WO2006129751A1 - メチオニンアミノペプチダーゼ遺伝子を含む高コピー高発現ベクター - Google Patents
メチオニンアミノペプチダーゼ遺伝子を含む高コピー高発現ベクター Download PDFInfo
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- WO2006129751A1 WO2006129751A1 PCT/JP2006/310983 JP2006310983W WO2006129751A1 WO 2006129751 A1 WO2006129751 A1 WO 2006129751A1 JP 2006310983 W JP2006310983 W JP 2006310983W WO 2006129751 A1 WO2006129751 A1 WO 2006129751A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
- C12N15/69—Increasing the copy number of the vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- the present invention relates to a vector containing a methionine aminopeptidase gene used for protein synthesis by genetic recombination technology. More specifically, it is a high-copy and high-expression type vector in which the N-terminal methionine residue (hereinafter sometimes referred to as first methionine) of the synthesized protein is removed in the synthesis reaction system.
- the present invention also relates to a vector for obtaining an unmodified protein, a method for producing the same, a transformant having the vector, and a method for producing a desired protein using the transformant.
- Non-Patent Document 1 A technique has been developed in which a MAP gene is incorporated into a vector and methionine residues are removed simultaneously by MAP in an expression system (see Non-Patent Document 1).
- High-copy high-expression vectors have already been developed in order to express a large amount of proteins with a wide variety of gene abilities derived from prokaryotes and eukaryotes.
- the target protein the protein to be produced, hereinafter referred to as the target protein
- the target protein performs its function by removing the first methionine
- the first methionine expressed using the high-copy high-expression vector A protein having a different function and activity from a natural protein.
- Patent Document 1 Japanese Patent Application Laid-Open No. 2004-208647
- Non-patent literature l Tong— Jian Shen et al., Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 8108— 8 112, September 1993, Biochemstry
- An object of the present invention is to provide a high-copy high-expression vector capable of easily mass-producing a protein having sufficient function 'activity similar to that of a natural type.
- A a target gene or a cloning site into which the target gene is inserted, (B) a sequence element necessary for high copy of the target gene, (C) a sequence element necessary for expression of the target gene and (D) a vector containing a methionine aminopeptidase gene;
- (C) The sequence elements necessary for the expression of the target gene are (C-1) promoter, (C-2) liposome binding site and (C 3) transcription termination site. (C-2) ribosome
- (A) the target gene or a cloning site into which the target gene is inserted exists between the binding site and (C3) the transcription termination site;
- the upstream transcriptional force includes, in order, a promoter, a ribosome binding site, a methionine aminopeptidase gene, a ribosome binding site, a target gene or target gene cloning site, and a base sequence having a transcription termination site. vector;
- the upstream transcription force is also inserted in this order: (C-1) T7 promoter sequence, (C-2) SD sequence, (D) methionine aminopeptidase gene, (C-2) SD sequence, and (A) target gene.
- the Ndel / BamHI cloning site and (C-3) transcription termination site the vector according to (1) above;
- Target gene force The vector according to (1) above, which is a gene encoding bemoglobin, albumin or a modified product thereof;
- the same protein as the natural form from which the first methionine has been removed can be easily mass-produced.
- human adult hemoglobin which is the main component of an artificial oxygen carrier, can be produced in large quantities with the first methionine removed as in the natural type.
- FIG. 1 shows the amino acid sequence and base sequence of human adult hemoglobin ⁇ chain.
- FIG. 2 shows the amino acid sequence and base sequence of human adult hemoglobin ⁇ chain.
- FIG. 3 shows the nucleotide sequence around the Ndel / BamHI cloning site of pET3a.
- FIG. 4 is a diagram showing the base sequence around the multicloning site of pBluescriptII KS (+).
- FIG. 5 is a diagram for explaining a method for producing a high-copy high-expression vector (pBEX) used in the production of the vector (pMAX) of the present invention in Examples.
