Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo

Bone morphogenetic protein-2 (rhBMP-2) is an osteoinductive protein factor which plays a dominant role in growth and regeneration of bone tissue. In clinical practice, bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials have not been produced yet. The fragment of the bmp-2 gene encoding a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on the basis of E. coli BL21(DE3). The level of rhBMP-2 production was approximately 25% of total cell protein. Biologically active dimeric form of rhBMP-2 was obtained as a result of isolation and purification of protein from inclusion bodies with subsequent refolding. The obtained rhBMP-2 sample contained more than 80% of the dimeric form and was able to interact with specific antibodies to BMP-2. Biological activity of the rhBMP-2 samples was verified in in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On a model of ectopic osteogenesis, it was shown that the obtained rhBMP-2 exhibited biological activity in vivo, causing tissue calcification in the site of injection. The protein activity in vivo depends on the way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The elaborated method of rhBMP-2 isolation and purification results in an increased common protein yield and ensures the maintenance of biologically active dimeric form compared to the analogues described in the literature.