Отправить статью по E-mail Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system
- Авторы: Grunina T.1, Demidenko A.1, Lyaschuk A.1, Poponova M.1, Galushkina Z.1, Soboleva L.1, Cherepushkin S.2, Polyakov N.1,3, Grumov D.1, Solovyev A.1, Zhukhovitsky V.1,4, Boksha I.1,5, Subbotina M.1,6, Gromov A.1, Lunin V.1,6, Karyagina A.1,6,7
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Учреждения:
- Gamaleya National Research Center of Epidemiology and Microbiology
- State Research Institute of Genetics and Selection of Industrial Microorganisms
- Vernadsky Institute of Geochemistry and Analytical Chemistry
- Sechenov Moscow State Medical University
- Mental Health Research Center
- All-Russia Research Institute of Agricultural Biotechnology
- Belozersky Institute of Physico-Chemical Biology
- Выпуск: Том 82, № 11 (2017)
- Страницы: 1285-1294
- Раздел: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151503
- DOI: https://doi.org/10.1134/S0006297917110062
- ID: 151503
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Аннотация
Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.
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Об авторах
T. Grunina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Россия, Moscow, 123098
A. Demidenko
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Россия, Moscow, 123098
A. Lyaschuk
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Россия, Moscow, 123098
M. Poponova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Россия, Moscow, 123098
Z. Galushkina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Россия, Moscow, 123098
L. Soboleva
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Россия, Moscow, 123098
S. Cherepushkin
State Research Institute of Genetics and Selection of Industrial Microorganisms
Email: akaryagina@gmail.com
Россия, Moscow, 117545
N. Polyakov
Gamaleya National Research Center of Epidemiology and Microbiology; Vernadsky Institute of Geochemistry and Analytical Chemistry
Email: akaryagina@gmail.com
Россия, Moscow, 123098; Moscow, 119991
D. Grumov
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Россия, Moscow, 123098
A. Solovyev
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Россия, Moscow, 123098
V. Zhukhovitsky
Gamaleya National Research Center of Epidemiology and Microbiology; Sechenov Moscow State Medical University
Email: akaryagina@gmail.com
Россия, Moscow, 123098; Moscow, 119991
I. Boksha
Gamaleya National Research Center of Epidemiology and Microbiology; Mental Health Research Center
Email: akaryagina@gmail.com
Россия, Moscow, 123098; Moscow, 115522
M. Subbotina
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology
Email: akaryagina@gmail.com
Россия, Moscow, 123098; Moscow, 127550
A. Gromov
Gamaleya National Research Center of Epidemiology and Microbiology
Автор, ответственный за переписку.
Email: alexander.v.gromov@gmail.com
Россия, Moscow, 123098
V. Lunin
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology
Email: akaryagina@gmail.com
Россия, Moscow, 123098; Moscow, 127550
A. Karyagina
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology; Belozersky Institute of Physico-Chemical Biology
Автор, ответственный за переписку.
Email: akaryagina@gmail.com
Россия, Moscow, 123098; Moscow, 127550; Moscow, 119992