- FIG. 6 A DNA fragment having an Xbal recognition sequence, SD sequence, MAP gene, SD sequence and Ndel recognition sequence in order of the upstream transcription force is incorporated into the Xbal / Ndel cloning site of pBEX, and the vector of the present invention (pMAX It is a figure explaining the method to manufacture.
- the vector of the present invention comprises a sequence element necessary for high copy of the target gene, It contains the sequence elements necessary for expression and the MAP gene.
- the vector of the present invention can simultaneously copy and express the target gene, and also simultaneously removes the N-terminal methionine from the expressed protein.
- the target gene is a gene that expresses the target protein, and the type thereof is not particularly limited, but is usually a gene encoding the target protein.
- the gene may be a gene that has been modified so as to be optimized for expression in the host, coding gene strength S of the target protein. Further, it may be any of a silkworm, a biological gene, or an artificially synthesized gene.
- target protein examples include proteins existing in the living body, and specific examples thereof include hemoglobin, albumin and modified products thereof.
- Preferred is a protein present in blood or a protein involved in oxygen transport, more preferred is hemoglobin, and most preferred is human adult hemoglobin.
- the protein in the present invention is not limited to a complete natural protein, but also includes a mutant protein and a partial polypeptide, but is preferably a natural protein having complete activity.
- the base sequence of the target gene does not include an intron (intervening sequence)! /, And the sequence is preferable.
- Specific examples of the target gene include genes having the base sequences described in SEQ ID NOs: 3 and 4.
- the sequence element necessary for high copy of the target gene is a base sequence necessary for replicating the target gene, and includes, for example, a base sequence including a replication origin suitable for the host.
- a replication origin suitable for the host include pUCori and fl (+) ori when the host is E. coli.
- the sequence element necessary for the replication is preferably a base sequence derived from pBluescriptll KS (+) (Stratagene), and more preferably included in a DNA fragment obtained by cleaving pBluescriptll KS (+) with EcoRI and Xbal. It is.
- sequence elements necessary for the expression of the target gene are sequences including a promoter, a ribosome binding site and a transcription termination site, and usually between the ribosome binding site and the transcription termination site. Is a sequence element in which a target gene or a cloning site exists.
- the ribosome binding site and transcription termination site are sequences derived from pET3a.
- the promoter is not particularly limited as long as it is operable in the host used for expression of the vector of the present invention.
- Examples of promoters operable in bacterial cells include E. coli T7, lac, trp or tac promoters, phage 'lambda PR or PL promoters, and the like.
- Examples of promoters that can operate in yeast host cells include promoters derived from yeast glycolytic genes, anorecordone dehydrogenase gene promoters, TPI1 promoters, and ADH2-4c promoters.
- promoters that can operate in filamentous fungal cells include ADH3 promoter and tpiA promoter.
- Promoters that can operate in mammalian cells include the SV40 promoter, the metamouthoneone gene promoter, or the adenovirus 2 major late promoter. Promoters that can operate in insect cells include polyhedrin promoter, P10 promoter, autographer 'Califor-force' polyhedrosis basic protein promoter, Bakiurovirus immediate early gene 1 promoter, or Bakiurovirus 39 K Examples include delayed early gene promoters.
- T7 promoter and tac promoter are preferred.
- T7 promoter is particularly preferred.
- Examples of the ribosome binding site include an SD sequence.
- a sequence derived from plasmid pET3a or the like can be used as a ribosome binding site.
- transcription termination sites include polyadenylation sites.
- a sequence derived from plasmid pET3a or the like can be used as a transcription termination site.
- the vector of the present invention is a vector produced by gene recombination technology, and is a vector capable of transforming host cells.
- the vector of the present invention usually contains a promoter and a ribosome binding site in order of upstream transcription force.
- MAP gene ribosome binding site, target gene or cloning part of target gene It has a position and a transcription termination site!
