EA201792179A1 - TECHNOLOGY FOR ISOLATION AND CLEANING OF RECOMBINANT FLAGELLIN - Google Patents

TECHNOLOGY FOR ISOLATION AND CLEANING OF RECOMBINANT FLAGELLIN

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Publication number
EA201792179A1
EA201792179A1 EA201792179A EA201792179A EA201792179A1 EA 201792179 A1 EA201792179 A1 EA 201792179A1 EA 201792179 A EA201792179 A EA 201792179A EA 201792179 A EA201792179 A EA 201792179A EA 201792179 A1 EA201792179 A1 EA 201792179A1
Authority
EA
Eurasian Patent Office
Prior art keywords
flagellin
cells
technology
protein
yield
Prior art date
Application number
EA201792179A
Other languages
Russian (ru)
Other versions
EA035920B1 (en
Inventor
Илья Владимирович ДУХОВЛИНОВ
Антон Иосифович ОРЛОВ
Original Assignee
Общество С Ограниченной Ответственностью "Активные Биосистемы" (Ооо "Абс")
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Общество С Ограниченной Ответственностью "Активные Биосистемы" (Ооо "Абс") filed Critical Общество С Ограниченной Ответственностью "Активные Биосистемы" (Ооо "Абс")
Priority to EA201792179A priority Critical patent/EA035920B1/en
Publication of EA201792179A1 publication Critical patent/EA201792179A1/en
Publication of EA035920B1 publication Critical patent/EA035920B1/en

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Classifications

    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Abstract

Изобретение относится к молекулярной биологии и биотехнологии и может быть использовано для производства рекомбинантного флагеллина. Предложен способ получения активного высокоочищенного рекомбинантного усеченного флагеллина, охарактеризованного последовательностью аминокислот SEQ ID NO:1, заключающийся в том, что используют субклон штамма-продуцента E.coli BL21(DE3)/pET151D-TOPO F27, содержащего ген, кодирующий такой флагеллин, оптимизированный по кодонному составу для экспрессии в клетках E.coli, обеспечивающий выход целевого белка до 45% от общего белка, культивируют клетки такого субклона с использованием индуктора экспрессии целевого гена 0,5 мМ ИПТГ, лизируют клетки, осаждают клеточные осадки, осуществляют выделение и очистку белка из супернатанта в денатурирующих условиях с использованием Ni-NTA сефарозы. Данная технология позволяет получить флагеллин с чистотой 98%, выход - 800 мг с 1 л питательной среды. Полученный с использованием предложенной технологии флагеллин демонстрирует эффективность в модели лабораторных животных как при введении до, так и после облучения.The invention relates to molecular biology and biotechnology and can be used for the production of recombinant flagellin. A method for obtaining highly purified active recombinant truncated flagellin, characterized by the amino acid sequence of SEQ ID NO: 1, is proposed. The method consists in using a codon composition for expression in E.coli cells, providing the yield of the target protein to 45% of the total protein, cultured cells of such a subclone using the inducer of expression of the target gene 0.5 mM IPTG, lyse cells, precipitated cells The precipitates isolate and purify the protein from the supernatant under denaturing conditions using Ni-NTA sepharose. This technology allows you to get flagellin with a purity of 98%, yield - 800 mg per 1 liter of nutrient medium. Obtained using the proposed technology, flagellin demonstrates effectiveness in a model of laboratory animals, both when administered before and after irradiation.

EA201792179A 2017-10-30 2017-10-30 Recombinant flagellin extraction and purification process EA035920B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EA201792179A EA035920B1 (en) 2017-10-30 2017-10-30 Recombinant flagellin extraction and purification process

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EA201792179A EA035920B1 (en) 2017-10-30 2017-10-30 Recombinant flagellin extraction and purification process

Publications (2)

Publication Number Publication Date
EA201792179A1 true EA201792179A1 (en) 2019-05-31
EA035920B1 EA035920B1 (en) 2020-08-31

Family

ID=66644961

Family Applications (1)

Application Number Title Priority Date Filing Date
EA201792179A EA035920B1 (en) 2017-10-30 2017-10-30 Recombinant flagellin extraction and purification process

Country Status (1)

Country Link
EA (1) EA035920B1 (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2012116413A (en) * 2012-04-24 2013-10-27 Общество с ограниченной ответственностью "Биоинженерные технологии" RECOMBINANT DNA ENCODING FLAGELLIN OF BACTERIA Salmonella typhimurium (FlagST) AND RECOMBINANT PLASMIDAS pAS222, PROVIDING SYNTHESIS OF FlagST IN Escherichia coli cells
RU2524133C2 (en) * 2012-11-15 2014-07-27 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт особо чистых биопрепаратов" Федерального медико-биологического агентства Escherichia coli BACTERIA STRAIN - PRODUCER OF RECOMBINANT FLAGELLIN

Also Published As

Publication number Publication date
EA035920B1 (en) 2020-08-31

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MM4A Lapse of a eurasian patent due to non-payment of renewal fees within the time limit in the following designated state(s)

Designated state(s): AM AZ BY KZ KG TJ TM RU