Optimization of rhBMP-2 active-form production in a heterologous expression system using microbiological and molecular genetic approaches

Autor: T. M. Grunina, Z. M. Galushkina, M. S. Poponova, A M Lyaschuk, E. O. Osidak, Anna S. Karyagina, L. A. Soboleva, Vladimir G. Lunin, I. S. Boksha, A. S. Semikhin, A. V. Gromov, A V Demidenko, O. V. Sergienko
Rok vydání: 2016
Předmět:
Zdroj: Molecular Genetics, Microbiology and Virology. 31:208-213
ISSN: 1934-841X
0891-4168
Popis: Recombinant bone morphogenetic protein-2 (rhBMP-2) has pronounced osteoinductive properties, as evidenced by the results of experimental and clinical practices. This applies to both the protein produced in eukaryotic cells and the protein synthesized in bacterial cells. In eukaryotic expression systems, production of the protein is extremely low and, consequently, the cost of materials on its basis is very high. Therefore, optimization of heterologous expression systems for rhBMP-2 production represents an important task. In the present work, optimization of codon composition of the rhBMP-2 gene nucleotide sequence and secondary structure of the transcript, as well as strain selection for efficient gene expression, were carried out. The producing strain based on Escherichia coli BL-21(DE3) provides a high level of rhBMP-2 synthesis (about 57% of total cell proteins). Biological activity of rhBMP-2 dimeric forms purified from the obtained producing strain was measured by induction of alkaline phosphatase activity in C2C12 cells. It is comparable with that of commercial rhBMP-2 expressed in E. coli (R&D Systems, United States). Purified rhBMP-2 does not contain impurities of E. coli endotoxin and can be used in experimental studies of osteoinduction in laboratory animals.
Databáze: OpenAIRE
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