EA025689B9 - Способ высокой экспрессии рекомбинантного белка в генно-инженерных бактериях и его применение - Google Patents

Способ высокой экспрессии рекомбинантного белка в генно-инженерных бактериях и его применение

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Publication number
EA025689B9
EA025689B9 EA201400607A EA201400607A EA025689B9 EA 025689 B9 EA025689 B9 EA 025689B9 EA 201400607 A EA201400607 A EA 201400607A EA 201400607 A EA201400607 A EA 201400607A EA 025689 B9 EA025689 B9 EA 025689B9
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EA
Eurasian Patent Office
Prior art keywords
bacteria
recombinant protein
engineering bacteria
escherichia coli
protein
Prior art date
Application number
EA201400607A
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English (en)
Other versions
EA025689B1 (ru
EA201400607A1 (ru
Inventor
Сяоцин Цю
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Протеин Дизайн Лаб, Лтд.
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Application filed by Протеин Дизайн Лаб, Лтд. filed Critical Протеин Дизайн Лаб, Лтд.
Publication of EA201400607A1 publication Critical patent/EA201400607A1/ru
Publication of EA025689B1 publication Critical patent/EA025689B1/ru
Publication of EA025689B9 publication Critical patent/EA025689B9/ru

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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
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

В изобретении представлен способ высокой экспрессии рекомбинантного белка в генно-инженерных бактериях и описание его применения. Этот метод включает в себя следующие этапы: (1) генно-инженерные бактерии Escherichia coli с системой экспрессии рЕТ трансфецируются рекомбинантными мутированными плазмидами для получения положительных моноклональных колоний; (2) положительные моноклональные колонии обогащаются для получения затравочного раствора бактерий, и производится индукция затравочного раствора бактерий для обогащения и роста в большом количестве среды; и (3) бактериальный супернатант, содержащий рекомбинантный белок, являющийся целью экспрессии, отделяется, затем производится экстракция и очистка рекомбинантного белка из бактериального супернатанта. Заявляемый способ характеризуется тем, что генно-инженерными бактериями Escherichia coli с экспрессионной системой рЕТ является штамм Е. coli B834 (DE3). Компоненты среды для обогащения массы и этапы очистки белка также оптимизированы таким образом, что достигается значительное улучшение выхода и чистоты белка, и данный способ пригоден для использования для крупномасштабного производства рекомбинантного белка, экспрессируемого генно-инженерными бактериями Escherichia coli.
EA201400607A 2011-11-25 2012-11-23 Способ высокой экспрессии рекомбинантного белка в генно-инженерных бактериях и его применение EA025689B9 (ru)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110380864.7A CN103131724B (zh) 2011-11-25 2011-11-25 一种高效表达工程菌重组蛋白的方法及应用
PCT/CN2012/085182 WO2013075660A1 (zh) 2011-11-25 2012-11-23 一种高效表达工程菌重组蛋白的方法及应用

Publications (3)

Publication Number Publication Date
EA201400607A1 EA201400607A1 (ru) 2014-10-30
EA025689B1 EA025689B1 (ru) 2017-01-30
EA025689B9 true EA025689B9 (ru) 2017-05-31

Family

ID=48469131

Family Applications (1)

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EA201400607A EA025689B9 (ru) 2011-11-25 2012-11-23 Способ высокой экспрессии рекомбинантного белка в генно-инженерных бактериях и его применение

Country Status (19)

Country Link
US (1) US9611299B2 (ru)
EP (1) EP2792749B1 (ru)
JP (1) JP5980342B2 (ru)
KR (1) KR101650869B1 (ru)
CN (1) CN103131724B (ru)
AP (1) AP2014007708A0 (ru)
AU (1) AU2012342993B2 (ru)
BR (1) BR112014012503A2 (ru)
CA (1) CA2856351C (ru)
CO (1) CO7230343A2 (ru)
EA (1) EA025689B9 (ru)
HK (1) HK1197265A1 (ru)
IL (1) IL232759A (ru)
IN (1) IN2014MN01278A (ru)
MY (1) MY183020A (ru)
PH (1) PH12014501154A1 (ru)
SG (1) SG11201402590RA (ru)
WO (1) WO2013075660A1 (ru)
ZA (1) ZA201403566B (ru)

