacterium belongs, the gene encoding metallopeptidase is abundant when the expression is increased in coryneform bacteria.
- a variant of the above base sequence may be used as long as it encodes a protein having the property of increasing the amount of secreted production of body protein compared to that of an unmodified strain.
- the Cgl0858 gene, nlpD gene, or ale-1 gene variant includes a homologue of the same gene.
- the homologue of the Cgl0858 gene, nlpD gene, or ale-1 gene is obtained by using the wild type Cgl0858 gene of C.
- glutamicum the wild type nlpD gene of E. coli, or the wild type ale-1 gene of S. capitis as the query sequence. It can be easily obtained from public databases by the BLAST search and FASTA search used, and based on these known gene sequences using chromosomes of microorganisms such as bacteria belonging to the family Enterobacteriaceae and coryneform bacteria as templates. It can be obtained by PCR using the prepared oligonucleotide as a primer.
- the gene encoding the metallopeptidase encodes a protein having the property of increasing the secretory production amount of the multimeric protein as compared with the unmodified strain when the expression is increased in coryneform bacteria, in the above amino acid sequence, It may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added.
- the property of increasing the secretory production amount of the multimeric protein compared to the unmodified strain is that before one or several substitutions, deletions, insertions or additions.
- the protein is usually maintained at 70% or more, preferably 80% or more, more preferably 90% or more.
- the above “one or several” differs depending on the position of the amino acid residue in the three-dimensional structure of the protein and the kind of amino acid residue, but specifically, preferably 1 to 20, more preferably 1 to 10 More preferably, it means 1 to 5.
- substitution, deletion, insertion, or addition of one or several amino acids described above is a conservative mutation that maintains the protein function normally.
- a typical conservative mutation is a conservative substitution.
- Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr.
- substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Trp to Phe or Tyr, substitution
- the gene having a conservative mutation as described above is 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably with respect to the entire encoded amino acid sequence.
- “homology” may refer to “identity”.
- a gene encoding a metallopeptidase hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence to all or part of the above base sequence, and increases expression in coryneform bacteria. It may be a DNA encoding a protein having the property of increasing the secreted production amount of a multimeric protein when compared with an unmodified strain. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more DNAs having homology.
- the probe used for the hybridization may be a part of a complementary sequence of a gene.
- a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template.
- hybridization washing conditions include 50 ° C., 2 ⁇ SSC, and 0.1% SDS.
- the gene encoding the metallopeptidase can be used as it is in the natural form, but it may be one obtained by replacing an arbitrary codon with an equivalent codon.
- a gene encoding a metallopeptidase may be modified to have an optimal codon depending on the codon usage frequency of the host to be used.
- the description regarding the said gene or protein variant can be applied mutatis mutandis to any protein such as cell surface protein, penicillin-binding protein, multimeric protein secreted and produced in the present invention, and genes encoding them.
- “gene expression is increased” means that the expression of a target gene is increased relative to an unmodified strain such as a wild strain or a parent strain.
- the expression of the gene is not particularly limited as long as it is higher than that of the non-modified strain, but is preferably 1.5 times or more, more preferably 2 times or more, more preferably 3 times or more compared to the non-modified strain.
- “increasing gene expression” means not only increasing the expression level of a target gene in a strain that originally expresses the target gene, but also in a strain that originally does not express the target gene. Including expressing a gene. That is, “increasing gene expression” includes, for example, introducing the gene into a strain that does not hold the target gene and expressing the gene.
- An increase in gene expression can be achieved, for example, by increasing the copy number of the gene.
- Increase in gene copy number can be achieved by introducing the target gene onto the chromosome of the host microorganism.
- Genes can be introduced into chromosomes by transposon or Mini-Mu random introduction onto chromosomes (Japanese Patent Laid-Open No. 2-109985, US5,882,888 EP805867B1), or sequences that exist in multiple copies on chromosomal DNA. This can be achieved by utilizing the target for homologous recombination.
- repetitive DNA inverted DNA
- inverted repeats present at both ends of the transposon can be used.
- a gene onto a chromosome by using the Red driven integration method (WO2005 / 010175). It is also possible to introduce a gene onto a chromosome by transduction using a phage or a conjugation transfer vector. Further, as described in WO03 / 040373, it is also possible to introduce a gene by targeting a gene unnecessary for secretory production of a heterologous protein on a chromosome. In this way, one or more copies of the gene can be introduced into the target sequence.
- the increase in the copy number of the gene can also be achieved by introducing a vector containing the target gene into the host bacterium.
- a DNA fragment containing a target gene is linked to a vector that functions in the host bacterium to construct an expression vector for the gene, and the host bacterium is transformed with the expression vector to increase the copy number of the gene.
- the vector a vector capable of autonomous replication in a host bacterial cell can be used.
- the vector is preferably a multicopy vector.
- the vector preferably has a marker such as an antibiotic resistance gene.
- the vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid.
- vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol.
- plasmids having improved drug resistance genes plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21 and pCRY2KE described in JP-A-2-72876 and US Pat. No. 5,185,262.
- the increase in gene expression can be achieved by improving the transcription efficiency of the gene.
- Improvement of gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a stronger promoter.
- strong promoter is meant a promoter that improves transcription of the gene over the native wild-type promoter.
- a stronger promoter for example, a known high expression promoter such as T7 promoter, trp promoter, lac promoter, tac promoter, PL promoter and the like can be used.
- a highly active promoter of a conventional promoter may be obtained by using various reporter genes.
- the activity of the promoter can be increased by bringing the ⁇ 35 and ⁇ 10 regions in the promoter region closer to the consensus sequence (WO 00/18935).
- Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotickpromoters in biotechnology. Biotechnol. Annu. Rev.,. 1, 105-128 (1995)).
- the increase in gene expression can be achieved by improving the translation efficiency of the gene.
- Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence.
- SD Shine-Dalgarno
- RBS ribosome binding site
- a stronger SD sequence is meant an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence.
- RBS of gene 10 derived from phage T7 can be mentioned (Olins P. O. et al, Gene, 1988, 73, 227-235).
- substitution of several nucleotides in the spacer region between the RBS and the start codon, particularly the sequence immediately upstream of the start codon (5'-UTR), or insertion or deletion contributes to mRNA stability and translation efficiency. It is known to have a great influence, and the translation efficiency of a gene can be improved by modifying them.
- a site that affects gene expression such as a promoter, an SD sequence, and a spacer region between the RBS and the start codon is also collectively referred to as an “expression control region”.
- the expression regulatory region can be determined using a promoter search vector or gene analysis software such as GENETYX.
- GENETYX gene analysis software
- These expression control regions can be modified by, for example, a method using a temperature sensitive vector or a Red driven integration method (WO2005 / 010175).
- the increase in gene expression can be achieved by amplifying a regulator that increases the expression of the target gene, or by deleting or weakening a regulator that decreases the expression of the target gene.
- the method of transformation is not particularly limited, and a conventionally known method can be used.
- a method for increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162) and methods for introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E .., 1997. Gene 1: 153-167) can be used.
- DNA-receptive cells such as those known for Bacillus subtilis, actinomycetes, and yeast, can be made into protoplasts or spheroplasts that readily incorporate recombinant DNA into recombinant DNA.
- Introduction method (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, M. J., Ward, J. M. and Hopwood, O. A. 1978. Nature 274: 398-400; Hinnen, A., Hicks, J. B. and Fink, G. R. 1978. Proc. Natl.Acad. Sci. USA 75: 1929-1933) can also be applied.
- transformation of coryneform bacteria can also be performed by an electric pulse method (Japanese Patent Laid-Open No. 2-207791).
- the increase in the expression of the target gene can be confirmed, for example, by confirming that the activity of the target protein expressed from the same gene has increased.
- An increase in the activity of the target protein can be confirmed by measuring the activity of the protein.
- the activity of the protein is preferably increased 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain, for example.
- the metallopeptidase activity can be measured by a method well known to those skilled in the art. Specifically, for example, it can be measured by a metalloprotease assay kit (manufactured by Oxford Biomedical Research).
- the increase in the expression of the target gene can be confirmed, for example, by confirming that the transcription amount of the gene has increased, or by confirming that the amount of the target protein expressed from the gene has increased. it can.
- the transcription amount of the target gene has increased by comparing the amount of mRNA transcribed from the same gene with an unmodified strain such as a wild strain or a parent strain.
- Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, etc. (Sambrook, J., et al., Molecular Cloning A Laboratory Manual / Third Edition, Cold spring Harbor Laboratory Press, Cold spring Harbor (USA ), 2001).
- the amount of mRNA is preferably increased 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain, for example.
- the amount of protein is preferably increased by 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to the unmodified strain.
- the bacterium of the present invention may further have a feature of improving the ability to produce and secrete heterologous proteins.
- the bacterium of the present invention may be modified so that the activity of penicillin binding protein is reduced.
- the bacterium of the present invention may have a reduced cell surface protein activity.
- the penicillin binding protein and the gene encoding it will be described below.
- Penicillin-binding proteins are proteins that bind to ⁇ -lactam antibiotics and whose enzyme functions are inhibited by binding to ⁇ -lactam antibiotics. .
- penicillin-binding proteins include high molecular weight PBPs (HMW-PBPs) and low molecular weight PBPs (LMW-PBPs).
- HMW-PBPs high molecular weight PBPs
- LMW-PBPs low molecular weight PBPs
- the high molecular weight PBPs include class A high molecular weight PBPs (class A HMW-PBPs) and class B high molecular weight PBPs (class B HMW-PBPs).
- the class AMW HMW-PBPs has both a transpeptidase active domain having a transpeptidase activity that crosslinks to the peptidoglycan part constituting the cell wall and a transglycosylase active domain having a transglycosylase activity that forms a polysaccharide chain from a disaccharide.
- class B HMW-PBPs has a transpeptidase active domain.
- examples of class A HMW-PBPs include PBP1a and PBP1b.
- class B HMW-PBPs include FtsI, PBP2a, and PBP2b.
- the penicillin-binding protein when the activity of the penicillin-binding protein is reduced, it is a penicillin-binding protein, and has the property of increasing the secretory production amount of a heterologous protein compared to an unmodified strain when the activity is reduced in a coryneform bacterium. Decrease the activity of the protein it has.
- a penicillin-binding protein for example, those selected from PBP1a, class B HMW-PBPs, and LMW-PBPs are preferable, those selected from PBP1a and class B HMW-PBPs are more preferable, and PBP1a is particularly preferable preferable.
- the property of increasing the secretory production of heterologous proteins when the activity is reduced in coryneform bacteria compared to non-modified strain means that a non-modified strain such as a wild strain is reduced in activity in coryneform bacteria. Alternatively, it refers to the property of giving coryneform bacteria the ability to secrete and produce a larger amount of heterologous protein than the parent strain.
- the secretory production of a larger amount of a heterologous protein than that of an unmodified strain is not particularly limited as long as the amount of secretory production of the heterologous protein is increased as compared with that of the unmodified strain, but for example, in the medium and / or on the cell surface
- the amount of accumulation is preferably 10% or more, more preferably 20% or more, particularly preferably 30% or more, and even more preferably 100% or more of the heterologous protein, as compared with the unmodified strain. Good.
- secretory production of a larger amount of heterologous protein than the unmodified strain means that the heterogeneous protein cannot be detected when the culture supernatant of the unmodified strain that has not been concentrated is subjected to SDS-PAGE and stained with CBB. It may be possible to detect a heterologous protein when the culture supernatant of a modified strain that has not been subjected to SDS-PAGE and stained with CBB.
