Enviar artigo por via de e-mail Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain
- Autores: 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
-
Afiliações:
- 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
- Edição: Volume 83, Nº 10 (2018)
- Páginas: 1207-1221
- Seção: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151738
- DOI: https://doi.org/10.1134/S0006297918100061
- ID: 151738
Citar
Acesso aberto
Acesso está concedido
Somente assinantes
Resumo
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.
Palavras-chave
Sobre autores
A. Karyagina
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology; Belozersky Institute of Physical and Chemical Biology
Autor responsável pela correspondência
Email: akaryagina@gmail.com
Rússia, Moscow, 123098; Moscow, 127550; Moscow, 119991
T. Grunina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
M. Poponova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
P. Orlova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
V. Manskikh
Gamaleya National Research Center of Epidemiology and Microbiology; Belozersky Institute of Physical and Chemical Biology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098; Moscow, 119991
A. Demidenko
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
N. Strukova
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
M. Manukhina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
K. Nikitin
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
A. Lyaschuk
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
Z. Galushkina
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
S. Cherepushkin
State Research Institute of Genetics and Selection of Industrial Microorganisms
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 117545
N. Polyakov
Gamaleya National Research Center of Epidemiology and Microbiology; Vernadsky Institute of Geochemistry and Analytical Chemistry
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098; Moscow, 119334
A. Solovyev
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
V. Zhukhovitsky
Gamaleya National Research Center of Epidemiology and Microbiology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
D. Tretyak
Moscow Technological University (Lomonosov Institute of Fine Chemical Technologies)
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 119571
I. Boksha
Gamaleya National Research Center of Epidemiology and Microbiology; Research Center of Mental Health
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098; Moscow, 115522
A. Gromov
Gamaleya National Research Center of Epidemiology and Microbiology
Autor responsável pela correspondência
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098
V. Lunin
Gamaleya National Research Center of Epidemiology and Microbiology; All-Russia Research Institute of Agricultural Biotechnology
Email: alexander.v.gromov@gmail.com
Rússia, Moscow, 123098; Moscow, 127550
Arquivos suplementares
