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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

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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Abbreviations

a.a.:

amino acid residue

BMP-2:

bone morphogenetic protein-2

DTT:

dithiothreitol

EPO:

erythropoietin

6His:

six histidine amino acid residues

MBP:

maltose-binding protein

rhEPO:

recombinant human erythropoietin

s-tag:

15-a.a. oligonucleotide derived from bovine pancreatic ribonuclease A

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Authors and Affiliations

  1. Gamaleya National Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

    T. M. Grunina, A. V. Demidenko, A. M. Lyaschuk, M. S. Poponova, Z. M. Galushkina, L. A. Soboleva, 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

  2. State Research Institute of Genetics and Selection of Industrial Microorganisms, 117545, Moscow, Russia

    S. A. Cherepushkin

  3. Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, 119991, Moscow, Russia

    N. B. Polyakov

  4. Sechenov Moscow State Medical University, 119991, Moscow, Russia

    V. G. Zhukhovitsky

  5. Mental Health Research Center, 115522, Moscow, Russia

    I. S. Boksha

  6. All-Russia Research Institute of Agricultural Biotechnology, 127550, Moscow, Russia

    M. E. Subbotina, V. G. Lunin & A. S. Karyagina

  7. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992, Moscow, Russia

    A. S. Karyagina

Authors
  1. T. M. Grunina
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  2. A. V. Demidenko
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  3. A. M. Lyaschuk
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  4. M. S. Poponova
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  5. Z. M. Galushkina
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  6. L. A. Soboleva
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  7. S. A. Cherepushkin
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  8. N. B. Polyakov
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  9. D. A. Grumov
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  10. A. I. Solovyev
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  11. V. G. Zhukhovitsky
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  12. I. S. Boksha
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  13. M. E. Subbotina
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  14. A. V. Gromov
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  15. V. G. Lunin
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  16. A. S. Karyagina
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Corresponding authors

Correspondence to A. V. Gromov or A. S. Karyagina.

Additional information

Original Russian Text © 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, 2017, published in Biokhimiya, 2017, Vol. 82, No. 11, pp. 1635–1646.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM17-304, September 25, 2017.

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Grunina, T.M., Demidenko, A.V., Lyaschuk, A.M. et al. Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system. Biochemistry Moscow 82, 1285–1294 (2017). https://doi.org/10.1134/S0006297917110062

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  • DOI: https://doi.org/10.1134/S0006297917110062

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