- a modified strain is prepared, and the amount of a heterologous protein secreted and produced when the modified strain is cultured in a medium is quantified, and secreted and produced when the unmodified strain (non-modified strain) is cultured in a medium. This can be confirmed by comparing with the amount of the heterologous protein.
- the Cgl0278 gene encoding the PBP1a protein of C. glutamicum ATCC 13032 corresponds to the complementary sequence of sequences 294001 to 296388 in the genome sequence registered as GenBank accession BA000036 (VERSION BA000036.3 GI: 42602314) in the NCBI database To do.
- GenBank accession BA000036 VERSION BA000036.3 GI: 42602314
- the nucleotide sequence of Cgl0278 gene of C. glutamicum ATCC 13032 and the amino acid sequence of PBP1a protein encoded by the same gene are shown in SEQ ID NOs: 99 and 100, respectively.
- the coryneform bacterium belongs, there may be differences in the base sequence of the gene encoding the penicillin-binding protein, so when the gene encoding the penicillin-binding protein decreases its activity in the coryneform bacterium As long as it encodes a protein having the property of increasing the secretory production amount of a heterologous protein compared to an unmodified strain, it may be a variant of the above base sequence.
- a gene encoding a penicillin-binding protein has the above amino acid sequence as long as it encodes a protein having the property of increasing the secretory production amount of a heterologous protein when compared with an unmodified strain when the activity is reduced in a coryneform bacterium.
- the above description of the metallopeptidase and the variant of the gene that encodes it can be applied mutatis mutandis.
- the cell surface protein and the gene encoding it will be described below.
- the cell surface protein is a protein constituting the cell surface layer (S layer) of bacteria and archaea.
- Examples of cell surface proteins of coryneform bacteria include PS1 and PS2 (also referred to as CspB) of C. glutamicum, and SlpA (also referred to as CspA) of C. stationis. In these, it is preferable to reduce the activity of PS2 protein.
- the base sequence of the cspB gene of C. ⁇ glutamicum ATCC 13869 and the amino acid sequence of the PS2 protein encoded by the gene are shown in SEQ ID NOs: 114 and 115, respectively.
- glutamicum ATCC14068 (AY525010) C. glutamicum ATCC14747 (AY525011) C. glutamicum ATCC14751 (AY524995) C. glutamicum ATCC14752 (AY524996) C. glutamicum ATCC14915 (AY524997) C. glutamicum ATCC15243 (AY524998) C. glutamicum ATCC15354 (AY524999) C. glutamicum ATCC17965 (AY525000) C. glutamicum ATCC17966 (AY525001) C. glutamicum ATCC19223 (AY525002) C. glutamicum ATCC19240 (AY525012) C. glutamicum ATCC21341 (AY525003) C. glutamicum ATCC21645 (AY525004) C.
- glutamicum ATCC31808 (AY525013) C. glutamicum ATCC31830 (AY525007) C. glutamicum ATCC31832 (AY525008) C. glutamicum LP-6 (AY525014) C. glutamicum DSM20137 (AY525015) C. glutamicum DSM20598 (AY525016) C. glutamicum DSM46307 (AY525017) C. glutamicum 22220 (AY525005) C. glutamicum 22243 (AY525006)
- the gene encoding the cell surface protein has a reduced activity in the coryneform bacterium.
- it may be a variant of the above base sequence.
- a gene encoding a cell surface protein has the above amino acid sequence as long as it encodes a protein having a property of increasing the secreted production amount of a heterologous protein when compared with an unmodified strain when the activity is reduced in a coryneform bacterium.
- a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added is substituted, deleted, inserted or added.
- the description regarding the metallopeptidase and the variant of the gene encoding the same can be applied mutatis mutandis.
- the activity of the cell surface protein is reduced means that the coryneform bacterium has been modified so that the activity of the cell surface protein is reduced, and that This includes cases where the activity is reduced.
- “When the activity of the cell surface protein is originally reduced in the coryneform bacterium” includes the case where the coryneform bacterium originally does not have the cell surface protein. That is, examples of coryneform bacteria in which the activity of cell surface proteins is reduced include coryneform bacteria that originally have no cell surface proteins. Examples of “when the coryneform bacterium originally has no cell surface protein” include a case where the coryneform bacterium originally does not have a gene encoding the cell surface protein.
- the coryneform bacterium originally has no cell surface protein means that the coryneform bacterium is selected from one or more cell surface proteins found in other strains of the species to which the coryneform bacterium belongs. It may be that there is no protein originally.
- C. glutamicum originally has no cell surface protein means that the C. glutamicum strain is one or more proteins selected from cell surface proteins found in other C. glutamicum strains, For example, it may be inherently free of PS1 and / or PS2 (CspB).
- Examples of coryneform bacteria that originally have no cell surface protein include C. glutamicum ATCC 13032 that originally does not have a cspB gene.
- Protein activity decreases means that the activity of the target protein is reduced compared to a non-modified strain such as a wild strain or a parent strain, and includes cases where the activity is completely lost. .
- the activity of the protein is decreased means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. That is, “activity” in the case of “decrease in protein activity” is not limited to the catalytic activity of the protein, and may mean the transcription amount (mRNA amount) of the gene encoding the protein or the amount of the protein. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all. Moreover, “the function per molecule of the protein is reduced” includes the case where the function per molecule of the protein is completely lost.
- the modification that reduces the activity of the protein is achieved, for example, by reducing the expression of a gene encoding the protein.
- “the expression of the gene is reduced” is also referred to as “the expression of the gene is weakened”.
- the decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof.
- Reduction of gene expression can be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter or Shine-Dalgarno (SD) sequence.
- SD Shine-Dalgarno
- the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted.
- Factors involved in expression control include small molecules (such as inducers and inhibitors) involved in transcription and translation control, proteins (such as transcription factors), nucleic acids (such as siRNA), and the like.
- the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein.
- Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome.
- the entire gene including the sequences before and after the gene on the chromosome may be deleted.
- the region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as a decrease in protein activity can be achieved.
- the longer region to be deleted can surely inactivate the gene.
- it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.
- gene disruption is, for example, introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. It can also be achieved by introducing a frameshift mutation (Journal of Biological Chemistry 272: 8611-8617 (1997) Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833 -20839 (1991)).
- gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome.
- the insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated.
- Other sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded protein, and examples include marker genes such as antibiotic resistance genes and genes useful for heterologous protein production.
- Modifying a gene on a chromosome as described above includes, for example, deleting a partial sequence of the gene and preparing a deleted gene modified so as not to produce a normally functioning protein. This can be achieved by replacing the gene on the chromosome with the deleted gene by transforming the bacterium with the recombinant DNA containing, and causing homologous recombination between the deleted gene and the gene on the chromosome. At this time, the recombinant DNA can be easily manipulated by including a marker gene in accordance with a trait such as auxotrophy of the host. Even if the protein encoded by the deletion-type gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost.
- the modification that reduces the activity of the protein may be performed by, for example, a mutation treatment.
- Mutation treatment includes X-ray irradiation or ultraviolet irradiation, or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methylmethanesulfonate (MMS), etc.
- MNNG N-methyl-N′-nitro-N-nitrosoguanidine
- EMS ethyl methanesulfonate
- MMS methylmethanesulfonate
- the decrease in the activity of the target protein can be confirmed by measuring the activity of the protein.
- a penicillin-binding protein for example, by measuring transpeptidase activity and / or transglycosylase activity according to the class to which the protein belongs, it can be confirmed whether the activity of the protein has decreased.
- the transpeptidase activity and / or transglycosylase activity can be measured, for example, by a method well known to those skilled in the art.
- the transpeptidase and transglycosylase activities of PBP1a can be measured by measuring the reaction of polymerizing lipid II into a polysaccharide chain (glycan ⁇ strand) to form a peptide cross-link (Born P, et al.
- the activity of the protein is, for example, 50% or less, preferably 20% or less, more preferably 10% or less, still more preferably 5% or less, and particularly preferably 0% compared to the unmodified strain. descend.
- the decrease in the expression of the target gene can be confirmed by confirming that the transcription amount of the gene has decreased, or by confirming that the amount of the target protein expressed from the gene has decreased.
- the amount of mRNA is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the unmodified strain.
- the amount of protein is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the unmodified strain.
- the destruction of the target gene can be confirmed by determining a part or all of the base sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.
- the method for reducing the activity of the protein as described above can be applied mutatis mutandis to any protein and the gene encoding them, in addition to the activity of penicillin-binding protein and cell surface protein.
- gene construct for secretory expression of multimeric protein and its introduction method will be described.
- the gene construct is also referred to as “gene construct used in the present invention”.
- secretory proteins are generally translated as preproteins (also called prepeptides) or preproproteins (also called prepropeptides), and then processed into mature proteins (mature proteins).
- secreted proteins are generally translated as preproteins or preproproteins, and then the signal peptide, which is the prepart, is cleaved by a protease (commonly called signal peptidase) to be converted into a mature protein or proprotein.
- protease commonly called signal peptidase
- Proprotein is further cleaved by protease to become mature protein. Therefore, in the method of the present invention, it is preferable to use a signal peptide for secretory production of multimeric protein.
- signal peptide refers to an amino acid sequence that is present at the N-terminus of a secretory protein precursor and is not normally present in a natural mature protein.
- the gene construct used in the present invention is not particularly limited as long as secretory expression of multimeric protein can be achieved, but preferably a promoter sequence that functions in a coryneform bacterium, and functions in a coryneform bacterium connected downstream of the promoter sequence. And a nucleic acid sequence encoding a multimeric protein connected downstream of the nucleic acid sequence encoding the signal peptide.
- the nucleic acid sequence encoding the signal peptide may be linked downstream of the promoter sequence so that the signal peptide is expressed under the control of the promoter.
- the nucleic acid sequence encoding the multimeric protein may be linked downstream of the nucleic acid sequence encoding the signal peptide so that the multimeric protein is expressed as a fusion protein with the signal peptide.
- the gene construct used in the present invention has control sequences (operators, terminators, etc.) effective for expressing multimeric protein genes in coryneform bacteria at appropriate positions so that they can function. May be.
- the promoter used in the present invention is not particularly limited as long as it is a promoter that functions in coryneform bacteria, and may be a promoter derived from coryneform bacteria or a promoter derived from a different species.
- the “promoter that functions in coryneform bacteria” refers to a promoter having promoter activity in coryneform bacteria.
- Specific examples of the heterologous promoter include promoters derived from E. coli such as tac promoter, lac promoter, trp promoter, araBAD promoter and the like. Among them, a strong promoter such as tac promoter is preferable, and an inducible promoter such as araBAD promoter is also preferable.
- promoters derived from coryneform bacteria include promoters of genes encoding cell surface proteins PS1, PS2 (also referred to as CspB), SlpA (also referred to as CspA), and promoters of various amino acid biosynthesis genes.
- Specific promoters for various amino acid biosynthesis genes include, for example, glutamate biosynthesis glutamate dehydrogenase gene, glutamine synthesis glutamine synthetase gene, lysine biosynthesis aspartokinase gene, threonine biosynthesis Homoserine dehydrogenase gene, isoleucine and valine biosynthesis acetohydroxy acid synthase gene, leucine biosynthesis 2-isopropylmalate synthase gene, proline and arginine biosynthesis glutamate kinase gene, histidine biosynthesis Phosphoribosyl-ATP pyrophosphorylase gene, aromatic amino acid biosynthetic deoxyarabinohepturonic acid phosphate (DAHP) synthase genes such as tryptophan, tyrosine and phenylalanine, such as inosinic acid and guanylic acid Examples include phosphoribosyl pyrophosphate (PRPP) amide transferase gene, ino
- a high activity type of a conventional promoter may be obtained and used by using various reporter genes.