- the vectors of the present invention may be derived from bacterial plasmids derived from chromosomes, episomes and viruses, yeast plasmids, papovaviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, fowlpox viruses, pseudorabies disease. Examples thereof include vectors derived from viruses, retroviruses, derived from nocteriophages, derived from transposons, and combinations thereof. Of these, vectors derived from bacterial plasmids are preferred.
- base sequences derived from two or more bacterial plasmids can be used in combination.
- the vector of the present invention may have a cloning site.
- the cloning site is preferably located between the ribosome binding site and the transcription termination site.
- Examples of the clawing site include Ndel / BamHI clawing site.
- the vector of the present invention may contain an introduction marker.
- the transfer marker include drug resistance genes and the like, for example, antibiotic resistance genes such as ampicillin resistance gene, neomycin resistance gene, and tetracycline resistance gene.
- Examples of the vector of the present invention include a vector comprising a T7 promoter sequence, an SD sequence, a MAP gene, an Ndel / BamHI cloning site and a transcription termination site.
- pET3a-derived nucleotide sequence including Ndel / BamHI cloning site and transcription termination site, and nucleotide sequence including SD sequence and MAP gene should be inserted into EcoRI and Xbal sites of pBluescriptll KS (+).
- a method for incorporating a DNA fragment containing a sequence element necessary for high copy of the target gene, a sequence element necessary for expression of the target gene, and a MAP gene The ability to use various methods used in gene recombination technology. For example, a method in which ligase is added to a mixture of DNA fragments treated with various restriction enzymes and vectors, and the vector fragments are bound to the DNA fragments. Is used.
- host cells include bacterial prokaryotic cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus and Staphylococcus, fungal cells such as yeast and Aspergillus, insect cells such as Drosophila S2 and Spodoptera S19.
- Escherichia coli having a high growth ability.
- the method for introducing a vector into a host cell can be carried out by a conventionally used method.
- a combinatorial cell method for example, a combinatorial cell method, a protoplast method, a calcium phosphate coprecipitation method, an electopore position method, Microinjection method, Ribosome fusion method, Particulate cancer method, DEAE—Dextran mediated transfection, Trans vection, Cationic lipid mediated transfection, Transduction, scrape profiling, bullet introduction, infection, etc.
- a combinatorial cell method for example, a combinatorial cell method, a protoplast method, a calcium phosphate coprecipitation method, an electopore position method, Microinjection method, Ribosome fusion method, Particulate cancer method, DEAE—Dextran mediated transfection, Trans vection, Cationic lipid mediated transfection, Transduction, scrape profiling, bullet introduction, infection, etc.
- Various methods can be mentioned, and any method can be used depending on the host
- the present invention also provides a method for producing a protein using the transformant.
- a medium for culturing the transformant is known, and a nutrient medium such as a YPD medium, a minimal medium such as an MB medium, a BMMY medium, a BMGY medium, or the like can be used.
- the transformant is usually cultured at about 16 to 42 ° C, preferably about 25 to 37 ° C for about 8 to 168 hours, preferably about 24 to 120 hours. Both shaking culture and stationary culture are possible. Agitation and aeration may be added as necessary.
- the E. coli is preferably cultured at 30 to 40 ° C. and can be cultured using a known medium such as TB medium.
- a synthetic substrate for the target protein is added to the medium. It is preferable to add to the culture.
- the target protein is hemoglobin, it is preferable to add aminolevulinic acid as a synthetic substrate to the medium.
- the protein of interest is obtained by isolating the protein from the above culture solution by a known method and purifying it as necessary.
- Isolation of the fusion protein produced in the culture 'Purification methods include methods that utilize differences in solubility such as known salting-out or solvent precipitation methods, and molecular weights such as dialysis, ultrafiltration, or gel electrophoresis. Differences in hydrophobicity such as methods using differences, methods using differences in charge such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, reverse phase high performance liquid chromatography, etc. Examples thereof include a method using the difference in isoelectric point such as isoelectric focusing method.
