Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain
| Autor: | Vasily N. Manskikh, Anna S. Karyagina, Vladimir G. Lunin, M S Manukhina, N. B. Polyakov, P A Orlova, M. S. Poponova, Z. M. Galushkina, V G Zhukhovitsky, A V Demidenko, Andrey I. Solovyev, I. S. Boksha, S A Cherepushkin, D A Tretyak, N V Strukova, A. V. Gromov, A M Lyaschuk, K E Nikitin, T. M. Grunina |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Recombinant Fusion Proteins Bone Morphogenetic Protein 2 Biochemistry Bone morphogenetic protein 2 law.invention 03 medical and health sciences Protein Domains In vivo law hemic and lymphatic diseases medicine Escherichia coli Animals Humans Amino Acid Sequence Rats Wistar Bone regeneration Erythropoietin 030102 biochemistry & molecular biology Chemistry Demineralized bone matrix Heparin Biological activity General Medicine Rats 030104 developmental biology Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Recombinant DNA Female Peptides medicine.drug Binding domain Half-Life |
| Zdroj: | Biochemistry. Biokhimiia. 83(10) |
| ISSN: | 1608-3040 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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