电邮这篇文章 Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system
- 作者: 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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隶属关系:
- 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
- 期: 卷 82, 编号 11 (2017)
- 页面: 1285-1294
- 栏目: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151503
- DOI: https://doi.org/10.1134/S0006297917110062
- ID: 151503
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详细
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.
作者简介
T. Grunina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098
A. Demidenko
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098
A. Lyaschuk
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098
M. Poponova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098
Z. Galushkina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098
L. Soboleva
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098
S. Cherepushkin
State Research Institute of Genetics and Selection of Industrial Microorganisms
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 117545
N. Polyakov
Gamaleya National Research Center of Epidemiology and Microbiology; Vernadsky Institute of Geochemistry and Analytical Chemistry
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098; Moscow, 119991
D. Grumov
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098
A. Solovyev
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098
V. Zhukhovitsky
Gamaleya National Research Center of Epidemiology and Microbiology; Sechenov Moscow State Medical University
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098; Moscow, 119991
I. Boksha
Gamaleya National Research Center of Epidemiology and Microbiology; Mental Health Research Center
Email: akaryagina@gmail.com
俄罗斯联邦, 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
俄罗斯联邦, Moscow, 123098; Moscow, 127550
A. Gromov
Gamaleya National Research Center of Epidemiology and Microbiology
编辑信件的主要联系方式.
Email: alexander.v.gromov@gmail.com
俄罗斯联邦, Moscow, 123098
V. Lunin
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology
Email: akaryagina@gmail.com
俄罗斯联邦, 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
编辑信件的主要联系方式.
Email: akaryagina@gmail.com
俄罗斯联邦, Moscow, 123098; Moscow, 127550; Moscow, 119992
