WO2011086447A2 - Procédé de fermentation pour la préparation de protéines hétérologues recombinantes - Google Patents

Procédé de fermentation pour la préparation de protéines hétérologues recombinantes Download PDF

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WO2011086447A2
WO2011086447A2 PCT/IB2011/000022 IB2011000022W WO2011086447A2 WO 2011086447 A2 WO2011086447 A2 WO 2011086447A2 IB 2011000022 W IB2011000022 W IB 2011000022W WO 2011086447 A2 WO2011086447 A2 WO 2011086447A2
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protein
fermentation
medium
vector
interest
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PCT/IB2011/000022
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WO2011086447A3 (fr
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Prakash Kotwal
Sampali Banerjee
Sriram Padmanabhan
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Lupin Limited
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention is related to fermentation process for the production of heterologous proteins in E: coli expression system.
  • the invention is also related to the process for the production of heterologous proteins in E. coli using pBAD24T7gl O vector.
  • the invention is further related to the vector pBAD24T7gl O.
  • the invention is also related to the process of preparation of the vector pBAD24T7g! 0.
  • IPTG isopropyl-P-D-thiogalactopyranoside
  • pBAD24 gives low yields of proteins for example aminopeptidase N, Staphylococcus aureus lysylphospliatidyl glycerol synthetase, glutamate transport protein and putative O-antigen flippase (Golich 2006, Oku 2004, Jin 2006, Nelson 2006, Marolda 2004).
  • the present inventors while working with the pBAD24 expression vector, did not observe any expression of proteins in spite of using the optimal inducer concentration as reported by Guzman et. al 1995.
  • the invention is related to an industrial process for the production of protein of interest from E. coli expression system comprising pBAD24T7g 10 vector. In another aspect the invention is related to an industrial process for the production of protein of interest in K12 cells of E. coli expression system comprising pBAD24T7gl O vector.
  • the invention is related to the modified pBAD24 vector called as pBAD24T7gl O having the following sequence in the 5 ' UTR of the translational start site shown in SEQ ID 1 .
  • the invention is related to the process of preparation of the vector pBAD24T7g l O.
  • the invention is related to the fermentation process for the production of protein of interest from E. coli, the process comprises the steps:
  • the fermentation is induced with a suitable inducer when the OD 6 oo is in the range of 30 to 80.
  • the feed solution is added to the fermentation mediurfi after 7 to 8 hrs from inoculation.
  • the process of the invention involves addition of the feed solution at such a rate that the OD 6 oo of fermentation solution is increased to more than 160 ⁇ 5 over a period of about 14 to about 1 5 hours.
  • the invention is further related to the production of pure protein of interest from the inclusion bodies obtained from the cell lysis after fermentation step.
  • the invention also relates to pharmaceutical composition
  • pharmaceutical composition comprising therapeutically effective amount of the biologically active protein of interest obtained according to the process of the present invention.
  • Fig. 1 Plasmid map of pBAD24T7gl O
  • Fig. 2 Expression of rhGCSF as an inclusion body
  • heterologous protein or “protein of interest” refers generally to peptides and proteins exogenous i.e. foreign to the E. coli cells.
  • proteins include molecules such as, colony stimulating factors (CSFs), for example -CSF, GM-CSF.
  • CSFs colony stimulating factors
  • growth hormone including human growth hormone
  • interferon such as interferon- alpha, -beta, and -gamma
  • interleukins ILs
  • IL-2 interleukins
  • IL-1 RA interleukins
  • reteplase staphylokinase
  • Streptokinase DPP-4, DPP-8, PTH PDGFAA, PDGFAB, PDGFBB and fragments of any of the above-listed polypeptides.
  • inclusion bodies refers to dense intracellular masses of aggregated polypeptide of interest, which constitute a significant portion of the total cell protein, including the cellular components. These aggregated polypeptides may be incorrectly folded or partially correctly folded proteins.
  • therapeutically effective amount refers to the amount of biologically active protein which has the therapeutic effect of biologically active protein.
  • biologically active protein refers to protein which is capable of promoting the differentiation and proliferation of hematopoietic precursor cells and the activation of mature cells of the hematopoietic system.
