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Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain

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Abstract

Recombinant human erythropoietin (EPO) with additional N-terminal heparin-binding protein domain (HBD) from bone morphogenetic protein 2 was synthesized in Escherichia coli cells. A procedure for HBD-EPO purification and refolding was developed for obtaining highly-purified HBD-EPO. The structure of recombinant HBD-EPO was close to that of the native EPO protein. HBD-EPO contained two disulfide bonds, as shown by MALDI-TOF mass spectrometry. The protein demonstrated in vitro biological activity in the proliferation of human erythroleukemia TF-1 cell test and in vivo activity in animal models. HBD-EPO increased the number of reticulocytes in the blood after subcutaneous injection and displayed local angiogenic activity after subcutaneous implantation of demineralized bone matrix (DBM) discs with immobilized HBD-EPO. We developed a quantitative sandwich ELISA method for measuring HBD-EPO concentration in solution using rabbit polyclonal serum and commercial monoclonal anti-EPO antibodies. Pharmacokinetic properties of HBD-EPO were typical for bacterially produced EPO. Under physiological conditions, HBD-EPO can reversibly bind to DBM, which is often used as an osteoplastic material for treatment of bone pathologies. The data on HBD-EPO binding to DBM and local angiogenic activity of this protein give hope for successful application of HBD-EPO immobilized on DBM in experiments on bone regeneration.

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Abbreviations

BCA:

2,2′-bicinchoninic acid

BMP-2:

bone morphogenetic protein 2

DBM:

demineralized bone matrix

DTT:

dithiothreitol

EPO:

erythropoietin

HBD:

heparinbinding domain

6His:

six-histidine tag

s-tag:

15-amino acid oligopeptide from bovine pancreas 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

    A. S. Karyagina, T. M. Grunina, M. S. Poponova, P. A. Orlova, V. N. Manskikh, A. V. Demidenko, N. V. Strukova, M. S. Manukhina, K. E. Nikitin, A. M. Lyaschuk, Z. M. Galushkina, N. B. Polyakov, A. I. Solovyev, V. G. Zhukhovitsky, I. S. Boksha, A. V. Gromov & V. G. Lunin

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

    A. S. Karyagina & V. G. Lunin

  3. Belozersky Institute of Physical and Chemical Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    A. S. Karyagina & V. N. Manskikh

  4. State Research Institute of Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Centre, 117545, Moscow, Russia

    S. A. Cherepushkin

  5. Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, 119334, Moscow, Russia

    N. B. Polyakov

  6. Moscow Technological University (Lomonosov Institute of Fine Chemical Technologies), 119571, Moscow, Russia

    D. A. Tretyak

  7. Research Center of Mental Health, 115522, Moscow, Russia

    I. S. Boksha

Authors
  1. A. S. Karyagina
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  2. T. M. Grunina
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  3. M. S. Poponova
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  4. P. A. Orlova
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  5. V. N. Manskikh
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  6. A. V. Demidenko
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  7. N. V. Strukova
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  8. M. S. Manukhina
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  9. K. E. Nikitin
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  10. A. M. Lyaschuk
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  18. A. V. Gromov
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  19. V. G. Lunin
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Correspondence to A. S. Karyagina or A. V. Gromov.

Additional information

Original Russian Text © A. S. Karyagina, T. M. Grunina, M. S. Poponova, P. A. Orlova, V. N. Manskikh, A. V. Demidenko, N. V. Strukova, M. S. Manukhina, K. E. Nikitin, A. M. Lyaschuk, Z. M. Galushkina, S. A. Cherepushkin, N. B. Polyakov, A. I. Solovyev, V. G. Zhukhovitsky, D. A. Tretyak, I. S. Boksha, A. V. Gromov, V. G. Lunin, 2018, published in Biokhimiya, 2018, Vol. 83, No. 10, pp. 1504–1522.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM18-141, August 13, 2018.

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10541_2018_673_MOESM1_ESM.pdf

Supplement to: A. S. Karyagina, T. M. Grunina, M. S. Poponova, P. A. Orlova, V. N. Manskikh, A. V. Demidenko, N. V. Strukova, M. S. Manukhina, K. E. Nikitin, A. M. Lyaschuk, Z. M. Galushkina, S. A. Cherepushkin, N. B. Polyakov, A. I. Solovyev, V. G. Zhukhovitsky, D. A. Tretyak, I. S. Boksha, A. V. Gromov, and V. G. Lunin, Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain (ISSN 0006-2979, Biochemistry (Moscow), 2018, Vol. 83, No. 10, pp. 1207-1221)

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Karyagina, A.S., Grunina, T.M., Poponova, M.S. et al. Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain. Biochemistry Moscow 83, 1207–1221 (2018). https://doi.org/10.1134/S0006297918100061

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