Enviar artigo por via de e-mail Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system
- Autores: Grunina T.M.1, Demidenko A.V.1, Lyaschuk A.M.1, Poponova M.S.1, Galushkina Z.M.1, Soboleva L.A.1, Cherepushkin S.A.2, Polyakov N.B.1,3, Grumov D.A.1, Solovyev A.I.1, Zhukhovitsky V.G.1,4, Boksha I.S.1,5, Subbotina M.E.1,6, Gromov A.V.1, Lunin V.G.1,6, Karyagina A.S.1,6,7
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Afiliações:
- 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
- Edição: Volume 82, Nº 11 (2017)
- Páginas: 1285-1294
- Seção: Article
- URL: https://journal-vniispk.ru/0006-2979/article/view/151503
- DOI: https://doi.org/10.1134/S0006297917110062
- ID: 151503
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Resumo
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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Sobre autores
T. Grunina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Rússia, Moscow, 123098
A. Demidenko
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Rússia, Moscow, 123098
A. Lyaschuk
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Rússia, Moscow, 123098
M. Poponova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Rússia, Moscow, 123098
Z. Galushkina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Rússia, Moscow, 123098
L. Soboleva
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Rússia, Moscow, 123098
S. Cherepushkin
State Research Institute of Genetics and Selection of Industrial Microorganisms
Email: akaryagina@gmail.com
Rússia, Moscow, 117545
N. Polyakov
Gamaleya National Research Center of Epidemiology and Microbiology; Vernadsky Institute of Geochemistry and Analytical Chemistry
Email: akaryagina@gmail.com
Rússia, Moscow, 123098; Moscow, 119991
D. Grumov
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Rússia, Moscow, 123098
A. Solovyev
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Rússia, Moscow, 123098
V. Zhukhovitsky
Gamaleya National Research Center of Epidemiology and Microbiology; Sechenov Moscow State Medical University
Email: akaryagina@gmail.com
Rússia, Moscow, 123098; Moscow, 119991
I. Boksha
Gamaleya National Research Center of Epidemiology and Microbiology; Mental Health Research Center
Email: akaryagina@gmail.com
Rússia, 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
Rússia, Moscow, 123098; Moscow, 127550
A. Gromov
Gamaleya National Research Center of Epidemiology and Microbiology
Autor responsável pela correspondência
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
V. Lunin
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology
Email: akaryagina@gmail.com
Rússia, 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
Autor responsável pela correspondência
Email: akaryagina@gmail.com
Rússia, Moscow, 123098; Moscow, 127550; Moscow, 119992
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