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RU2005127888A - METHOD FOR OBTAINING HUMAN GENERAL GENERAL OBSTRUCIUM CHEMISTRINE - Google Patents

METHOD FOR OBTAINING HUMAN GENERAL GENERAL OBSTRUCIUM CHEMISTRINE Download PDF

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Publication number
RU2005127888A
RU2005127888A RU2005127888/13A RU2005127888A RU2005127888A RU 2005127888 A RU2005127888 A RU 2005127888A RU 2005127888/13 A RU2005127888/13 A RU 2005127888/13A RU 2005127888 A RU2005127888 A RU 2005127888A RU 2005127888 A RU2005127888 A RU 2005127888A
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RU
Russia
Prior art keywords
cells
escherichia coli
per
human glucagon
strain
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RU2005127888/13A
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Russian (ru)
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RU2302465C2 (en
Inventor
Роман Станиславович Есипов (RU)
Роман Станиславович Есипов
Василий Николаевич Степаненко (RU)
Василий Николаевич Степаненко
Лариса Анатольевна Чупова (RU)
Лариса Анатольевна Чупова
Александр Ильич Гуревич (RU)
Александр Ильич Гуревич
Анатолий Иванович Мирошников (RU)
Анатолий Иванович Мирошников
Original Assignee
Институт биоорганической химии им. академиков М.М. Шем кина и Ю.А. Овчинникова Российской Академии Наук (RU)
Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук
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Application filed by Институт биоорганической химии им. академиков М.М. Шем кина и Ю.А. Овчинникова Российской Академии Наук (RU), Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук filed Critical Институт биоорганической химии им. академиков М.М. Шем кина и Ю.А. Овчинникова Российской Академии Наук (RU)
Priority to RU2005127888/13A priority Critical patent/RU2302465C2/en
Publication of RU2005127888A publication Critical patent/RU2005127888A/en
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Publication of RU2302465C2 publication Critical patent/RU2302465C2/en

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (3)