- the activity of the promoter can be increased by bringing the ⁇ 35 and ⁇ 10 regions in the promoter region closer to the consensus sequence (WO 00/18935).
- Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotickpromoters in biotechnology. Biotechnol. Annu. Rev.,. 1, 105-128 (1995)).
- substitution of several nucleotides in the spacer region between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately upstream of the start codon (5'-UTR), insertion or deletion makes the mRNA stable. It is known to greatly affect sex and translation efficiency, and these can be modified.
- the signal peptide used in the present invention is not particularly limited as long as it is a signal peptide that functions in a coryneform bacterium, and may be a signal peptide derived from a coryneform bacterium or a signal peptide derived from a different species.
- the “signal peptide that functions in coryneform bacteria” refers to a peptide that can be secreted by coryneform bacteria when linked to the N-terminus of the target protein.
- the signal peptide is preferably a signal peptide of a secretory protein of a coryneform bacterium as a host, and more preferably a signal peptide of a cell surface protein of a coryneform bacterium.
- PS1 and PS2 CspB derived from C. glutamicum (Special Table hei 6-502548), and SlpA (CspA) derived from C. ammoniagenes (C. 10-108675).
- the amino acid sequence of the signal peptide of PS1 is shown in SEQ ID NO: 101
- the amino acid sequence of the signal peptide of PS2 (CspB) is shown in SEQ ID NO: 102
- amino acid sequence of the signal peptide of SlpA (CspA) is shown in SEQ ID NO: 103.
- a DNase derived from coryneform bacteria also has a signal peptide, and such a signal peptide can also be used in the present invention.
- the signal peptide has a certain common sequence characteristic across species, but a signal peptide that exhibits a secretory function in a certain species does not necessarily exhibit a secretory function in other species. Therefore, when using a heterologous signal peptide, one that functions in coryneform bacteria may be selected as appropriate. Whether or not a signal peptide functions in coryneform bacteria can be confirmed by, for example, expressing the target protein by fusing it with the signal peptide and confirming whether the protein is secreted.
- a part of the N-terminal amino acid sequence of the secretory protein from which the signal peptide is derived may be added.
- the signal sequence is generally cleaved by a signal peptidase when the translation product is secreted outside the cell.
- the gene encoding the signal peptide can be used in its natural form, but may be modified so as to have an optimal codon according to the codon usage frequency of the host to be used.
- Examples of the multimeric protein secreted and produced by the method of the present invention include bioactive proteins, receptor proteins, antigenic proteins used as vaccines, and enzymes that are multimeric proteins.
- Examples of the physiologically active protein include growth factors (growth factors), hormones, cytokines, and antibody-related molecules.
- the antibody-related molecule refers to a protein containing a molecular species consisting of a single domain selected from domains constituting a complete antibody or a combination of two or more domains. Domains constituting a complete antibody include VH, CH1, CH2, and CH3, which are heavy chain domains, and VL and CL, which are light chain domains.
- the antibody-related molecule may be a monomeric protein or a multimeric protein as long as it contains the above-described molecular species. When the antibody-related molecule is a multimeric protein, it may be a homomultimer composed of a single type of subunit or a heteromultimer composed of two or more types of subunits. Also good.
- antibody-related molecules include, for example, complete antibodies, Fab, F (ab ′), F (ab ′) 2 , Fc, dimer consisting of heavy chain (H chain) and light chain (L chain) , Fc fusion protein, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc (Fv) 2 , disulfide bond Fv (sdFv), Diabody.
- antibody-related molecules that are multimeric proteins include, for example, complete antibodies, Fab, F (ab ′), F (ab ′) 2 , Fc, dimer consisting of heavy chain (H chain) and light chain (L chain) Body, Fc fusion protein, sc (Fv) 2 , and Diabody. Of these, Fab, F (ab ′) 2 and Fc fusion proteins are preferred.
- Fab fragment, “antigen binding” is a portion obtained by removing the F chain region of the H chain from a complete antibody, and is an antibody fragment having only an antigen binding region.
- Fab is a dimer composed of one molecule of the H chain Fab portion and one molecule of the L chain, and is associated by a C-terminal disulfide bond.
- the complete antibody is a tetramer of H2L2 and has a molecular weight of about 150 kDa, whereas Fab has a small molecular weight of about 50 kDa. Therefore, Fab is considered to have excellent permeability to the target tissue.
- Fab does not have an Fc region, so it does not have complement activity or crystallization ability, but retains ability to bind to antigen and is mainly used for antigen neutralization.
- Fab has attracted attention in recent years.
- F (ab ′) is a portion obtained by removing the H chain Fc ′ region from a complete antibody.
- F (ab ′) is a dimer composed of an F (ab ′) portion of one molecule of H chain and one molecule of L chain, and is associated by a disulfide bond at the C terminal.
- the remaining portion of the H chain in F (ab ′) is longer than the remaining portion of the H chain in Fab, and a disulfide bond portion that connects the H chains remains. Therefore, two molecules of F (ab ′) can form F (ab ′) 2 by a disulfide bond.
- F (ab ′) and F (ab ′) 2 can also be used as antibody drugs in the same manner as Fab fragments.
- Fc fragment, crystallizable
- a protein obtained by fusing an F chain region of an H chain and another functional protein is referred to as an Fc fusion protein.
- growth factors that are multimeric proteins include, for example, vascular endothelial growth factor (VEGF).
- VEGF vascular endothelial growth factor
- hormones that are multimeric proteins include insulin.
- cytokines that are multimeric proteins include interleukin-5, interferon- ⁇ , and tumor necrosis factor (Tumor Necrosis Factor; TNF).
- TNF Tumor Necrosis Factor
- growth factors growth factors
- hormones and cytokines
- the bioactive protein may belong to any one group selected from growth factors (growth factors), hormones, and cytokines, and belongs to a plurality of groups selected from them. Also good.
- the receptor protein is not particularly limited as long as it is a multimeric protein.
- it may be a receptor protein for a physiologically active protein or other physiologically active substance.
- physiologically active substances include neurotransmitters such as dopamine.
- the receptor protein may also be an orphan receptor for which the corresponding ligand is not known.
- the antigenic protein used as a vaccine is not particularly limited as long as it is a multimeric protein that can elicit an immune response, and may be appropriately selected according to the target of the assumed immune response.
- enzymes that are multimeric proteins include reverse transcriptase.
- genes encoding these proteins can be modified depending on the host used and to obtain the desired activity.
- the genes encoding these proteins may be modified so that these proteins include additions, deletions, substitutions, etc. of one or several amino acids.
- the above description concerning the metallopeptidase and the variant of the gene encoding the same can be applied mutatis mutandis to the multimeric protein secreted and produced by the method of the present invention and the gene encoding the same.
- the genes encoding these proteins may be those obtained by replacing an arbitrary codon with an equivalent codon.
- the genes encoding these proteins may be converted into optimal codons depending on the codon usage frequency of the host, if necessary.
- the N-terminal region of the multimeric protein finally obtained by the method of the present invention may or may not be the same as the natural protein.
- the N-terminal region of the finally obtained multimeric protein may have one or several extra amino acids added or deleted compared to the natural protein.
- the “one or several” may vary depending on the total length, structure, etc. of the target multimeric protein, but specifically, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to Mean 5.
- the multimeric protein that is secreted and produced may be a protein added with a pro-structure part (pro-protein).
- the finally obtained multimeric protein may or may not be a proprotein. That is, the proprotein may be cleaved from the prostructure to become a mature protein.
- the cleavage can be performed by, for example, a protease.
- a protease from the viewpoint of the activity of the finally obtained protein, it is generally preferable that the proprotein is cleaved at almost the same position as the natural protein, and cleaved at the same position as the natural protein. More preferably, a mature protein identical to the natural one is obtained.
- proteases that cleave proproteins at positions that produce proteins identical to naturally occurring mature proteins are most preferred.
- the N-terminal region of the finally obtained multimeric protein may not be the same as the natural protein.
- Proteases that can be used in the present invention include those obtained from a culture solution of microorganisms, such as a culture solution of actinomycetes, in addition to those commercially available such as Dispase (manufactured by Boehringer Mannheim). Such a protease can be used in an unpurified state, or may be used after being purified to an appropriate purity as required.
- the method for introducing the gene construct used in the present invention into coryneform bacteria is not particularly limited.
- the gene construct used in the present invention may be present on a vector that autonomously proliferates outside the chromosome, such as a plasmid, or may be integrated on the chromosome.
- the introduction of the genetic construct used in the present invention, the impartation or enhancement of the protein secretory production ability, the expression increase of the gene encoding the metallopeptidase, the penicillin binding protein Modifications such as a decrease in activity and a decrease in activity of cell surface protein can be performed in any order.
- the gene construct used in the present invention can be introduced into a host using, for example, a vector containing the gene construct.
- the vector is not particularly limited as long as it can autonomously replicate in coryneform bacteria, and may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid.
- a plasmid derived from coryneform bacteria is preferable.
- Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric, Biol.
- plasmids having improved drug resistance genes plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21 and pCRY2KE described in JP-A-2-72876 and US Pat. No. 5,185,262.
- artificial transposons can be used.
- a transposon a multimeric protein gene is introduced onto the chromosome by homologous recombination or its own transposability.
- an introduction method using homologous recombination for example, linear DNA, a plasmid containing a temperature-sensitive replication origin, a plasmid capable of conjugation transfer, or a suspension vector that does not have a replication origin that functions in the host is used.
- a method is mentioned.
- a promoter sequence and a nucleic acid sequence encoding a signal peptide contained in the gene construct are included.
- Either or both may be originally present on the host chromosome.
- a nucleic acid sequence encoding a signal peptide connected to the downstream of the promoter sequence that is originally present on the host chromosome is used as it is, and downstream of the nucleic acid sequence encoding the signal peptide.
- the gene construct used in the present invention is constructed on the chromosome, and the bacterium of the present invention can be constructed.
- the gene construct for secretory expression of each subunit may be retained in the bacterium of the present invention so that the secretory expression of the target multimeric protein can be achieved. That's fine.
- the gene construct for secretory expression of each subunit may be all retained on a single expression vector, or all may be retained on a chromosome.
- the gene construct for secretory expression of each subunit may be separately held on a plurality of expression vectors, or may be separately held on a single or a plurality of expression vectors and on a chromosome. . “When two or more types of subunits are expressed” means, for example, when two or more types of multimeric proteins are secreted and produced, or heteromultimeric proteins are secreted and produced. .
- the method of introducing the gene construct used in the present invention into the coryneform bacterium is not particularly limited, and a commonly used method such as a protoplast method (Gene, 39, 281-286 (1985)), an electroporation method (Bio / Technology, 7, 1067-1070) (1989)).
- the present invention relates to a method for producing a multimeric protein (hereinafter referred to as “the present invention”), which comprises culturing the bacterium of the present invention and recovering the secreted multimeric protein. Or a “method for producing a multimeric protein of the present invention”). That is, by culturing the bacterium of the present invention obtained as described above and expressing the multimeric protein, a large amount of multimeric protein secreted outside the bacterial cell can be obtained.
- the bacterium of the present invention can be cultured according to commonly used methods and conditions.
