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- Title
Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain.
- Authors
Karyagina, A. S.; Grunina, T. M.; Poponova, M. S.; Orlova, P. A.; Manskikh, V. N.; Demidenko, A. V.; Strukova, N. V.; Manukhina, M. S.; Nikitin, K. E.; Lyaschuk, A. M.; Galushkina, Z. M.; Cherepushkin, S. A.; Polyakov, N. B.; Solovyev, A. I.; Zhukhovitsky, V. G.; Tretyak, D. A.; Boksha, I. S.; Gromov, A. V.; Lunin, V. G.
- Abstract
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.
- Subjects
ESCHERICHIA coli; RECOMBINANT erythropoietin; HEPARIN; BONE morphogenetic proteins; ERYTHROPOIETIN
- Publication
Biochemistry (00062979), 2018, Vol 83, Issue 10, p1207
- ISSN
0006-2979
- Publication type
Article
- DOI
10.1134/S0006297918100061