  • the invention provides a process for industrial preparation of recombinant protein of interests from E. coli using pBAD24T7gl O vector.
  • a high cell density fermentation process for the production of protein of interests from E. coli using pBAD24T7gl O vector for the production of protein of interests from E. coli using pBAD24T7gl O vector.
  • the proteins synthesized using the process of the invention includes but not limited to GCSF, reteplase, interleukins, human growth hormone, DPP-4, DPP-8, staphylokinase, interferon, teriparatide.
  • the pBAD24 vector is digested with Nhel/EcoRI and ligated with the annealed oligo carrying T7gl 0 sequence elements which are already been digested with the same set of enzymes. This construction allows to retain all the MCS present in pBAD24 along with translational enhancer sequence of T7 phage.
  • the protein of interest is cloned as EcoRI/Hindlll fragment with an internally created Ndel site which provides initiation codon for translation.
  • the Figure 1 shows the plasmid map of pBAD24T7gl O.
  • pBAD24T7gl O contains an inherent ampicillin (antibiotic) marker for selection of transformants
  • other antibiotic markers like chloramphenicol (coded by chloramphenicol acetyl trasnferase- CAT), Kanamycin (APH, phosphotransferases), tetracycline resistance gene etc. can be used as antibiotic markers. These markers can be cloned either under a constitutive promoter or under their own respective promoters at suitable restriction sites of the pBAD24T7gl O vector. Fermentation
  • E. coli cells are transformed with suitable vector containing gene to synthesize rh-protein.
  • suitable vector containing gene to synthesize rh-protein Various strains of E. coli may be used for the process of the present invention for example cells which are protease deficient strains such as BL21 , ER2566 and the protease expressing strains of K 12 derivatives such as HB I OI , JM109, LE392, C600, TOP 10, DH5 alpha and the like.
  • the culture of E. coli is used for the fermentation.
  • High expression of the proteins using the pBAD24T7gl 0 vector ⁇ of the present invention depends on various parameters of the fermentation process. Some of the parameters are fermentation media, concentration of the inducer, nutrient feed rate. Culture medium is prepared for different stages of the fermentation as described below:
  • the batch medium of the fermentation process of the invention has carbon to nitrogen ratio from 1 : 0.5 to 1 : 2.
  • the fed medium of the fermentation process of the invention has carbon to nitrogen ratio from 1 : 0.05 to 1 : 1 .
  • the feed medium comprises 1 to 30 % carbon source and 1 to 20% nitrogen source.
  • the carbon source may comprise glucose, glycerol, sorbitol, maltose, sucrose or starch, mannitol.
  • the carbon source is mannitol, glucose or glycerol or mixture thereof.
  • the nitrogen source may comprise ammonia, nitrate, peptone, soy peptone, yeast extract or tryptone.
  • the nitrogen source is yeast extract, soya peptone or tryptone or mixtures thereof.
  • the feed medium comprises of 0.005 to 0.02 % antibiotics and 1 to 10 % of inorganic phosphates and trace elements.
  • the feed medium may comprise antibiotics such as ampicillin, tetracycline, any other antibiotic such as kanamycin, tetracycline, chloramphenicol, hygromycin and the like depending on the antibiotic marker of the vector.
  • antibiotics such as ampicillin, tetracycline, any other antibiotic such as kanamycin, tetracycline, chloramphenicol, hygromycin and the like depending on the antibiotic marker of the vector.
  • the expression of the protein of interests is induced at a cell density when OD 6 oo of the culture is 30 to 80.
  • the production is induced with the L-(+)-arabmose as inducer.
  • the percentage of dissolved oxygen is adjusted between 20%-60%.
  • the temperature of the fermentation broth is maintained between 30 0 C to 44 0 C and preferably at 37 °C.
  • pH of the fermentation broth is maintained at pH 6.5 - pH 7.5 and preferably at pH 7.0.
  • the fermentation may be carried out for a period of 12 to 24 hours.
  • seed medium is inoculated with glycerol stock of the culture. Inoculated flask is incubated at 37 °C, 200 rpm for 16 to 18 h.
