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Biochemistry (Moscow)

, Volume 83, Issue 10, pp 1207–1221 | Cite as

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

  • A. S. KaryaginaEmail author
  • 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. GromovEmail author
  • V. G. Lunin
Article

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.

Keywords

erythropoietin Escherichia coli heparin-binding domain 

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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Supplementary material

10541_2018_673_MOESM1_ESM.pdf (137 kb)
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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Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • A. S. Karyagina
    • 1
    • 2
    • 3
    Email author
  • T. M. Grunina
    • 1
  • M. S. Poponova
    • 1
  • P. A. Orlova
    • 1
  • V. N. Manskikh
    • 1
    • 3
  • A. V. Demidenko
    • 1
  • N. V. Strukova
    • 1
  • M. S. Manukhina
    • 1
  • K. E. Nikitin
    • 1
  • A. M. Lyaschuk
    • 1
  • Z. M. Galushkina
    • 1
  • S. A. Cherepushkin
    • 4
  • N. B. Polyakov
    • 1
    • 5
  • A. I. Solovyev
    • 1
  • V. G. Zhukhovitsky
    • 1
  • D. A. Tretyak
    • 6
  • I. S. Boksha
    • 1
    • 7
  • A. V. Gromov
    • 1
    Email author
  • V. G. Lunin
    • 1
    • 2
  1. 1.Gamaleya National Research Center of Epidemiology and MicrobiologyMinistry of Health of the Russian FederationMoscowRussia
  2. 2.All-Russia Research Institute of Agricultural BiotechnologyMoscowRussia
  3. 3.Belozersky Institute of Physical and Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  4. 4.State Research Institute of Genetics and Selection of Industrial MicroorganismsKurchatov Institute National Research CentreMoscowRussia
  5. 5.Vernadsky Institute of Geochemistry and Analytical ChemistryRussian Academy of SciencesMoscowRussia
  6. 6.Moscow Technological University (Lomonosov Institute of Fine Chemical Technologies)MoscowRussia
  7. 7.Research Center of Mental HealthMoscowRussia

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