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.
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Abbreviations
- a.a.:
-
amino acid residue
- BMP-2:
-
bone morphogenetic protein-2
- DBM:
-
demineralized bone matrix
- DTT:
-
dithiothreitol
- EPO(R):
-
erythropoietin (receptor)
- HBD:
-
heparin-binding domain
- 6His:
-
six histidine amino acid residues
- s-tag:
-
15-a.a. oligopeptide from bovine pancreatic ribonuclease A
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Original Russian Text © A. S. Karyagina, T. M. Grunina, A. M. Lyaschuk, E. V. Voronina, R. A. Marigin, S. A. Cherepushkin, I. N. Trusova, A. V. Grishin, M. S. Poponova, P. A. Orlova, V. N. Manskikh, N. V. Strukova, M. S. Generalova, K. E. Nikitin, L. A. Soboleva, I. S. Boksha, A. V. Gromov, 2019, published in Biokhimiya, 2019, Vol. 84, No. 1, pp. 85–99.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM18-197, November 12, 2018.
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Karyagina, A.S., Grunina, T.M., Lyaschuk, A.M. et al. Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities. Biochemistry Moscow 84, 20–32 (2019). https://doi.org/10.1134/S0006297919010036
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DOI: https://doi.org/10.1134/S0006297919010036