Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system
- Authors: 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
-
Affiliations:
- 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
- Issue: Vol 82, No 11 (2017)
- Pages: 1285-1294
- Section: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151503
- DOI: https://doi.org/10.1134/S0006297917110062
- ID: 151503
Cite item
Abstract
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.
About the authors
T. M. Grunina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098
A. V. Demidenko
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098
A. M. Lyaschuk
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098
M. S. Poponova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098
Z. M. Galushkina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098
L. A. Soboleva
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098
S. A. Cherepushkin
State Research Institute of Genetics and Selection of Industrial Microorganisms
Email: akaryagina@gmail.com
Russian Federation, Moscow, 117545
N. B. Polyakov
Gamaleya National Research Center of Epidemiology and Microbiology; Vernadsky Institute of Geochemistry and Analytical Chemistry
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098; Moscow, 119991
D. A. Grumov
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098
A. I. Solovyev
Gamaleya National Research Center of Epidemiology and Microbiology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098
V. G. Zhukhovitsky
Gamaleya National Research Center of Epidemiology and Microbiology; Sechenov Moscow State Medical University
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098; Moscow, 119991
I. S. Boksha
Gamaleya National Research Center of Epidemiology and Microbiology; Mental Health Research Center
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098; Moscow, 115522
M. E. Subbotina
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098; Moscow, 127550
A. V. Gromov
Gamaleya National Research Center of Epidemiology and Microbiology
Author for correspondence.
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
V. G. Lunin
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098; Moscow, 127550
A. S. Karyagina
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology; Belozersky Institute of Physico-Chemical Biology
Author for correspondence.
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098; Moscow, 127550; Moscow, 119992