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

A. S. Karyagina1,2,3,a*, T. M. Grunina1, M. S. Poponova1, P. A. Orlova1, V. N. Manskikh1,3, A. V. Demidenko1, N. V. Strukova1, M. S. Manukhina1, K. E. Nikitin1, A. M. Lyaschuk1, Z. M. Galushkina1, S. A. Cherepushkin4, N. B. Polyakov1,5, A. I. Solovyev1, V. G. Zhukhovitsky1, D. A. Tretyak6, I. S. Boksha1,7, A. V. Gromov1,b*, and V. G. Lunin1,2

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

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

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

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

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

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

7Research Center of Mental Health, 115522 Moscow, Russia

* To whom correspondence should be addressed.

Received May 18, 2018
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.
KEY WORDS: erythropoietin, Escherichia coli, heparin-binding domain

DOI: 10.1134/S0006297918100061