Abstract

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

A. S. Karyagina^1,2,3,a, T. M. Grunina¹, M. S. Poponova¹, P. A. Orlova¹, V. N. Manskikh^1,3, A. V. Demidenko¹, N. V. Strukova¹, M. S. Manukhina¹, K. E. Nikitin¹, A. M. Lyaschuk¹, Z. M. Galushkina¹, S. A. Cherepushkin⁴, N. B. Polyakov^1,5, A. I. Solovyev¹, V. G. Zhukhovitsky¹, D. A. Tretyak⁶, I. S. Boksha^1,7, A. V. Gromov^1,b, and V. G. Lunin^1,2

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