  • Seed medium used herein is composed of 1% Tryptone or soy peptone, 0.5% yeast extract, 1 % sodium chloride suspended in water and pH of the seed medium is in the range, of 7.2 to 7.4 Preparation and inoculation of fermenter medium:
  • the fermentation medium is the medium required for the growth and expression of rh-proteins at fermenter scale.
  • the fermentation medium comprises of suitable salts, carbon source and nitrogen source while antifoam (at 0.05%) may also be added.
  • suitable salts include ammonium chloride, potassium di-hydrogen phosphate, di-sodium hydrogen phosphate, sodium chloride, magnesium sulphate and the like.
  • Suitable carbon sources include but not limited to one or more of mannitol, glucose, arabinose or glycerol.
  • Nitrogen sources which may be used include but not limited to one or more are yeast extract, tryptone, soy peptone and the like.
  • the fermentation is carried out in the presence of zinc ions.
  • zinc ions help prevent bacteriophage contaminations. Additionally the presence of zinc may help in the activation of the methionine aminopeptidase activity in E.coli as described in BBRC, 2003, 307, 172-79; Protein Science, 1998, 7, 2684-87; both the references are incorporated herein in their entirety.
  • non toxic surfactants may be added such as Tween 20, Tween-80.
  • Tween-20 at concentration of 0.05% is added in the medium, considering its role to prevent bacteriophage contamination in -bacterial fermentations.
  • the ferm enter medium is prepared using 1 to 3 % tryptone or soy peptone, 1.5 to 6 % yeast extract, 1 to 5 % mannitol, 0.05 to 0.2 % ammonium chloride, 0.1 to 0.5 % potassium di-hydrogen phosphate, 0.5 to 3 % di-sodium hydrogen phosphate, 0.01 to 0.1 % sodium chloride, 0.01 to 0.1 % magnesium sulphate, 0.00025% zinc, 0.01 to 0.2 % Tween- 20.
  • the suitable salts are dissolved in water in a transfer flask.
  • the required quantities of yeast extract and tryptone or soy peptone are mixed in water and added to a consumeriiter vessel.
  • required quantity of mannitol is dissolved in water in a transfer flask:
  • Magnesium sulphate is dissolved in another transfer flask. All the transfer flasks and the fermenter are sterilized at 121 °C for 30 minutes in an autoclave. Fermenter media is reconstituted aseptically after all the solutions in transfer flasks are cooled.
  • Feed solution used for growth and expression of rh-proteins at fermenter scale is prepared as follows.
  • the feed solution comprises of suitable salts, carbon source, nitrogen source, inducer and antibiotic.
  • the concentration of the feed solution comprises of 1 to 5 % Tryptone or soy peptone, 5 to 20 % Yeast Extract, 5 to 30 % Mannitol, 0.1 to 0.5 % Ammonium chloride, 0.5 to 2 % Potassium di-hydrogen phosphate, 2 to 8 % di-Sodium hydrogen phosphate, 0.025 to 0.3 % Sodium chloride, 0:025 to 0.3 % Magnesium sulphate. L-(+)-arabinose is added as inducer. Inducer is used in the range of about 3 to 25 mM.
  • the feed solution is prepared by dissolving required quantities of salts in water as solution A.
  • the required quantities of yeast extract and tryptone or soy peptone are mixed and dissolved in water as solution B.
  • the solution of carbon source, mannitol is prepared by dissolving in water as solution C.
  • Solution D is prepared by dissolving magnesium sulphate in water. All the solutions A, B, C and D in separate flasks are sterilized separately at 121 °C in an autoclave and mixed together.
  • L-(+)-arabinose and antibiotic such as ampicillin is also added.
  • the fermenter used for the invention is microbial fermenter having provision for oxygen enrichment and is equipped with control devices comprising of sensors & controllers for temperature, pH and oxygen while pumps for addition of feed medium, acid/base, inducer and antifoam solutions.
  • the pH is maintained using buffers preferably 30% Phosphoric acid or 12% Ammonia solution.
  • Antifoam used is 1510-US from Dow corning & is added drop wise as per requirement.
