Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain
- Authors: Karyagina A.S.1,2,3, Grunina T.M.1, Poponova M.S.1, Orlova P.A.1, Manskikh V.N.1,3, Demidenko A.V.1, Strukova N.V.1, Manukhina M.S.1, Nikitin K.E.1, Lyaschuk A.M.1, Galushkina Z.M.1, Cherepushkin S.A.4, Polyakov N.B.1,5, Solovyev A.I.1, Zhukhovitsky V.G.1, Tretyak D.A.6, Boksha I.S.1,7, Gromov A.V.1, Lunin V.G.1,2
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Affiliations:
- Gamaleya National Research Center of Epidemiology and Microbiology
- All-Russia Research Institute of Agricultural Biotechnology
- Belozersky Institute of Physical and Chemical Biology
- State Research Institute of Genetics and Selection of Industrial Microorganisms
- Vernadsky Institute of Geochemistry and Analytical Chemistry
- Moscow Technological University (Lomonosov Institute of Fine Chemical Technologies)
- Research Center of Mental Health
- Issue: Vol 83, No 10 (2018)
- Pages: 1207-1221
- Section: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151738
- DOI: https://doi.org/10.1134/S0006297918100061
- ID: 151738
Cite item
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.
Keywords
About the authors
A. S. Karyagina
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology; Belozersky Institute of Physical and Chemical Biology
Author for correspondence.
Email: akaryagina@gmail.com
Russian Federation, Moscow, 123098; Moscow, 127550; Moscow, 119991
T. M. Grunina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
M. S. Poponova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
P. A. Orlova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
V. N. Manskikh
Gamaleya National Research Center of Epidemiology and Microbiology; Belozersky Institute of Physical and Chemical Biology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098; Moscow, 119991
A. V. Demidenko
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
N. V. Strukova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
M. S. Manukhina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
K. E. Nikitin
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
A. M. Lyaschuk
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
Z. M. Galushkina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
S. A. Cherepushkin
State Research Institute of Genetics and Selection of Industrial Microorganisms
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 117545
N. B. Polyakov
Gamaleya National Research Center of Epidemiology and Microbiology; Vernadsky Institute of Geochemistry and Analytical Chemistry
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098; Moscow, 119334
A. I. Solovyev
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
V. G. Zhukhovitsky
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
D. A. Tretyak
Moscow Technological University (Lomonosov Institute of Fine Chemical Technologies)
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 119571
I. S. Boksha
Gamaleya National Research Center of Epidemiology and Microbiology; Research Center of Mental Health
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098; Moscow, 115522
A. V. Gromov
Gamaleya National Research Center of Epidemiology and Microbiology
Author for correspondence.
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098
V. G. Lunin
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology
Email: alexander.v.gromov@gmail.com
Russian Federation, Moscow, 123098; Moscow, 127550