Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in  Escherichia coli  heterologous expression system

T. M. Grunina; A. V. Demidenko; A. M. Lyaschuk; M. S. Poponova; Z. M. Galushkina; L. A. Soboleva; S. A. Cherepushkin; N. B. Polyakov; D. A. Grumov; A. I. Solovyev; V. G. Zhukhovitsky; I. S. Boksha; M. E. Subbotina; A. V. Gromov; V. G. Lunin; A. S. Karyagina

doi:10.1134/S0006297917110062

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:
1. Gamaleya National Research Center of Epidemiology and Microbiology
2. State Research Institute of Genetics and Selection of Industrial Microorganisms
3. Vernadsky Institute of Geochemistry and Analytical Chemistry
4. Sechenov Moscow State Medical University
5. Mental Health Research Center
6. All-Russia Research Institute of Agricultural Biotechnology
7. 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

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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.

Keywords

erythropoietin, heterologous expression, Escherichia coli

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Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system

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Abstract

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About the authors

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