- the bacterium of the present invention can be cultured in a normal medium containing a carbon source, a nitrogen source, and inorganic ions.
- organic micronutrients such as vitamins and amino acids can be added as necessary.
- carbon source carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and the like can be used.
- nitrogen source ammonia gas, aqueous ammonia, ammonium salt, and others can be used.
- inorganic ions calcium ions, magnesium ions, phosphate ions, potassium ions, iron ions and the like are appropriately used as necessary. Cultivation is carried out under aerobic conditions in an appropriate range of pH 5.0 to 8.5 and 15 ° C. to 37 ° C., and cultured for about 1 to 7 days.
- culture conditions for L-amino acid production of coryneform bacteria and other conditions described in the method for producing proteins using Sec and Tat signal peptides can be used (see WO01 / 23591 and WO2005 / 103278).
- an inducible promoter when used for the expression of multimeric protein, it can also be cultured by adding a promoter inducer to the medium.
- the target protein is produced in large quantities in the microbial cells and efficiently secreted outside the microbial cells.
- the produced multimeric protein is secreted outside the microbial cells, so that proteins that are generally lethal when accumulated in large quantities in the microbial cells are not affected by lethal effects. Can be produced continuously.
- the protein secreted into the medium by the method of the present invention can be separated and purified from the cultured medium according to a method well known to those skilled in the art. For example, after removing cells by centrifugation, etc., salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium / high pressure liquid chromatography, reverse phase chromatography And can be separated and purified by a known appropriate method such as hydrophobic chromatography, or a combination thereof. In some cases, the culture or culture supernatant may be used as it is.
- the protein secreted into the cell surface by the method of the present invention is the same as that secreted into the medium after being solubilized by methods well known to those skilled in the art, for example, by increasing the salt concentration or using a surfactant. And can be separated and purified.
- the protein secreted into the surface of the bacterial cell may be used as, for example, an immobilized enzyme without solubilizing the protein.
- the secreted production of the desired multimeric protein is confirmed by conducting SDS-PAGE using the fraction containing the culture supernatant and / or cell surface as a sample and confirming the molecular weight of the separated protein band. be able to.
- the fraction containing the culture supernatant and / or cell surface layer can be used as a sample for confirmation by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press,) Cold spring Harbor (USA), 2001)).
- it can confirm by determining the N terminal amino acid sequence of the structural component of the target multimeric protein using a protein sequencer.
- it can confirm by determining the mass of the structural component of the target multimeric protein using a mass spectrometer.
- the target multimeric protein has an enzyme or some measurable physiological activity
- the fraction containing the culture supernatant and / or cell surface is used as a sample, and the enzymatic activity of the target multimeric protein Alternatively, it can be confirmed that the target multimeric protein is secreted and produced by measuring the physiological activity.
- Reference Example 1 Construction of Corynebacterium glutamicum lacking penicillin-binding protein PBP1a (1) Construction of Cgl0278 gene deletion vector pBS ⁇ Cgl0278 encoding PBP1a Genomic sequence of C. glutamicum ATCC13032 strain and Cgl0278 gene encoding penicillin-binding protein PBP1a Has already been determined (GenBank Accession No. BA000036, NCBI gene entry NCgl0274). With reference to this sequence, primers described in ⁇ SEQ ID NO: 26>, ⁇ SEQ ID NO: 27>, ⁇ SEQ ID NO: 28>, and ⁇ SEQ ID NO: 29> were synthesized. Using the chromosomal DNA of C.
- glutamicum ATCC13869 strain prepared in accordance with a conventional method (the method of Saito and Miura [Biochim. Biophys. Acta, 72, 619 (1963)]), ⁇ SEQ ID NO: 26> and ⁇ SEQ ID NO: 27> PCR was used to amplify a region of about 1 kbp 5 ′ upstream of Cgl0278 encoding PBP1a using primers, and a region of about 1 kbp 3 ′ downstream using primers ⁇ SEQ ID NO: 28> and ⁇ SEQ ID NO: 29>, respectively. .
- an about 2 kbp DNA fragment was obtained by fusion of both DNA fragments by PCR using the amplified DNA fragments as templates and the DNAs shown in ⁇ SEQ ID NO: 26> and ⁇ SEQ ID NO: 29> as primers.
- the primers ⁇ SEQ ID NO: 26> and ⁇ SEQ ID NO: 29> are designed with recognition sequences for restriction enzymes Bam HI and Xba I, respectively. Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) was used for PCR, and the reaction conditions followed the protocol recommended by the manufacturer.
- This DNA fragment was treated with restriction enzymes Bam HI and Xba I and inserted into the Bam HI-Xba I site of pBS4 described in WO2005 / 113744 to obtain a vector pBS ⁇ Cgl0278 for Cgl0278 gene deletion.
- DNA Ligation Kit Ver.2.1 manufactured by Takara Bio Inc. was used, and the reaction conditions followed the protocol recommended by the manufacturer.
- C. glutamicum YDK010 strain described in WO2004 / 029254 was transformed with the constructed pBS ⁇ Cgl0278.
- the C. glutamicum YDK010 strain is a cell surface protein PS2 deficient strain of C. glutamicum AJ12036 (FERM BP-734) (WO2004 / 029254). From the obtained transformant, a strain was selected according to the method described in WO2005 / 113744 and WO2006 / 057450 to obtain a YDK010 ⁇ PBP1a strain lacking the Cgl0278 gene.
- Example 1 Cloning of a metalloendopeptidase-like gene Cgl0858 homolog from C. glutamicum ATCC13869
- the genomic sequence of C. glutamicum ATCC13032 and the base sequence of the Cgl0858 gene encoding a metalloendopeptidase-like protein have already been determined (GenBank Accession No. BA000036, NCBI gene entry NCgl0824).
- primers described in ⁇ SEQ ID NO: 01> and ⁇ SEQ ID NO: 02> were synthesized.
- Using the chromosomal DNA of C. glutamicum ATCC13869 strain prepared in accordance with a conventional method the method of Saito and Miura [Biochim. Biophys.
- the amplified DNA fragment of about 1.1 kbp was collected by agarose gel electrophoresis using Wizard (registered trademark) SV-Gel-and PCR-Clean-Up System (Promega).
- the recovered fragment is inserted into the Sma I site of pVC7 (shuttle vector that can be replicated in both E. coli and coryneform bacteria) described in JP-A-9-070291, and then competent cells of Escherichia coli JM109 (Takara Bio) Introduced.
- pVMEP1 A strain carrying the plasmid from which the Cgl0858 homolog was cloned was obtained, from which the plasmid was recovered and named pVMEP1.
- the nucleotide sequence of the fragment cloned in pVMEP1 was determined using BigDye (registered trademark) Terminator v3.1 Sequencing Kit (Applied Biosystems) and 3130 Genetic Analyzer (Applied Biosystems). As a result of the nucleotide sequence determination, it was found that the nucleotide sequence of the Cgl0858 homologue of cloned C.Cglutamicum ATCC13869 was partially different from the nucleotide sequence of Cgl0858 of C. glutamicum ATCC13032. The base sequence of Cgl0858 homologue derived from C.
- FIG. 1 shows the alignment of the amino acid sequences of the protein encoded by Cgl0858 of C. glutamicum ATCC13032 and the protein encoded by the Cgl0858 homolog of ATCC13869.
- the homology between the protein encoded by Cgl0858 of ATCC13032 and the protein encoded by the Cgl0858 homolog of ATCC13869 was 97.9% of the total amino acid sequence. Creation of alignment and calculation of homology were performed using Genetyx_Version9 (manufactured by Genetics).
- Example 2 Secretion expression of Fab (H & L) fragment of antibody trastuzumab using C. glutamicum with enhanced expression of Cgl0858 homolog
- the gene sequence of the variable region of the heavy chain of a typical antibody trastuzumab has already been determined (GenBank Accession No. AY513484).
- the DNA shown in ⁇ SEQ ID NO: 30> to ⁇ SEQ ID NO: 63> was synthesized in consideration of the codon usage frequency of C. glutamicum with reference to this sequence and the general variable region of the antibody H chain.
- pPKSPTG1 is a secretory expression vector of protransglutaminase (transglutaminase with prostructure) described in WO01 / 23591, which is a promoter derived from the PS2 gene of C.Cglutamicum ATCC13869 strain, downstream of the promoter.
- a region containing the promoter region and the signal peptide region was amplified by PCR to obtain a DNA fragment of about 0.7 kbp. It was.
- DNAs described in ⁇ SEQ ID NO: 65> and ⁇ SEQ ID NO: 67> A DNA fragment of about 2.0 kbp fused with both DNA fragments was obtained by PCR using as a primer.
- the primer of ⁇ SEQ ID NO: 67> is designed with a recognition sequence for the restriction enzyme Kpn I.
- ⁇ SEQ ID NO: 69>, ⁇ SEQ ID NO: 70>, ⁇ SEQ ID NO: 71>, ⁇ SEQ ID NO: 72>, ⁇ SEQ ID NO: 73 >, ⁇ SEQ ID NO: 74>, and ⁇ SEQ ID NO: 75> primers are designed with recognition sequences for stop codon and restriction enzyme Kpn I, respectively. PyrobestPDNA polymerase (Takara Bio Inc.) was used for PCR, and the reaction conditions followed the protocol recommended by the manufacturer.
- the nucleotide sequence was determined using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130 Genetic Analyzer (Applied Biosystems).
- ammoniagenes ATCC6872 strain was used as a template, and shown in ⁇ SEQ ID NO: 95> and ⁇ SEQ ID NO: 96> Using a primer, a region containing the promoter region and the signal peptide region was amplified by the PCR method to obtain a DNA fragment of about 0.7 kbp.
- This fusion DNA fragment was treated with the restriction enzyme BamHI and inserted into the BamHI site of pPK4 described in JP-A-9-322774 to obtain a plasmid pPKStrast-FabL that secreted and expressed the L chain region of the Fab portion of trastuzumab.
- the base sequence was determined using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130 Genetic Analyzer (Applied Biosystems).
- glutamicum YDK010 strain was transformed. Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, phosphate, containing 5 mg / l chloramphenicol and 25 mg / l kanamycin. Potassium dihydrogen 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 450 ⁇ g, biotin 450 ⁇ g, DL-methionine 0.15 g, calcium carbonate 50 g, 1 L with water Each was cultured at 30 ° C. for 96 hours.
- the Fab (H & L) fragment of the antibody trastuzumab Comparison of secretion amount was performed.
- the Fab (H & L) fragment secretion expression plasmids compared to the control strain into which pVC7 was introduced, in the strain into which pVMEP1 was introduced, the Fab (H & L of antibody trastuzumab, which is a heterodimer, was introduced.
- the amount of fragment secretion was significantly improved (FIG. 2).
- the PVDF membrane was subjected to Western blotting using an alkaline phosphatase-labeled anti-human IgG [H & L] antibody (ROCKLAND) and Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad), and F (ab ′) 2 of the antibody trastuzumab 2 was detected.
- pPKStrast-FabH (1-2P) is a plasmid that co-expresses the H chain gene and the L chain gene containing cysteine residues that form disulfide bonds that connect the H chains.
- glutamicum YDK010 ⁇ PBP1a strain constructed in Reference Example 1 was transformed. Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, diphosphate phosphate containing 5 mg / l chloramphenicol and 25 mg / l kanamycin.
- MM liquid medium glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, diphosphate phosphate containing 5 mg / l chloramphenicol and 25 mg / l kanamycin.
