Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain


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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.

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

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