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2000  0,477
1999  0,762
1998  0,785
1997  0,507
1996  0,518
1995  0,502
Vol 44(2010) N 6 p. 923-930;
N.E. Sharapova1,2, A.P. Kotnova2, Z.M. Galushkina2, N.V. Lavrova1,2, N.N. Poletaeva2, A.E. Tukhvatulin2, A.S. Semikhin1,2, A.V. Gromov2, L.A. Soboleva2, S.A. Ershova1,2,3, V.V. Zaitsev4, O.V. Sergienko1,2, V.G. Lunin1,2, A.S. Karyagina1,2,3*

Production of the Recombinant Human Bone Morphogenetic Protein-2 in Escherichia coliand Testing of Its Biological Activity in vitro and in vivo

1Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow, 127550
2Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, 123098
3Belozersky Institute of Physical and Chemical Biology, Moscow State University, Moscow, 119899
4Priorov Central Institute of Traumatology and Orthopedics, Rosmedtechnologii, Moscow, 125299

*akaryagina@gmail.com
Received - 2010-04-05; Accepted - 2010-05-13

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.

recombinant human bone morphogenetic protein-2, BMP-2, homodimer, refolding, Escherichia coli, biological activity, alkaline phosphatase, ectopic osteogenesis



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