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US9200251B1 (en) 2011-03-31 2015-12-01 David Gordon Bermudes Bacterial methionine analogue and methionine synthesis inhibitor anticancer, antiinfective and coronary heart disease protective microcins and methods of treatment therewith
CN105695385B (zh) * 2016-03-30 2017-05-10 通化东宝药业股份有限公司 重组人胰岛素基因工程菌高表达株筛选培养基及制备方法
CN107266540A (zh) * 2017-07-19 2017-10-20 福州大学 一种结核分枝杆菌延伸因子EF‑Tu蛋白的制备方法
CN109387581B (zh) * 2017-09-06 2020-01-24 申联生物医药（上海）股份有限公司 一种口蹄疫抗原的定量方法
KR102140531B1 (ko) * 2018-08-07 2020-08-04 (주)휴온스 Gly-Tβ4의 제조방법
CN113528410A (zh) * 2021-06-23 2021-10-22 云舟生物科技（广州）有限公司 一种用于快速高效诱导表达外源蛋白的大肠杆菌工程菌及其制备方法与应用

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US5439795A (en) * 1990-01-31 1995-08-08 American Cyanamid Company Monoclonal antibodies that specifically bind somatotropin binding proteins but not to corresponding somatotropin receptors and methods of use in immunoassays
JP3737157B2 (ja) * 1994-07-11 2006-01-18 協和醗酵工業株式会社 光学活性γ−ヒドロキシ−Ｌ−グルタミン酸の製造法
US6586207B2 (en) 2000-05-26 2003-07-01 California Institute Of Technology Overexpression of aminoacyl-tRNA synthetases for efficient production of engineered proteins containing amino acid analogues
CA2652558A1 (en) * 2006-05-18 2007-11-29 Biobalance Llc Bacterial strains, compositions including same and probiotic use thereof
CN100999553B (zh) * 2006-11-28 2010-11-03 中国农业大学 Hr2模拟蛋白及其编码基因与应用
CN101643501B (zh) * 2008-11-07 2012-06-20 畿晋庆三联（北京）生物技术有限公司 一种新型抗生素及其核苷酸序列、制备方法与应用
CN101633699B (zh) * 2009-09-02 2012-02-15 畿晋庆堂国际生物技术有限公司 一种含抗体模拟物的新型抗生素及其制备方法与应用
US9073989B2 (en) 2011-01-25 2015-07-07 Protein Design Lab, Ltd Antibiotic comprising an antibody mimetic, its preparation methods and uses thereof
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CN102653779B (zh) 2011-03-04 2014-02-19 北京科润三联生物技术有限责任公司 一种新型重组抗菌多肽药物的制备方法

Also Published As

Publication number Publication date
KR20140093740A (ko) 2014-07-28
EP2792749A1 (en) 2014-10-22
CA2856351C (en) 2017-05-23
JP5980342B2 (ja) 2016-08-31
CO7230343A2 (es) 2015-03-31
IL232759A (en) 2017-11-30
HK1197265A1 (en) 2015-01-09
MY183020A (en) 2021-02-05
EP2792749B1 (en) 2017-11-22
CN103131724B (zh) 2015-02-25
JP2014533511A (ja) 2014-12-15
AU2012342993B2 (en) 2015-11-26
US20140322754A1 (en) 2014-10-30
PH12014501154A1 (en) 2014-08-11
BR112014012503A2 (pt) 2017-06-06
IN2014MN01278A (ru) 2015-07-03
KR101650869B1 (ko) 2016-08-24
SG11201402590RA (en) 2014-10-30
NZ625595A (en) 2016-11-25
AP2014007708A0 (en) 2014-06-30
EA025689B1 (ru) 2017-01-30
IL232759A0 (en) 2014-07-31
ZA201403566B (en) 2015-12-23
EA201400607A1 (ru) 2014-10-30
US9611299B2 (en) 2017-04-04
AU2012342993A1 (en) 2014-06-19
WO2013075660A1 (zh) 2013-05-30
EP2792749A4 (en) 2015-07-29
CA2856351A1 (en) 2013-05-30
CN103131724A (zh) 2013-06-05

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