  • Fermenter with sterilized media is connected to its control panel; pH and DO probes and feed pumps are checked for their calibration. Initially agitation is set at 300 rpm and is increased to maximum of 800 rpm as to maintain dissolved oxygen in a range of 20 to 60 %. Aeration is maintained at I vvm of air. If required, air is supplemented with oxygen gas in the proportion set by automatic controller of the instrument so as to maintain dissolved oxygen in a range of 20 to 60 %. Temperature is maintained at 37°C by PID controller of the instrument. Acid, base and sterile antifoam solution bottles are connected to respective ports on to a fermenter with the help of silicon tubing and pumps. pH is maintained at 7.00 by automatic addition of acid or base with the help of PID controller of the instrument.
  • Filter sterilized ampicillin solution is added at concentration of 100 ⁇ g/ml.
  • the feed solution is added at such a rate that the OD 60 o of fermentation solution is increases to more than 1 60 ⁇ 1 0 over a period of about 14 to 15 hours.
  • Cell pellet of 2 liter fermented broth is suspended in 800 ml to 1 liter of 10 mM Tris pH 8.0 and is mixed so as to get uniform mixture, having initial OD 6 oo between 100 to 1 50.
  • Suspended cell pellet is subjected to homogenization in a homogenizer. Homogenization is carried out at a pressure of 800 to 900 bars and is done for 3 to 4 cycles, which is for about 20 minutes. The entire operation is done under cooling conditions. The fall in optical density of cell suspension is between 80 to 85%.
  • the invention further provides a process for production of pure protein of interest from the inclusion bodies.
  • the process for the production of pure protein of interest from the inclusion bodies includes solubilizing the inclusion bodies of proteins; refolding the said solubilized proteins; purifying the refolded proteins; and isolating pure proteins. There are various methods reported in the literature, one skilled in the art can follow one or more methods to obtain protein of interest.
  • Purification methods include but not limited to aqueous two phase extraction, various chromatography techniques.
  • the purification of GCSF is disclosed in our copending patent application No. IN 865/KOL/2009 filed on June 16, 2009, which is incorporated herein by reference in its entirety).
  • the protein obtained using the aqueous two phase extraction process may be further processed, to provide the protein or polypeptide having high purity. Further purification may be necessary to remove related impurities.
  • the impurities may include oxidized forms, deamidated forms, aggregated proteins and also degraded forms such as biologically inactive monomeric forms, incorrectly folded protein molecules, denaturated forms of protein, host cell proteins, host cell substances such as DNAs, (lipo)polysaccharides etc and additives which had been used in the preparation and processing of proteins.
  • Such higher purity may be required depending on the use for which the protein or polypeptide is intended.
  • therapeutic uses of the protein will typically require further purification following the extraction methods of the invention. All protein purification methods known to the skilled artisan may be used for further purification. Such techniques have been extensively described in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Volume 152, Academic Press, San Diego, Calif.
  • the yield of the protein of interest from E. coli using pBAD24T7gl 0 vector using the processes of the invention are in the range of 20 to 35% of the total cellular protein in the bacterial cell.
  • the process of obtaining pure protein of interest as described herein further comprises of forming the pure proteins into a finished dosage form for clinical use.
  • the biologically active protein obtained by the entire process for the purification and/or isolation of the present invention is suitable for the preparation of pharmaceutical composition, which comprises the therapeutically effective amount of biologically active protein and one or more pharmaceutical excipients and is suitable for clinical use.
  • pharmaceutical composition which comprises the therapeutically effective amount of biologically active protein and one or more pharmaceutical excipients and is suitable for clinical use.
  • Suitable pharmaceutically acceptable excipients include but not limited to suitable diluents, adjuvants and/or carriers and the similar useful in protein therapy.
  • the invention relates to pharmaceutical compositions containing the proteins obtained according to the present invention.
  • the proteins obtained can either be stored in the form of a lyophilized powder or in liquid form. It is administered either subcutaneously or intravenously.
  • pBAD24 plasmid was digested with Nhel/EcoRI and ligated with the annealed oligo carrying T7gl0 sequence elements that has already been digested with the same set of enzymes. This construction would allow retaining all the MCS present in pBAD24 along with translational enhancer sequence of T7 phage.
  • human GCSF gene was cloned as a EcoRI/Hindlll fragment with an internally created Ndel site that would provide initiation codon for translation.