- Example 2 (4) and Example 2 (6) were subjected to non-reducing SDS-PAGE using the same gel, and then applied to CBB-R250 (Bio-Rad). Then, the amount of secretory production of Fab (H & L) fragment of antibody trastuzumab was compared. As a result, the amount of secreted production of the Fab (H & L) fragment of the antibody trastuzumab, which is a heterodimer, in the strain that combined the enhanced expression of the Cgl0858 homolog and the deletion of PBP1a, compared to the strain that enhanced the expression of the Cgl0858 homolog alone. was confirmed to be further synergistically improved (FIG. 4).
- Example 3 Secretion expression of VEGF-A using Corynebacterium glutamicum with enhanced expression of Cgl0858 homolog (1) Construction of secretion expression plasmid for VEGF-A Vascular endothelial growth factor-A (vascular endothelial growth factor-A; The gene sequence of (VEGF-A) has already been determined (GenBank Accession No. NP_001165097). Considering this sequence and the codon usage of C. glutamicum, DNAs shown in ⁇ SEQ ID NO: 05> to ⁇ SEQ ID NO: 18> were synthesized.
- VEGF-A The full-length amino acid sequence of VEGF-A encoded by the DNA shown in ⁇ SEQ ID NO: 21> is shown in ⁇ SEQ ID NO: 106>, and the amino acid sequence of mature VEGF-A is shown in ⁇ SEQ ID NO: 107>.
- pPKSPTG1 described in WO01 / 23591 (including CspB (PS2) promoter region derived from C. glutamicum ATCC13869 strain and DNA encoding CspA (SlpA) signal peptide derived from C.
- ammoniagenes ATCC6872 strain Using the primers shown in SEQ ID NO: 24> and ⁇ SEQ ID NO: 25>, the promoter region and the signal peptide region were amplified by the PCR method to obtain a DNA fragment of about 0.7 kbp. Next, both amplified DNA fragments (mature VEGF gene sequence fragment, promoter region and signal peptide region fragment) are used as templates, and DNAs described in ⁇ SEQ ID NO: 24> and ⁇ SEQ ID NO: 23> are used as primers. According to the PCR method, a DNA fragment of about 1.2 kbp fused with both DNA fragments was obtained.
- primers ⁇ SEQ ID NO: 24> and ⁇ SEQ ID NO: 23> recognition sequences for restriction enzymes Kpn I and Xba I are designed.
- PrimeSTAR registered trademark
- HS DNA polymerase manufactured by Takara Bio Inc.
- This fusion DNA fragment was treated with restriction enzymes Kpn I and Xba I and then inserted into the Kpn I-Xba I site of pPK4 described in JP-A-9-322774 to obtain a plasmid pPKSVEGF for expressing VEGF-A.
- the base sequence was determined using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130 Genetic Analyzer (Applied Biosystems).
- the intensity of the protein band having the same molecular weight as that of the target VEGF-A homodimer was increased in the strain introduced with pVMEP1 compared to the control strain introduced with pVC7 (FIG. 5).
- the N-terminal sequence of this protein band was determined using the protein sequencer PPSQ-21A (manufactured by Shimadzu Corporation), it was consistent with the N-terminal sequence of VEGF-A, and VEGF-A homodimer was secreted into the culture supernatant. It was confirmed that it was expressed.
- PPSQ-21A protein sequencer
- Example 4 Secretory expression of Fab (H & L) fragment of antibody adalimumab using C. glutamicum with enhanced expression of Cgl0858 homolog (1) Construction of secretion expression plasmid of Fab (H & L) fragment of antibody adalimumab Tumor necrosis factor ⁇ The amino acid sequence of the specific antibody adalimumab (tumor necrosis factor- ⁇ ; TNF- ⁇ ) has already been determined (Independent Administrative Institution Pharmaceuticals and Medical Devices Agency Review Report (February 14, 2008)). Using this sequence as a reference, a promoter derived from the PS2 gene of the C.
- glutamicum ATCC13869 strain a DNA encoding a signal peptide derived from the SlpA of the C. stationis ATCC6872 strain operably linked downstream of the promoter, and the signal peptide DNA encoding the amino acid sequence from the 1st to 230th cysteine residues of the H chain of adalimumab ligated to be expressed as a fusion protein of C. glutamicum ATCC13869, a promoter derived from the PS2 gene of the C. glutamicum ATCC13869 strain, DNA encoding the signal peptide derived from SlpA of C.
- a promoter derived from the PS2 gene of C. glutamicum ATCC13869 strain a DNA encoding a signal peptide derived from SlpA of C. stationis ATCC6872 strain operably linked downstream of the promoter, and a fusion protein with the signal peptide DNA encoding the L chain of adalimumab ligated to be expressed as a promoter, further downstream from the PS2 gene promoter of C.
- the DNA fragment shown in SEQ ID NO: 109> was totally synthesized.
- the synthetic DNAs of ⁇ SEQ ID NO: 108> and ⁇ SEQ ID NO: 109> contain recognition sequences for the restriction enzyme BamHI at the 5 ′ end and the restriction enzyme Xba I at the 3 ′ end, respectively.
- the DNA encoding the adalimumab H chain and L chain in the synthetic DNA was designed in consideration of the codon usage of C. glutamicum.
- the DNA sequence encoding the 1st to 230th amino acid sequences of the adalimumab H chain in the synthetic DNA is shown in ⁇ SEQ ID NO: 110>, and the same amino acid sequence is shown in ⁇ SEQ ID NO: 111>.
- the DNA sequence encoding the L chain of adalimumab in the synthetic DNA is shown in ⁇ SEQ ID NO: 112>, and the amino acid sequence of the L chain of adalimumab is shown in ⁇ SEQ ID NO: 113>.
- the total synthesized DNA fragments of about 2.7 kbp were digested with restriction enzymes BamH I and Xba I, and inserted into the BamH I-Xba I site of pPK4 described in JP-A-9-322774, so that the Fab fragment of antibody adalimumab Plasmids pPKSada-FabHL and pPKSada-FabLH that co-express H chain (1-230C) and L chain were obtained.
- “FabHL” and “FabLH” in the respective plasmid names indicate the loading order of the adalimumab H chain gene and L chain gene in the expression plasmid.
- Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, phosphate, containing 5 mg / l chloramphenicol and 25 mg / l kanamycin. Potassium dihydrogen 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 450 ⁇ g, biotin 450 ⁇ g, DL-methionine 0.15 g, calcium carbonate 50 g to 1 L with water Each was cultured at 30 ° C. for 96 hours.
- the culture supernatant obtained by centrifuging each culture solution is subjected to non-reducing SDS-PAGE, followed by staining with CBB-R250 (Bio-Rad), and antibody adalimumab Fab (H & L)
- CBB-R250 Bio-Rad
- antibody adalimumab Fab H & L
- the amount of secreted fragments was compared.
- the secretion amount of the Fab (H & L) fragment of the antibody adalimumab which is a heterodimer, was significantly higher in the strain introduced with pVMEP1 than in the control strain introduced with pVC7, regardless of which secretory expression plasmid was used. It was improved (Fig. 6).
- Example 5 Secretory expression of Fab (H & L) fragment of antibody trastuzumab in C. glutamicum ATCC13869 strain with enhanced expression of Cgl0858 homologue Expression plasmid pVMEP1 of Cgl0858 homolog constructed in Example 1 and Example 2 (3) Using the constructed expression plasmid pPKStrast-FabH (1-229C) + L of the Fab (H & L) fragment of the antibody trastuzumab, the cell surface protein PS2 (CspB) of the YDK010 strain (C. glutamicum AJ12036 (FERM BP-734)) C.
- CspB cell surface protein PS2
- glutamicum ATCC13869 strain which is a wild strain of the deletion strain (WO2004 / 029254), was transformed.
- Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin.
- the secretion amount of the Fab (H & L) fragment of antibody trastuzumab was significantly improved in the strain introduced with pVMEP1 compared to the control strain introduced with pVC7 (FIG. 7). Therefore, even when the wild strain ATCC13869 was used as an expression host, the secretion amount of the antibody Fab (H & L) fragment was improved by enhancing the expression of the Cgl0858 homolog.
- a coryneform bacterium capable of efficiently secreting and producing a multimeric protein can be provided. Further, by using the coryneform bacterium provided by the present invention as an expression host, a multimeric protein, for example, an industrially useful multimeric protein can be efficiently secreted and produced.
- SEQ ID Nos: 1, 2 Primer
- SEQ ID NO: 3 Base sequence of Cgl0858 homologue of C. glutamicum ATCC13869
- SEQ ID NO: 4 Amino acid sequence of the protein encoded by Cgl0858 homologue of C.
- VEGF-A total synthesis DNA base sequence sequence numbers 19 and 20: primer sequence number 21: VEGF-A gene base sequence sequence numbers 22 to 25: primer sequence numbers 26 to 29: primer sequence numbers 30 to 63: for total synthesis of trastuzumab heavy chain DNA base sequence sequence numbers 64 and 65: primer sequence number 66: base sequence number 67 to 75 of trastuzumab H chain gene: primer sequence number 76 to 91: base sequence sequence number 92 of DNA for total synthesis of trastuzumab L chain 93: Primer SEQ ID NO: 94: Base sequence sequence number of the L chain gene of trastuzumab 95 to 97: primer SEQ ID NO: 98: amino acid sequence of the protein encoded by Cgl0858 of C.
- glutamicum ATCC13032 SEQ ID NO: 99 nucleotide sequence of Cgl0278 of C. glutamicum ATCC13032 SEQ ID NO: 100: of the protein encoded by Cgl0278 of C. glutamicum ATCC13032 Amino acid sequence SEQ ID NO: 101: Amino acid sequence of C. glutamicum-derived PS1 signal peptide SEQ ID NO: 102: Amino acid sequence of C. glutamicum-derived PS2 (CspB) signal peptide SEQ ID NO: 103: Signal peptide of C.
