Abstract

Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities

A. S. Karyagina^1,2,3,a, T. M. Grunina¹, A. M. Lyaschuk¹, E. V. Voronina⁴, R. A. Marigin⁴, S. A. Cherepushkin⁴, I. N. Trusova⁴, A. V. Grishin^1,2, M. S. Poponova¹, P. A. Orlova¹, V. N. Manskikh^1,3, N. V. Strukova¹, M. S. Generalova¹, K. E. Nikitin¹, L. A. Soboleva¹, I. S. Boksha^1,5, and A. V. Gromov^1,b

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

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

³Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

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

⁵Research Center of Mental Health, 115522 Moscow, Russia

^* To whom correspondence should be addressed.

Received July 10, 2018; Revised September 14, 2018; Accepted September 14, 2018
The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.
KEY WORDS: erythropoietin, heparin-binding domain, proliferation of human erythroleukemia TF-1 cells, vascularization of surrounding tissues
DOI: 10.1134/S0006297919010036