  • Culture used was genetically engineered Escherichia coli K 12 cells transformed with suitable vector containing gene to synthesize rh-proteins.
  • tactca insects tactttagat tgatttacgc gccctgtagc ggcgcattaa gcgcggcggg
  • Seed medium was the medium used for initial growth of the culture from the glycerol stock of it. Seed medium was composed of 1% Tryptone or soy peptone, 0.5% Yeast Extract 1 % Sodium chloride suspended in water while pH of this medium falls in the range of 7.2 to 7.4.
  • Fermenter medium used for growth and expression of rh-GCSF at fermenter scale is composed of 1 .8% tryptone or soy peptone, 3.6% yeast Extract, 1% (1.5 %) Mannitol, 0.1% Ammonium chloride, 0.3% Potassium di-hydrogen phosphate, 1 .2% di-Sodium hydrogen phosphate, 0.05% Sodium chloride, 0.05% Magnesium sulphate, 0.00025% zinc and 0.05% Tween-20 " .
  • Feed solution used for growth and expression of rh-GCSF at fermenter scale is composed of 3.9 % Tryptone or soy peptone ,' 7.8 % Yeast Extract, 20 % mannitol, 0.325% Ammonium chloride, 0.976% Potassium di-hydrogen phosphate, 4.162% di-Sodium hydrogen phosphate, 0.162% Sodium chloride, 0.162%) Magnesium sulphate.
  • Inducer used was 20mM L-(+)-arabinose.
  • Fermenter used was Biostat B plus (M/s Sartorius) microbial fermenter having provision for oxygen enrichment. For maintaining the pH during process either 30% Phosphoric acid or 12% Ammonia solution was used.
  • Antifoam used was 151 0- US from Dow corning & was added drop wise as per requirement
  • Preparation of seed culture 200ml seed medium, composition as described above was inoculated with glycerol stock of the culture. Inoculated flask was incubated on rotary shaker at 37°C, 200 rpm for 16 to 1 8 hours.
  • A-Required quantities of salts as described in the fermenter medium were dissolved in 700 ml of water and were added to a transfer flask.
  • yeast extract B- Required quantities of yeast extract, tryptone or soy peptone & Tween-20 were mixed and dissolved in 850ml of water. This solution was prepared in a fermenter vessel.
  • A, B, C & D were sterilized separately at 121 °C for 30 minutes in an autoclave, after cooling all three were mixed aseptically in a fermenter.
  • E-Required quantities of salts as described in the 1 feed solution were dissolved in 200ml of water
  • F- Required quantities of Yeast extract and Tryptone or soy peptone were mixed and dissolved in 280ml of water.
  • E, F, G & H were prepared in the separate flasks, sterilized separately at 121°C for 30 minutes in an autoclave and were pooled together to make 800ml feed solution.
  • 2.4 g inducer L-(+)-arabinose was dissolved in 20ml of water, filter sterilized & was added to feed solution.
  • Temperature was maintained at 37 °C by PID controller of the instrument. Acid, base and sterile antifoam solution bottles were connected to respective ports on to a fermenter with the help of silicon tubing and pumps. pH was maintained at 7.00 by automatic addition of acid or base with the help of PID controller of the instrument.
  • OD 6 oo of culture was gradually increased to 160 over a period 14 to 15 hours.
  • Cell pellet of 2 liter fermented broth was suspended in 800 ml to 1 liter of 10 niM Tris pH 8.0 and was mixed so as to get uniform mixture of it having initial OD 6 oo between 100 to 50. Suspended cell pellet was subjected to homogenization.
  • Homogenization was carried out at a pressure of 800 to 900 bars and was done for 3 to 4 cycles, which was about 20 minutes. The entire operation was done under cooling conditions. Optical cell density falls in a range of 20 to 30. Cell lysate was chilled to about 5 to 8 °C on ice for 30 mins.
  • Chilled cell lysate was subjected to centrifugation at 9000 rpm for 30 minutes at 4 °C. The supernatant obtained is separated and discarded while the pellet fraction i.e. inclusion bodies are preserved at -20 °C.