- SEQ ID NO: 104 amino acid sequence of trastuzumab H chain
- SEQ ID NO: 105 amino acid sequence of L chain of trastuzumab
- SEQ ID NO: 106 amino acid sequence of VEGF-A
- SEQ ID NO: 107 amino acid sequence of mature VEGF-A
- SEQ ID NO: 108 109: Fab (H & L) fragment of adalimumab
- SEQ ID NO: 110 Base sequence of adalimumab H chain gene (1-230C coding region)
- SEQ ID NO: 112 L of adalimumab
- SEQ ID NO: 113 amino acid sequence of L chain of adalimumab
- SEQ ID NO: 114 nucleotide sequence of cspB gene of
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Abstract
Description
[1]多量体タンパク質を分泌生産する能力を有し、かつ、メタロペプチダーゼをコードする遺伝子の発現が上昇するように改変されたコリネ型細菌。
[2]前記遺伝子のコピー数を高めること、または、前記遺伝子の発現調節配列を改変することにより、前記遺伝子の発現が上昇した、前記コリネ型細菌。
[3]前記メタロペプチダーゼがM23/M37メタロペプチダーゼ、またはM23/M37メタロペプチダーゼが有するモチーフと相同な領域を含むタンパク質である、前記コリネ型細菌。
[4]前記メタロペプチダーゼが、下記(A)または(B)に示すタンパク質である、前記コリネ型細菌。
(A)配列番号4に示すアミノ酸を有するタンパク質、
(B)配列番号4に示すアミノ酸配列において、1~10個のアミノ酸の置換、欠失、挿入、または付加を含むアミノ酸配列を有し、かつ、コリネ型細菌で発現を上昇させたときに多量体タンパク質の分泌生産量を非改変株と比べて上昇させる性質を有するタンパク質。
[5]さらに、ペニシリン結合タンパク質の活性が低下するように改変されている、前記コリネ型細菌。
[6]細胞表層タンパク質の活性が低下している、前記コリネ型細菌。
[7]前記コリネ型細菌が、コリネバクテリウム属またはブレビバクテリウム属に属する細菌である、前記コリネ型細菌。
[8]前記コリネ型細菌がコリネバクテリウム・グルタミカムである、前記コリネ型細菌。
[9]前記コリネ型細菌が多量体タンパク質の分泌発現用の遺伝子構築物を有し、
前記遺伝子構築物が、コリネ型細菌で機能するプロモーター配列、該プロモーター配列の下流に接続されたコリネ型細菌で機能するシグナルペプチドをコードする核酸配列、および該シグナルペプチドをコードする核酸配列の下流に接続された前記多量体タンパク質をコードする核酸配列を含む、前記コリネ型細菌。
[10]前記多量体タンパク質が抗体関連分子である、前記コリネ型細菌。
[11]前記抗体関連分子がFab、F(ab’)2、およびFc融合タンパク質から選ばれる1またはそれ以上のタンパク質である、前記コリネ型細菌。
[12]前記多量体タンパク質が血管内皮細胞増殖因子-A(VEGF-A)である、前記コリネ型細菌。
[13]前記コリネ型細菌を培養し、分泌生産された多量体タンパク質を回収することを含む、多量体タンパク質の製造方法。
本発明は、多量体タンパク質(multimer protein)を分泌生産する能力を有するコリネ型細菌であって、メタロペプチダーゼをコードする遺伝子の発現が上昇するように改変された、コリネ型細菌(以下、「本発明の細菌」または「本発明のコリネ型細菌」ともいう)を提供する。
コリネバクテリウム・アセトアシドフィラム(Corynebacterium acetoacidophilum)
コリネバクテリウム・アセトグルタミカム(Corynebacterium acetoglutamicum)
コリネバクテリウム・アルカノリティカム(Corynebacterium alkanolyticum)
コリネバクテリウム・カルナエ(Corynebacterium callunae)
コリネバクテリウム・グルタミカム(Corynebacterium glutamicum)
コリネバクテリウム・リリウム(Corynebacterium lilium)
コリネバクテリウム・メラセコーラ(Corynebacterium melassecola)
コリネバクテリウム・サーモアミノゲネス(コリネバクテリウム・エフィシエンス)(Corynebacterium thermoaminogenes (Corynebacterium efficiens))
コリネバクテリウム・ハーキュリス(Corynebacterium herculis)
ブレビバクテリウム・ディバリカタム(Brevibacterium divaricatum)
ブレビバクテリウム・フラバム(Brevibacterium flavum)
ブレビバクテリウム・イマリオフィラム(Brevibacterium immariophilum)
ブレビバクテリウム・ラクトファーメンタム(コリネバクテリウム・グルタミカム)(Brevibacterium lactofermentum (Corynebacterium glutamicum))
ブレビバクテリウム・ロゼウム(Brevibacterium roseum)
ブレビバクテリウム・サッカロリティカム(Brevibacterium saccharolyticum)
ブレビバクテリウム・チオゲニタリス(Brevibacterium thiogenitalis)
コリネバクテリウム・アンモニアゲネス(コリネバクテリウム・スタティオニス)(Corynebacterium ammoniagenes (Corynebacterium stationis))
ブレビバクテリウム・アルバム(Brevibacterium album)
ブレビバクテリウム・セリナム(Brevibacterium cerinum)
ミクロバクテリウム・アンモニアフィラム(Microbacterium ammoniaphilum)
Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium acetoglutamicum ATCC 15806
Corynebacterium alkanolyticum ATCC 21511
Corynebacterium callunae ATCC 15991
Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060,ATCC 13869,FERM BP-734
Corynebacterium lilium ATCC 15990
Corynebacterium melassecola ATCC 17965
Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC 13868
Brevibacterium divaricatum ATCC 14020
Brevibacterium flavum ATCC 13826, ATCC 14067, AJ12418(FERM BP-2205)
Brevibacterium immariophilum ATCC 14068
Brevibacterium lactofermentum ATCC 13869
Brevibacterium roseum ATCC 13825
Brevibacterium saccharolyticum ATCC 14066
Brevibacterium thiogenitalis ATCC 19240
Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872
Brevibacterium album ATCC 15111
Brevibacterium cerinum ATCC 15112
Microbacterium ammoniaphilum ATCC 15354
C. glutamicum ATCC13058(AY524990)
C. glutamicum ATCC13744(AY524991)
C. glutamicum ATCC13745(AY524992)
C. glutamicum ATCC14017(AY524993)
C. glutamicum ATCC14020(AY525009)
C. glutamicum ATCC14067(AY524994)
C. glutamicum ATCC14068(AY525010)
C. glutamicum ATCC14747(AY525011)
C. glutamicum ATCC14751(AY524995)
C. glutamicum ATCC14752(AY524996)
C. glutamicum ATCC14915(AY524997)
C. glutamicum ATCC15243(AY524998)
C. glutamicum ATCC15354(AY524999)
C. glutamicum ATCC17965(AY525000)
C. glutamicum ATCC17966(AY525001)
C. glutamicum ATCC19223(AY525002)
C. glutamicum ATCC19240(AY525012)
C. glutamicum ATCC21341(AY525003)
C. glutamicum ATCC21645(AY525004)
C. glutamicum ATCC31808(AY525013)
C. glutamicum ATCC31830(AY525007)
C. glutamicum ATCC31832(AY525008)
C. glutamicum LP-6(AY525014)
C. glutamicum DSM20137(AY525015)
C. glutamicum DSM20598(AY525016)
C. glutamicum DSM46307(AY525017)
C. glutamicum 22220(AY525005)
C. glutamicum 22243(AY525006)
本発明は、本発明の細菌を培養し、分泌生産された多量体タンパク質を回収することを含む、多量体タンパク質の製造方法(以下、「本発明の方法」または「本発明の多量体タンパク質の製造方法」ともいう)を提供する。すなわち、上記のようにして得られる本発明の細菌を培養し、多量体タンパク質を発現させることにより、菌体外に分泌された多量の多量体タンパク質が得られる。
(1)PBP1aをコードするCgl0278遺伝子欠損用ベクターpBSΔCgl0278の構築
C. glutamicum ATCC13032株のゲノム配列および、ペニシリン結合タンパク質PBP1aをコードするCgl0278遺伝子の塩基配列は既に決定されている(GenBank Accession No. BA000036, NCBI gene entry NCgl0274)。この配列を参考にして<配列番号26>、<配列番号27>、<配列番号28>、および<配列番号29>に記載のプライマーを合成した。常法に従って(斉藤、三浦の方法[Biochim. Biophys. Acta, 72, 619(1963)])調製したC. glutamicum ATCC13869株の染色体DNAを鋳型として、<配列番号26>と<配列番号27>のプライマーを用いてPBP1aをコードするCgl0278の5'側上流約1kbpを、<配列番号28>と<配列番号29>のプライマーを用いて3'側下流約1kbpの領域を、それぞれPCR法によって増幅した。次に、増幅した両DNA断片を鋳型に、<配列番号26>と<配列番号29>に示すDNAをプライマーとして用いたPCR法により両DNA断片が融合した約2kbpのDNA断片を得た。<配列番号26>と<配列番号29>のプライマーにはそれぞれ制限酵素Bam HIとXba Iの認識配列がデザインしてある。PCRにはPyrobest DNA polymerase(タカラバイオ社製)を用い、反応条件は業者の推奨するプロトコルに従った。このDNA断片を制限酵素Bam HIおよびXba Iで処理し、WO2005/113744に記載のpBS4のBam HI-Xba I部位に挿入することによって、Cgl0278遺伝子欠損用のベクターpBSΔCgl0278を得た。ライゲーション反応にはDNA Ligation Kit Ver.2.1(タカラバイオ社製)を用い、反応条件は業者の推奨するプロトコルに従った。
次に、構築したpBSΔCgl0278でWO2004/029254に記載のC. glutamicum YDK010株を形質転換した。なお、C. glutamicum YDK010株は、C. glutamicum AJ12036(FERM BP-734)の細胞表層タンパク質PS2の欠損株である(WO2004/029254)。得られた形質転換体からWO2005/113744およびWO2006/057450記載の方法に従って菌株の選択を行い、Cgl0278遺伝子が欠損したYDK010ΔPBP1a株を得た。
