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Biochemistry (Mosc). 2019 Jan;84(1):20-32. doi: 10.1134/S0006297919010036.

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

Author information

1
Gamaleya National Research Center of Epidemiology and Microbiology, Ministry of Healthcare of the Russian Federation, Moscow, 123098, Russia. akaryagina@gmail.com.
2
All-Russia Research Institute of Agricultural Biotechnology, Moscow, 127550, Russia.
3
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
4
Gamaleya National Research Center of Epidemiology and Microbiology, Ministry of Healthcare of the Russian Federation, Moscow, 123098, Russia.
5
State Research Institute of Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center, Moscow, 117545, Russia.
6
Research Center of Mental Health, Moscow, 115522, Russia.
7
Gamaleya National Research Center of Epidemiology and Microbiology, Ministry of Healthcare of the Russian Federation, Moscow, 123098, Russia. alexander.v.gromov@gmail.com.

Abstract

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.

PMID:
30927522
DOI:
10.1134/S0006297919010036
[Indexed for MEDLINE]

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