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Abstract

La présente invention concerne un procédé de fermentation à densité de cellules élevée pour la production de protéines hétérologues dans un système d'expression de E. coli. L'invention concerne en outre le procédé pour la production de protéines hétérologues dans E. coli en utilisant un vecteur pBAD24T7g10. L'invention concerne en outre le vecteur pBAD24T7g10. L'invention concerne en outre le procédé de préparation du vecteur pBAD24T7g10. La fermentation peut être effectuée en utilisant des vecteurs de E. coli K12.
PCT/IB2011/000022 2010-01-12 2011-01-10 Procédé de fermentation pour la préparation de protéines hétérologues recombinantes WO2011086447A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017537641A (ja) * 2014-12-29 2017-12-21 アグリカルチュラル テクノロジー リサーチ インスティテュート 発現エレメント、発現カセット、及びそれらを含むベクター
CN114015628A (zh) * 2021-12-17 2022-02-08 河南兴华生物技术有限公司 表达pmt蛋白的培养基及培养方法
CN114107264A (zh) * 2021-12-07 2022-03-01 武汉新华扬生物股份有限公司 一种发酵生产氨肽酶的方法
CN114350588A (zh) * 2021-12-31 2022-04-15 山东新时代药业有限公司 一种rhG-CSF的发酵培养基及其发酵方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004001056A1 (fr) 2002-06-24 2003-12-31 Dr. Reddy's Laboratories Ltd. Procede de preparation de g-csf
US20070015248A1 (en) 2003-12-22 2007-01-18 Andreas Anton Expression vector and use thereof
WO2007102174A2 (fr) 2006-03-06 2007-09-13 Cadila Healthcare Limited Méthode de préparation du g-csf humain
WO2008096368A2 (fr) 2007-02-05 2008-08-14 Natco Pharma Limited Nouveau procédé de production du facteur g-csf humain de recombinaison

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2406955A1 (fr) * 2000-04-19 2001-10-25 Ovita Limited Pou du mouton, bovicola ovis, traitement par allergene
CN101809146B (zh) * 2007-05-22 2013-12-11 Ls9公司 产烃基因及其使用方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004001056A1 (fr) 2002-06-24 2003-12-31 Dr. Reddy's Laboratories Ltd. Procede de preparation de g-csf
US20070015248A1 (en) 2003-12-22 2007-01-18 Andreas Anton Expression vector and use thereof
WO2007102174A2 (fr) 2006-03-06 2007-09-13 Cadila Healthcare Limited Méthode de préparation du g-csf humain
WO2008096368A2 (fr) 2007-02-05 2008-08-14 Natco Pharma Limited Nouveau procédé de production du facteur g-csf humain de recombinaison

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", JOHN WILEY & SONS
"New Protein Techniques: Methods in Molecular Biology", 1988, HUMANA PRESS
BBRC, vol. 307, 2003, pages 172 - 79
BERGER; KIMMEL: "Guide to Molecular Cloning Techniques, Methods in Enzymology", vol. 152, 1987, ACADEMIC PRESS
D. PERLMAN; WAYNE W.: "Advances in Applied Microbiology", article "Umbreit Bacteriophages of the genus clostridium in a journal"
GUZMAN ET AL., J. BACTERIOL., vol. 177, 1995, pages 41214230
GUZMAN, J. BACTERIOL, vol. 177, 1995, pages 4121 - 30
HARRISON, INNOVATIONS, vol. 11, 2000, pages 4 - 7
KHALILZADEH ET AL., IRAN. J. BIOTECHNOL., vol. 6, no. 2, 2008, pages 63 - 84
PROTEIN SCIENCE, vol. 7, 1998, pages 2684 - 87
R. C. VAN DAM MIERAS: "Biotechnological Innovations in chemical synthesis", pages: 248
SAMBROOK, J.; FRITSCH, E. F.; MANIATIS, T.: "Molecular Cloning: A Laboratory Manual", 1989
SCOPES, R. K.: "Protein Purification: Principles and Practice", 1987, SPRINGER-VERLAG
YEE; BLANCH, BIOTECHNOLOGY AND BIOENGINEERING, vol. 41, 1993, pages 221 - 230

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