C. glutamicum ATCC13032のゲノム配列および、メタロエンドペプチダーゼ様タンパク質をコードするCgl0858遺伝子の塩基配列は既に決定されている(GenBank Accession No. BA000036, NCBI gene entry NCgl0824)。この配列を参考にして<配列番号01>および<配列番号02>に記載のプライマーを合成した。常法に従って(斉藤、三浦の方法[Biochim. Biophys. Acta, 72, 619(1963)])調製したC. glutamicum ATCC13869株の染色体DNAを鋳型として、<配列番号01>および<配列番号02>のプライマーを用いて、メタロエンドペプチダーゼ様タンパク質をコードするCgl0858ホモログを含む約1.1kbpの領域をPCR法によって増幅した。PCR反応にはPyrobest DNA polymerase(タカラバイオ社製)を用い、反応条件は業者の推奨するプロトコルに従った。
(1)抗体トラスツズマブのFab断片のH鎖領域の分泌発現プラスミドの構築
乳癌細胞特異的な抗体トラスツズマブのH鎖の可変領域の遺伝子配列は既に決定されている(GenBank Accession No.AY513484)。この配列と一般的な抗体H鎖の不可変領域の配列を参考にし、C. glutamicumのコドン使用頻度を考慮して<配列番号30>~<配列番号63>に示したDNAを合成した。これらのDNAを鋳型とし、別途合成した<配列番号64>と<配列番号65>に示したDNAをプライマーとして用いて、トラスツズマブの完全長H鎖領域をPCR法によって増幅し、約1.4kbpの<配列番号66>に示したDNA断片を得た。<配列番号66>に示したDNAにコードされる抗体トラスツズマブのH鎖のアミノ酸配列を<配列番号104>に示した。
乳癌細胞特異的な抗体トラスツズマブのL鎖の可変領域の遺伝子配列は既に決定されている(GenBank Accession No. AY513485)。この配列と一般的な抗体L鎖の不可変領域の配列を参考にし、C. glutamicumのコドン使用頻度を考慮して<配列番号76>~<配列番号91>に示したDNAを合成した。これらのDNAを鋳型とし、別途合成した<配列番号92>と<配列番号93>に示したDNAをプライマーとして用いて、トラスツズマブの完全長L鎖領域をPCR法によって増幅し、約0.6kbpの<配列番号94>に示したDNA断片を得た。<配列番号94>に示したDNAにコードされる抗体トラスツズマブのL鎖のアミノ酸配列を<配列番号105>に示した。次にWO01/23591記載のpPKSPTG1(C. glutamicum ATCC13869株由来のプロモーター領域とC. ammoniagenes ATCC6872株由来のシグナルペプチド領域を含む)を鋳型に、<配列番号95>と<配列番号96>に示したプライマーを用いて、上記プロモーター領域と上記シグナルペプチド領域とを含む領域をPCR法にて増幅し、約0.7kbpのDNA断片を得た。次に増幅した両DNA断片(トラスツズマブのL鎖領域を含む断片と、プロモーター領域及びシグナルペプチド領域を含む断片)を鋳型に、<配列番号95>と<配列番号97>に記載のDNAをプライマーとして用いたPCR法により、両DNA断片が融合した約1.3kbpのDNA断片を得た。<配列番号95>と<配列番号97>のプライマーにはそれぞれ制限酵素BamH Iの認識配列がデザインしてある。PCRにはPyrobest DNA polymerase(タカラバイオ社製)を用い、反応条件は業者の推奨するプロトコルに従った。この融合DNA断片を制限酵素BamH I処理し、特開平9-322774記載のpPK4のBamH I部位に挿入することによって、トラスツズマブのFab部分のL鎖領域を分泌発現させるプラスミドpPKStrast-FabLを得た。挿入断片の塩基配列決定の結果、予想通りの遺伝子が構築されていることを確認した。尚、塩基配列の決定はBigDye Terminator v3.1 Cycle Sequencing Kit(アプライドバイオシステムズ社製)と3130 ジェネティックアナライザ(アプライドバイオシステムズ社製)を用いて行った。
実施例2(1)で構築した抗体トラスツズマブのFab断片のH鎖領域の各発現プラスミドを制限酵素Kpn Iで消化することにより得られたそれぞれ約1.4kbのDNA断片を、実施例2(2)で構築した抗体トラスツズマブのFab断片のL鎖領域の発現プラスミドであるpPKStrast-FabLのKpn I部位に挿入することによって、トラスツズマブのFab断片のH鎖領域とL鎖領域を共発現させるプラスミドpPKStrast-FabH(1-223C)+L、pPKStrast-FabH(1-228T)+L、pPKStrast-FabH(1-229C)+L、pPKStrast-FabH(1-230P)+L、 pPKStrast-FabH(1-231P)+L、pPKStrast-FabH(1-232C)+L、pPKStrast-FabH(1-233P)+Lを得た。
実施例1で構築したCgl0858ホモログの発現プラスミドpVMEP1と、実施例2(3)で構築した各抗体トラスツズマブのFab(H&L)断片の分泌発現プラスミドpPKStrast-FabH(1-223C)+L、pPKStrast-FabH(1-228T)+L、pPKStrast-FabH(1-229C)+L、pPKStrast-FabH(1-230P)+L、pPKStrast-FabH(1-231P)+L、pPKStrast-FabH(1-232C)+L、pPKStrast-FabH(1-233P)+Lを用いて、WO2004/029254に記載のC. glutamicum YDK010株を形質転換した。得られた各形質転換体を、5 mg/lのクロラムフェニコールと25 mg/lのカナマイシンを含むMM液体培地(グルコース 120 g、硫酸マグネシウム七水和物 3 g、硫酸アンモニウム 30 g、リン酸二水素カリウム 1.5 g、硫酸鉄七水和物 0.03 g、硫酸マンガン五水和物 0.03 g、チアミン塩酸塩 450 μg、ビオチン 450 μg、DL-メチオニン 0.15 g、炭酸カルシウム 50 g、水で1LにしてpH7.0に調整)でそれぞれ30 ℃、96時間培養した。培養終了後、各培養液を遠心分離して得られた培養上清を非還元SDS-PAGEに供してからSYPRO Orange(インビトロジェン社製)にて染色を行い、抗体トラスツズマブのFab(H&L)断片の分泌量の比較を行った。その結果、いずれの抗体トラスツズマブのFab(H&L)断片分泌発現プラスミドを用いた場合でも、pVC7を導入した対照株と比較して、pVMEP1を導入した株では、ヘテロダイマーである抗体トラスツズマブのFab(H&L)断片の分泌量が有意に向上していた(図2)。
実施例2(4)で得られた各培養上清を非還元SDS-PAGEに供してからiBlot(登録商標)Gel Transfer Stacks PVDF, Mini(インビトロジェン社製)とiBlotTMゲルトランスファーシステム(インビトロジェン社製)を用いてPVDF膜に転写した。このPVDF膜に対してアルカリフォスファターゼ標識抗ヒトIgG[H&L]抗体(ROCKLAND社製)とAlkaline Phosphatase Conjugate Substrate Kit(バイオラッド社製)を用いたウェスタンブロッティングを行い、抗体トラスツズマブのF(ab’)2の検出を行った。その結果、H鎖同士をつなぐジスルフィド結合を形成するシステイン残基が含まれているH鎖遺伝子とL鎖遺伝子を共発現するプラスミドであるpPKStrast-FabH(1-229C)+L、pPKStrast-FabH(1-230P)+L、 pPKStrast-FabH(1-231P)+L、pPKStrast-FabH(1-232C)+L、pPKStrast-FabH(1-233P)+Lを有する形質転換体の培養上清において、抗体トラスツズマブのF(ab’)2の分子量のタンパク質バンドが検出された。さらに、これらいずれの分泌発現プラスミドを用いた場合にも、pVC7を導入した対照株と比較して、pVMEP1を導入した株では、ヘテロ四量体であるF(ab’)2の分子量のタンパク質バンドが有意に向上していた(図3)。
実施例1で構築したCgl0858ホモログの発現プラスミドpVMEP1と、実施例2(3)で構築した各抗体トラスツズマブのFab(H&L)断片の分泌発現プラスミドpPKStrast-FabH(1-223C)+L、pPKStrast-FabH(1-228T)+L、pPKStrast-FabH(1-229C)+L、pPKStrast-FabH(1-230P)+L、pPKStrast-FabH(1-231P)+L、pPKStrast-FabH(1-232C)+L、pPKStrast-FabH(1-233P)+Lを用いて、参考例1で構築したC. glutamicum YDK010ΔPBP1a株を形質転換した。得られた各形質転換体を、5mg/lのクロラムフェニコールと25 mg/lのカナマイシンを含むMM液体培地(グルコース 120 g、硫酸マグネシウム七水和物 3 g、硫酸アンモニウム 30 g、リン酸二水素カリウム 1.5 g、硫酸鉄七水和物 0.03 g、硫酸マンガン五水和物 0.03 g、チアミン塩酸塩 450 μg、ビオチン 450 μg、DL-メチオニン 0.15 g、炭酸カルシウム 50 g、水で1LにしてpH7.0に調整)でそれぞれ30 ℃、96時間培養した。培養終了後、各培養液を遠心分離して培養上清を得た。
(1)VEGF-Aの分泌発現プラスミドの構築
血管内皮細胞増殖因子-A(vascular endothelial growth factor-A;VEGF-A)の遺伝子配列は既に決定されている(GenBank Accession No. NP_001165097)。この配列と、C. glutamicumのコドン使用頻度を考慮して、<配列番号05>~<配列番号18>に示したDNAを合成した。これらのDNAを鋳型とし、別途合成した<配列番号19>と<配列番号20>に示したDNAをプライマーとして用いて、VEGF-A遺伝子配列の全長をPCR法によって増幅し、約0.6kbpの<配列番号21>に示したDNA断片を得た。次にこのDNA断片を鋳型とし、<配列番号22>と<配列番号23>に示したDNAをプライマーとして用いて、成熟型VEGF-A遺伝子配列をPCR法によって増幅し、約0.5kbpのDNA断片を得た。<配列番号21>に示したDNAにコードされるVEGF-Aの全長のアミノ酸配列を<配列番号106>に、成熟型VEGF-Aのアミノ酸配列を<配列番号107>に示した。次にWO01/23591記載のpPKSPTG1(C. glutamicum ATCC13869株由来のCspB(PS2)のプロモーター領域と、C. ammoniagenes ATCC6872株由来のCspA(SlpA)シグナルペプチドをコードするDNAを含む)を鋳型として、<配列番号24>と<配列番号25>に示したプライマーを用いて、プロモーター領域とシグナルペプチド領域をPCR法にて増幅し、約0.7kbpのDNA断片を得た。次に増幅させた両DNA断片(成熟型VEGF遺伝子配列の断片と、プロモーター領域及びシグナルペプチド領域の断片)を鋳型に、<配列番号24>と<配列番号23>に記載のDNAをプライマーとして用いたPCR法により、両DNA断片が融合した約1.2kbpのDNA断片を得た。<配列番号24>と<配列番号23>のプライマーにはそれぞれ制限酵素Kpn IおよびXba Iの認識配列がデザインしてある。PCR反応にはPrimeSTAR(登録商標) HS DNA polymerase(タカラバイオ社製)を用い、反応条件は業者の推奨するプロトコルに従った。この融合DNA断片を制限酵素Kpn I及びXba I処理後に、特開平9-322774記載のpPK4のKpn I-Xba I部位に挿入することによって、VEGF-Aを発現させるプラスミドpPKSVEGFを得た。挿入断片の塩基配列決定の結果、予想通りの遺伝子が構築されていることを確認した。尚、塩基配列の決定はBigDye Terminator v3.1 Cycle Sequencing Kit(アプライドバイオシステムズ社製)と3130 ジェネティックアナライザ(アプライドバイオシステムズ社製)を用いて行った。
実施例1(1)で構築したCgl0858ホモログの発現プラスミドpVMEP1と実施例3(1)で構築したVEGF-Aの分泌発現プラスミドpPKSVEGFを用いて、WO2004/029254に記載のC. glutamicum YDK010株を形質転換した。得られた形質転換体を、5mg/lのクロラムフェニコールと25 mg/lのカナマイシンを含むMM液体培地(グルコース 120 g、硫酸マグネシウム七水和物 3 g、硫酸アンモニウム 30 g、リン酸二水素カリウム 1.5 g、硫酸鉄七水和物 0.03 g、硫酸マンガン五水和物 0.03 g、チアミン塩酸塩 450 μg、ビオチン 450 μg、DL-メチオニン 0.15 g、炭酸カルシウム 50 g、水で1LにしてpH7.0に調整)でそれぞれ30 ℃、96時間培養した。培養終了後、各培養液を遠心分離して得られた培養上清を非還元SDS-PAGEに供してからSYPRO Orange(インビトロジェン社製)にて染色を行い、VEGF-Aの分泌量の比較を行った。その結果、pVC7を導入した対照株と比較して、pVMEP1を導入した株では、目的のVEGF-Aのホモダイマーと同じ分子量のタンパク質バンドの強度が増加していた(図5)。このタンパク質バンドのN末端配列をプロテインシークエンサーPPSQ-21A(島津製作所製)を用いて決定したところ、VEGF-AのN末端配列と一致した事から、培養上清中にVEGF-Aのホモダイマーが分泌発現している事が確認できた。以上の通り、Cgl0858ホモログの発現を上昇させることで、VEGF-Aのホモダイマーの分泌生産量を向上させることができた。