1. Рекомбинантная плазмидная ДНК pER-Gl, обеспечивающая синтез гибридного полипептида, содержащего глюкагон человека и интеин, в клетках Escherichia coli, имеющая молекулярную массу 4,38 МДа, состоящая из NruI/BamHI-фрагмента ДНК плазмиды pTWIN-1; NruI/BamHI-фрагмента ДНК, содержащего адаптированную к этим сайтам последовательность гена рекомбинантного человеческого глюкагона, приведенную на рис.1; и содержащая в качестве генетического маркера ген β-лактамазы, детерминирующий устойчивость трансформированных плазмидой pER-Gl клеток E. coli к пенициллиновым антибиотикам; уникальные сайты узнавания рестрикционных эндонуклеаз, расположенные на следующем расстоянии влево от сайта BamHI-NruI-118 п.о., NdeI-773 п.о., XbaI-812 п.о., EcoRV-2847 п.о., HpaI-2903 п.о.1. Recombinant plasmid DNA pER-Gl, providing synthesis of a hybrid polypeptide containing human glucagon and intein in Escherichia coli cells, having a molecular weight of 4.38 MDa, consisting of a NruI / BamHI DNA fragment of plasmid pTWIN-1; NruI / BamHI DNA fragment containing the recombinant human glucagon gene sequence adapted to these sites, shown in Fig. 1; and containing, as a genetic marker, a β-lactamase gene that determines the resistance of E. coli cells transformed with plasmid pER-Gl to penicillin antibiotics; unique restriction endonuclease recognition sites located at the following distance to the left of the BamHI-NruI-118 bp site, NdeI-773 bp, XbaI-812 bp, EcoRV-2847 bp, HpaI-2903 by. 2. Штамм Escherichia coli ER2566/pER-Gl, продуцирующий гибридный полипептид, содержащий глюкагон человека и интеин, полученный путем трансформации клеток штамма Escherichia coli ER2566 рекомбинантной плазмидной ДНК pER-Gl по п.1.2. The strain Escherichia coli ER2566 / pER-Gl producing a hybrid polypeptide containing human glucagon and intein obtained by transformation of cells of the Escherichia coli strain ER2566 recombinant plasmid DNA pER-Gl according to claim 1. 3. Способ получения рекомбинантного глюкагона человека, включающий, трансформацию клеток Escherichia coli экспрессионной плазмидной ДНК, кодирующей гибридный белок глюкагона человека, культивирование полученного штамма, разрушение бактериальных клеток в буферном растворе с помощью ультразвука с последующим выделением и расщеплением гибридного белка и выделением и очисткой целевого продукта, отличающийся тем, что в качестве клеток используют клетки штамма Escherichia coli ER2566, в качестве плазмидной ДНК используют ДНК pER-Gl по п.1, выделение и расщепление гибридного белка глюкагона проводят путем отделения телец включения, растворения их в буфере, содержащем 2М мочевину, разбавление и доведение полученного раствора до рН до 5,5-6,0 с последующим инкубированием 24 ч при 25°С, целевой продукт очищают обращено-фазовой хроматографией.3. A method for producing recombinant human glucagon, including transforming Escherichia coli cells with expression plasmid DNA encoding a human glucagon hybrid protein, culturing the obtained strain, destroying bacterial cells in a buffer solution using ultrasound, followed by isolation and digestion of the hybrid protein and isolation and purification of the target product characterized in that cells of the Escherichia coli strain ER2566 are used as cells, pER-Gl DNA according to claim 1 is used as plasmid DNA, isolation and cleaved glucagon fusion protein is carried out by separating inclusion bodies, dissolving them in a buffer containing 2M urea, diluting and adjusting the resulting solution to pH 5.5-6.0, followed by incubation for 24 hours at 25 ° C, the target product is purified by reversed-phase chromatography.
RU2005127888/13A 2005-09-07 2005-09-07 METHOD FOR PRODUCTION OF HUMAN GENE ENGINEERED GLUCAGONE, RECOMBINANT PLASMIDE DNA pER-Gl, ENCODING AUTOCATALITICALLY CLEAVABLE HYBRID PROTEIN FORMING HUMAN GLUCAGONE AND STRAIN OF Escherichia coli ER-2566/pER-Gl AS PRODUCER OF SAID PROTEIN RU2302465C2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
RU2005127888/13A RU2302465C2 (en) 2005-09-07 2005-09-07 METHOD FOR PRODUCTION OF HUMAN GENE ENGINEERED GLUCAGONE, RECOMBINANT PLASMIDE DNA pER-Gl, ENCODING AUTOCATALITICALLY CLEAVABLE HYBRID PROTEIN FORMING HUMAN GLUCAGONE AND STRAIN OF Escherichia coli ER-2566/pER-Gl AS PRODUCER OF SAID PROTEIN

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
RU2005127888/13A RU2302465C2 (en) 2005-09-07 2005-09-07 METHOD FOR PRODUCTION OF HUMAN GENE ENGINEERED GLUCAGONE, RECOMBINANT PLASMIDE DNA pER-Gl, ENCODING AUTOCATALITICALLY CLEAVABLE HYBRID PROTEIN FORMING HUMAN GLUCAGONE AND STRAIN OF Escherichia coli ER-2566/pER-Gl AS PRODUCER OF SAID PROTEIN

Publications (2)

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RU2005127888A true RU2005127888A (en) 2007-03-20
RU2302465C2 RU2302465C2 (en) 2007-07-10

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RU2005127888/13A RU2302465C2 (en) 2005-09-07 2005-09-07 METHOD FOR PRODUCTION OF HUMAN GENE ENGINEERED GLUCAGONE, RECOMBINANT PLASMIDE DNA pER-Gl, ENCODING AUTOCATALITICALLY CLEAVABLE HYBRID PROTEIN FORMING HUMAN GLUCAGONE AND STRAIN OF Escherichia coli ER-2566/pER-Gl AS PRODUCER OF SAID PROTEIN

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* Cited by examiner, † Cited by third party
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RU2642260C1 (en) * 2016-10-21 2018-01-24 Федеральное государственное бюджетное учреждение "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Национального исследовательского центра "Курчатовский институт" (НИЦ "Курчатовский институт" - ГосНИИгенетика) Polypeptide for blood sugar level reducing on basis of human glucagon-like peptide-1, recombinant producing strain e. coli and method of obtaining this polypeptide

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JP3254692B2 (en) * 1991-09-04 2002-02-12 松下電器産業株式会社 Air conditioner wind direction control method
FI20021726A0 (en) * 2002-09-27 2002-09-27 Ctt Cancer Targeting Tech Oy Process for producing peptides

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Effective date: 20160908