- isolation method for example, after collecting the cells from the culture solution by centrifugation, suspending the cells in a phosphate buffer or the like, disrupting the cells by sonication, The disrupted cells are centrifuged and the supernatant is recovered, dialyzed with a phosphate buffer, etc., purified with a cation exchange ram, passed through an anion exchange column at PH7.4, and further An example is a method in which the pH is set to 8.5 and purification is performed with an anion exchange column.
- the obtained target protein is preferably stored after being concentrated, for example, in a buffer Z-glycerin mixed solution.
- the isolation / purification process for the target protein described above must be performed under temperature conditions that do not cause denaturation of the target protein. However, since MAP is highly active at temperatures higher than room temperature, optimal temperature conditions are set. There is a need to.
- Examples of methods for confirming the isolated / purified fusion protein include known Western blotting methods and activity measurement methods.
- the structure of the purified fusion protein can be revealed by amino acid analysis, amino terminal analysis, primary structure analysis, and the like.
- the protein obtained using the vector of the present invention can be used as it is or after chemical modification as necessary, and then used for various uses of known proteins. For example, it is administered to a living body for medical use. be able to.
- the protein is a protein that functions as an oxygen carrier such as hemoglobin, it can be administered as a substitute for red blood cells.
- the protein can be administered by encapsulating it in ribosomes or dispersed in emulsion.
- the protein produced using the vector of the present invention can be administered as a pharmaceutical composition containing an electrolyte, Z or plasma expander, Z or PH regulator, and Z or antioxidant.
- the target gene was designed for human adult hemoglobin as an oxygen transport protein.
- the base sequence of the target gene was designed using codons that succeeded in high expression in Escherichia coli without changing the amino acid sequence of human adult hemoglobin.
- the amino acid sequence and base sequence (SEQ ID NOs: 3 and 4) of the designed target gene are shown in Fig. 1 (a chain) and Fig. 2 ( ⁇ chain).
- each oligomer Afl, Af2, Af3, Af4, Af5, Bfl, Bf2, Bf3, Bf4, and Bf5 shown in Fig. 1 are equimolar. After mixing, the first PCR was performed. Take a small amount of this solution, add Afl5 and Arl5, and perform the second PCR.
- Fragments amplified by PCR were excised using restriction enzyme sites Ndel and Xhel introduced at the ends, and connected to plasmid pBluescriptll SK (+) (Stratagene) treated with the same enzymes.
- E. coli XL-1 Blue MRF '(Stratagene) as a host was transformed and spread on an LB plate containing ampicillin. After 1-cm culture at 37 ° C, E. coli colonies were picked and liquid-cultured using LB medium containing ampicillin. After collecting the cells, the plasmid was extracted (using Miniprep manufactured by Promega) and the nucleotide sequence was examined using a DNA sequencer (manufactured by LI-COR). It was confirmed that the expected fragment with no unexpected mutation was obtained.
- FIGS. 1-10 An outline of the pMAX construction method is shown in FIGS.
- primers pXEf and pXEr (Fig. 3) containing the Xbal and EcoRV sites of pET3a were designed, and the target sequence was amplified by PCR using these primers.
- EcoRI site was incorporated in pXEr instead of EcoRV. This is because EcoRV gives a blunt end, whereas EcoR I gives a sticky end, which makes subsequent ligation easier.
- PCR yields a fragment containing the SD sequence of pET3a, the Ndel / BamHI cloning site, and a terminator. This is treated with Xbal and EcoRI and ligated to pBluescriptl I KS (+) treated with the same restriction enzyme to obtain a vector (pBEX).
- FIG. 3 shows the nucleotide sequence around the Ndel / BamHI cloning site of pET3a.
- DNA from the Xbal site to EcoRI site in Fig. 3 containing the ribosome binding site (SD sequence) necessary for translation and the terminator part necessary for termination of transcription is used with primers (XEife and XEr in Fig. 3). Amplified by PCR.