(1)抗体アダリムマブのFab(H&L)断片の分泌発現プラスミドの構築
腫瘍壊死因子α(tumor necrosis factor-α;TNF-α)特異的な抗体アダリムマブのアミノ酸配列は既に決定されている(独立行政法人医薬品医療機器総合機構審査報告書(平成20年2月14日))。この配列を参考にし、C. glutamicum ATCC13869株のPS2遺伝子由来のプロモーター、同プロモーターの下流に発現可能に連結されたC. stationis ATCC6872株のSlpA由来のシグナルペプチドをコードするDNA、および同シグナルペプチドとの融合タンパク質として発現するよう連結されたアダリムマブのH鎖の1番目から230番目のシステイン残基までのアミノ酸配列をコードするDNA、その下流にさらにC. glutamicum ATCC13869株のPS2遺伝子由来のプロモーター、同プロモーターの下流に発現可能に連結されたC. stationis ATCC6872株のSlpA由来のシグナルペプチドをコードするDNA、および同シグナルペプチドとの融合タンパク質として発現するよう連結されたアダリムマブのL鎖をコードするDNAを含む、<配列番号108>に示すDNA断片を全合成した。同様に、C. glutamicum ATCC13869株のPS2遺伝子由来のプロモーター、同プロモーターの下流に発現可能に連結されたC. stationis ATCC6872株のSlpA由来のシグナルペプチドをコードするDNA、および同シグナルペプチドとの融合タンパク質として発現するよう連結されたアダリムマブのL鎖をコードするDNA、その下流にさらにC. glutamicum ATCC13869株のPS2遺伝子由来のプロモーター、同プロモーターの下流に発現可能に連結されたC. stationis ATCC6872株のSlpA由来のシグナルペプチドをコードするDNA、および同シグナルペプチドとの融合タンパク質として発現するよう連結されたアダリムマブのH鎖の1番目から230番目のシステイン残基までのアミノ酸配列をコードするDNAを含む、<配列番号109>に示すDNA断片を全合成した。<配列番号108>、<配列番号109>の合成DNAにはそれぞれ5'末端に制限酵素BamH I、3'末端に制限酵素Xba Iの認識配列が含まれている。なお、合成DNA中のアダリムマブのH鎖およびL鎖をコードするDNAは、C. glutamicumのコドン使用頻度を考慮して設計した。合成DNA中のアダリムマブのH鎖の1番目から230番目のアミノ酸配列をコードするDNA配列を<配列番号110>、同アミノ酸配列を<配列番号111>に示した。また、合成DNA中のアダリムマブのL鎖をコードするDNA配列を<配列番号112>、アダリムマブのL鎖のアミノ酸配列を<配列番号113>に示した。全合成したそれぞれ約2.7kbpのDNA断片を制限酵素BamH IおよびXba Iで消化し、特開平9-322774に記載のpPK4のBamH I-Xba I部位に挿入することによって、抗体アダリムマブのFab断片のH鎖(1-230C)とL鎖を共発現させるプラスミドpPKSada-FabHLおよびpPKSada-FabLHを得た。それぞれのプラスミド名中の「FabHL」および「FabLH」は、発現プラスミド中のアダリムマブのH鎖遺伝子およびL鎖遺伝子の搭載順序を示している。
実施例1で構築したCgl0858ホモログの発現プラスミドpVMEP1と、実施例4(1)で構築した抗体アダリムマブのFab(H&L)断片の分泌発現プラスミドpPKSada-FabHLおよびpPKSada-FabLHを用いて、参考例1で構築したYDK010ΔPBP1a株を形質転換した。得られた各形質転換体を、5 mg/lのクロラムフェニコールと25 mg/lのカナマイシンを含むMM液体培地(グルコース 120 g、硫酸マグネシウム七水和物 3 g、硫酸アンモニウム 30 g、リン酸二水素カリウム 1.5 g、硫酸鉄七水和物 0.03 g、硫酸マンガン五水和物 0.03 g、チアミン塩酸塩 450 μg、ビオチン 450 μg、DL-メチオニン 0.15 g、炭酸カルシウム 50 gを水で1LにしてpH7.0に調整)でそれぞれ30 ℃、96時間培養した。培養終了後、各培養液を遠心分離して得られた培養上清を非還元SDS-PAGEに供してからCBB-R250(バイオラッド社製)にて染色を行い、抗体アダリムマブのFab(H&L)断片の分泌量の比較を行った。その結果、いずれの分泌発現プラスミドを用いた場合でも、pVC7を導入した対照株と比較して、pVMEP1を導入した株では、ヘテロダイマーである抗体アダリムマブのFab(H&L)断片の分泌量が有意に向上していた(図6)。このことから、トラスツズマブのFab(H&L)断片を発現させた場合に限られず、アダリムマブのFab(H&L)断片を発現させた場合でも、Cgl0858ホモログの発現を増強させることによって、抗体Fab(H&L)断片の分泌量が向上することが明らかとなった。
実施例1で構築したCgl0858ホモログの発現プラスミドpVMEP1と、実施例2(3)で構築した抗体トラスツズマブのFab(H&L)断片の分泌発現プラスミドpPKStrast-FabH(1-229C)+Lを用いて、YDK010株(C. glutamicum AJ12036(FERM BP-734)の細胞表層タンパク質PS2(CspB)の欠損株(WO2004/029254))の野生株であるC. glutamicum ATCC13869株を形質転換した。得られた各形質転換体を、25 mg/lのカナマイシンを含むMM液体培地(グルコース 120 g、硫酸マグネシウム七水和物 3 g、硫酸アンモニウム 30 g、リン酸二水素カリウム 1.5 g、硫酸鉄七水和物 0.03 g、硫酸マンガン五水和物 0.03 g、チアミン塩酸塩 450 μg、ビオチン 450 μg、DL-メチオニン 0.15 g、炭酸カルシウム 50 gを水で1LにしてpH7.0に調整)でそれぞれ30 ℃、96時間培養した。培養終了後、各培養液を遠心分離して得られた培養上清を非還元SDS-PAGEに供してからSYPRO Orange(インビトロジェン社製)にて染色を行い、抗体トラスツズマブのFab(H&L)断片の分泌量の比較を行った。その結果、pVC7を導入した対照株と比較して、pVMEP1を導入した株では、ヘテロダイマーである抗体トラスツズマブのFab(H&L)断片の分泌量が有意に向上していた(図7)。このことから、野生株であるATCC13869株を発現宿主として用いた場合でも、Cgl0858ホモログの発現を増強させることによって、抗体Fab(H&L)断片の分泌量が向上した。
配列番号1、2:プライマー
配列番号3:C. glutamicum ATCC13869のCgl0858ホモログの塩基配列
配列番号4:C. glutamicum ATCC13869のCgl0858ホモログがコードするタンパク質のアミノ酸配列
配列番号5~18:VEGF-A全合成用DNAの塩基配列
配列番号19、20:プライマー
配列番号21:VEGF-A遺伝子の塩基配列
配列番号22~25:プライマー
配列番号26~29:プライマー
配列番号30~63:トラスツズマブのH鎖全合成用DNAの塩基配列
配列番号64、65:プライマー
配列番号66:トラスツズマブのH鎖遺伝子の塩基配列
配列番号67~75:プライマー
配列番号76~91:トラスツズマブのL鎖全合成用DNAの塩基配列
配列番号92、93:プライマー
配列番号94:トラスツズマブのL鎖遺伝子の塩基配列
配列番号95~97:プライマー
配列番号98:C. glutamicum ATCC13032のCgl0858がコードするタンパク質のアミノ酸配列
配列番号99:C. glutamicum ATCC13032のCgl0278の塩基配列
配列番号100:C. glutamicum ATCC13032のCgl0278がコードするタンパク質のアミノ酸配列
配列番号101:C. glutamicum由来PS1のシグナルペプチドのアミノ酸配列
配列番号102:C. glutamicum由来PS2(CspB)のシグナルペプチドのアミノ酸配列
配列番号103:C. ammoniagenes由来SlpA(CspA)のシグナルペプチドのアミノ酸配列
配列番号104:トラスツズマブのH鎖のアミノ酸配列
配列番号105:トラスツズマブのL鎖のアミノ酸配列
配列番号106:VEGF-Aのアミノ酸配列
配列番号107:成熟型VEGF-Aのアミノ酸配列
配列番号108、109:アダリムマブのFab(H&L)断片発現用全合成DNAの塩基配列
配列番号110:アダリムマブのH鎖遺伝子(1-230Cコード領域)の塩基配列
配列番号111:アダリムマブのH鎖(1-230C)のアミノ酸配列
配列番号112:アダリムマブのL鎖遺伝子の塩基配列
配列番号113:アダリムマブのL鎖のアミノ酸配列
配列番号114:C. glutamicum ATCC13869のcspB遺伝子の塩基配列
配列番号115:C. glutamicum ATCC13869のcspB遺伝子がコードするタンパク質のアミノ酸配列
Claims (13)
- 多量体タンパク質を分泌生産する能力を有し、かつ、メタロペプチダーゼをコードする遺伝子の発現が上昇するように改変されたコリネ型細菌。
- 前記遺伝子のコピー数を高めること、または、前記遺伝子の発現調節配列を改変することにより、前記遺伝子の発現が上昇した、請求項1に記載のコリネ型細菌。
- 前記メタロペプチダーゼがM23/M37メタロペプチダーゼ、またはM23/M37メタロペプチダーゼが有するモチーフと相同な領域を含むタンパク質である、請求項1または2に記載のコリネ型細菌。
- 前記メタロペプチダーゼが、下記(A)または(B)に示すタンパク質である、請求項1~3のいずれか1項に記載のコリネ型細菌。
(A)配列番号4に示すアミノ酸を有するタンパク質、
(B)配列番号4に示すアミノ酸配列において、1~10個のアミノ酸の置換、欠失、挿入、または付加を含むアミノ酸配列を有し、かつ、コリネ型細菌で発現を上昇させたときに多量体タンパク質の分泌生産量を非改変株と比べて上昇させる性質を有するタンパク質。 - さらに、ペニシリン結合タンパク質の活性が低下するように改変されている、請求項1~4のいずれか1項に記載のコリネ型細菌。
- 細胞表層タンパク質の活性が低下している、請求項1~5のいずれか1項に記載のコリネ型細菌。
- 前記コリネ型細菌が、コリネバクテリウム属またはブレビバクテリウム属に属する細菌である、請求項1~6のいずれか1項に記載のコリネ型細菌。
- 前記コリネ型細菌がコリネバクテリウム・グルタミカムである、請求項1~7のいずれか1項に記載のコリネ型細菌。
- 前記コリネ型細菌が多量体タンパク質の分泌発現用の遺伝子構築物を有し、
前記遺伝子構築物が、コリネ型細菌で機能するプロモーター配列、該プロモーター配列の下流に接続されたコリネ型細菌で機能するシグナルペプチドをコードする核酸配列、および該シグナルペプチドをコードする核酸配列の下流に接続された前記多量体タンパク質をコードする核酸配列を含む、請求項1~8のいずれか1項に記載のコリネ型細菌。 - 前記多量体タンパク質が抗体関連分子である、請求項1~9のいずれか1項に記載のコリネ型細菌。
- 前記抗体関連分子がFab、F(ab’)2、およびFc融合タンパク質から選ばれる1またはそれ以上のタンパク質である、請求項10に記載のコリネ型細菌。
- 前記多量体タンパク質が血管内皮細胞増殖因子-A(VEGF-A)である、請求項1~9のいずれか1項に記載のコリネ型細菌。
- 請求項1~12のいずれか1項に記載のコリネ型細菌を培養し、分泌生産された多量体タンパク質を回収することを含む、多量体タンパク質の製造方法。
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IN2014CN04002A (ja) | 2015-10-23 |
EP2774982A4 (en) | 2015-03-18 |
US9404138B2 (en) | 2016-08-02 |
CN103946372A (zh) | 2014-07-23 |
JPWO2013065772A1 (ja) | 2015-04-02 |
AU2012333515B2 (en) | 2015-07-16 |
JP6136930B2 (ja) | 2017-05-31 |
EP2774982B1 (en) | 2017-06-21 |
AU2012333515A1 (en) | 2014-06-12 |
CN103946372B (zh) | 2016-06-08 |
EP2774982A1 (en) | 2014-09-10 |
US20140234901A1 (en) | 2014-08-21 |
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