- primer XEr uses the EcoRI recognition sequence of pET3a as the recognition sequence of EcoRI so that it can be switched to pBluescriptll KS (+). This fragment was digested with Xbal and EcoRI and inserted into the cloning site of pBluescriptll KS (+).
- Figure 4 shows the base sequence around the multicloning site of pBluescriptll KS (+).
- pBluescriptll KS (+) By incorporating the fragment grown by pET3a into pBluescriptll KS (+), the pBlues criptll KS (+)-derived T7 promoter, pET3a-derived SD sequence, Ndel recognition sequence, BamHI recognition sequence and transcription termination site were linked.
- pBluescriptll KS (+) is frequently used in cloning as a high-copy vector, and has a multiple cloning site into which various restriction enzyme cleavage sites are incorporated (Fig. 4). on the other hand
- the pET vector has the T7 promoter, SD sequence, and terminator necessary for protein induction.
- the target gene is cloned at the Ndel / BamHI cloning site downstream of the SD sequence to achieve high protein expression. ( Figure 3).
- Figure 3 the number of vector copies per cell is low.
- pBEX is a high-copy high-expression vector that takes advantage of both.
- the chromosomal strength of E. coli JM109 was also extracted by PCR, and a DNA fragment having an Xbal recognition sequence, an SD sequence, a MAP gene, an SD sequence, and an Ndel recognition sequence in order from the upstream side of the transcription (see Fig. 6).
- This DNA fragment was used as a saddle and amplified by PCR.
- pMAX restriction enzymes
- the obtained pMAX was introduced into XL-1 Blue MRF ', and bacterial cells containing pMAX were selected and used as a stock.
- E. coli BL21 (DE3) expressing T7 RNA polymerase as a host.
- the human hemoglobin (human Hb) gene was easily synthesized in E. coli!
- a gene modified so that it can be easily expressed in E. coli means a gene in which the codon of the human Hb gene is replaced with a codon with a high expression frequency in E. coli among multiple triplet codons corresponding to one amino acid. . This is known to increase the expression level of the target protein.
- the cells were suspended in 20 mM phosphate buffer (pH 7.0), and disrupted by ultrasonic treatment.
- the disrupted cells were centrifuged at 15,000 rpm for 60 minutes, and the supernatant was collected. This is dialyzed with 20 mM phosphate buffer (pH 6), purified with SP Sepharose column (Amersham Biosciences), and passed through Q- Sepharose column (Amersham Biosciences) with PH7.4. Further purify with Q- Sepharose column at pH 8.5. The eluate was concentrated to 20 mM phosphate buffer (pH 7.4) by buffer exchange, and then CO was blown and stored at 4 ° C.
- the final sample was subjected to SDS polyacrylamide gel electrophoresis and a single band with an expected molecular weight of approximately 15,00 0 was detected.
- N-terminal amino acid analysis was performed and it was the same as the expected rHb, and the first methionine was not detected.
- the molecular weights of the ⁇ chain and j8 chain were 15126.0 and 15867.5, respectively, which were consistent with the calculated values.
- the vector of the present invention is used to transform various host cells so as to mass-produce the target protein.
- the protein obtained by the production method of the present invention can be used for various protein applications, and since there is no possibility of contamination with viruses or the like, it can be administered to a living body especially for medical use.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002610780A CA2610780A1 (en) | 2005-06-03 | 2006-06-01 | High-copy-number, high-expression vector having methionine aminopeptidase gene therein |
US11/921,329 US20080166769A1 (en) | 2005-06-03 | 2006-06-01 | High-Copy-Number, High-Expression Vector Having Methionine Aminopeptidase Gene |
JP2007519059A JPWO2006129751A1 (ja) | 2005-06-03 | 2006-06-01 | メチオニンアミノペプチダーゼ遺伝子を含む高コピー高発現ベクター |
EP06747085A EP1889906A4 (en) | 2005-06-03 | 2006-06-01 | VECTOR WITH HIGH COPY AND HIGH EXPRESSION AND WITH METHIONINAMINOPEPTIDASE GENE INCLUDED |
Applications Claiming Priority (2)
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JP2005-164552 | 2005-06-03 | ||
JP2005164552 | 2005-06-03 |
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WO2006129751A1 true WO2006129751A1 (ja) | 2006-12-07 |
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PCT/JP2006/310983 WO2006129751A1 (ja) | 2005-06-03 | 2006-06-01 | メチオニンアミノペプチダーゼ遺伝子を含む高コピー高発現ベクター |
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US (1) | US20080166769A1 (ja) |
EP (1) | EP1889906A4 (ja) |
JP (1) | JPWO2006129751A1 (ja) |
CA (1) | CA2610780A1 (ja) |
WO (1) | WO2006129751A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10088488B2 (en) | 2009-11-18 | 2018-10-02 | Bio-Rad Laboratories, Inc. | Multiplex immunoassays for hemoglobin, hemoglobin variants, and glycated forms |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002253270A (ja) * | 2000-07-31 | 2002-09-10 | Takeda Chem Ind Ltd | 組換え蛋白質の製造方法 |
JP2004208647A (ja) * | 2003-01-08 | 2004-07-29 | Kumamoto Technology & Industry Foundation | 高コピー高発現ベクター |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US5753465A (en) * | 1994-08-30 | 1998-05-19 | Carnegie Mellon University | Unmodified recombinant human adult hemoglobin production |
-
2006
- 2006-06-01 WO PCT/JP2006/310983 patent/WO2006129751A1/ja active Application Filing
- 2006-06-01 US US11/921,329 patent/US20080166769A1/en not_active Abandoned
- 2006-06-01 JP JP2007519059A patent/JPWO2006129751A1/ja active Pending
- 2006-06-01 CA CA002610780A patent/CA2610780A1/en not_active Abandoned
- 2006-06-01 EP EP06747085A patent/EP1889906A4/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002253270A (ja) * | 2000-07-31 | 2002-09-10 | Takeda Chem Ind Ltd | 組換え蛋白質の製造方法 |
JP2004208647A (ja) * | 2003-01-08 | 2004-07-29 | Kumamoto Technology & Industry Foundation | 高コピー高発現ベクター |
Non-Patent Citations (3)
Title |
---|
CRAIG S.P. ET AL.: "High level expression in Escherichia coli of soluble enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase", PROC. NATL. ACAD. SCI., vol. 88, 1991, pages 2500 - 2504, XP002313723 * |
HWANG D.D.W. ET AL.: "Co-expression of glutathione S-transferase with methionine aminopeptidase: a system of producing enriched N-terminal processed proteins in Escherichia coli", BIOCHEM. J., vol. 338, 1999, pages 335 - 342, XP002297186 * |
SHEN T.-J. ET AL.: "Production of unmodified human adult hemoglobin in Escherichia coli", PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 8108 - 8112, XP002077005 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10088488B2 (en) | 2009-11-18 | 2018-10-02 | Bio-Rad Laboratories, Inc. | Multiplex immunoassays for hemoglobin, hemoglobin variants, and glycated forms |
US11262366B2 (en) | 2009-11-18 | 2022-03-01 | Bio-Rad Laboratories, Inc. | Multiplex immunoassays for hemoglobin, hemoglobin variants, and glycated forms |
Also Published As
Publication number | Publication date |
---|---|
CA2610780A1 (en) | 2006-12-07 |
EP1889906A1 (en) | 2008-02-20 |
US20080166769A1 (en) | 2008-07-10 |
EP1889906A4 (en) | 2009-06-03 |
JPWO2006129751A1 (ja) | 2009-01-08 |
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