In this work the recombinant human bone morphogenetic protein 2 (rhBMP-2) gene was cloned from MG-63 cells by RT-PCR and the protein was expressed in Escherichia cob expression system purified by Ni-NTA column under denaturing conditions and refolded at 4 degrees C by urea gradient dialysis We found that the protein refolding yield was increased with the increase of pH value from pH 6 0 to pH 90 The yield was 42% and 96% at pH 74 and pH 9 0 respectively while that at pH 60 was only 3 4% The cell culture results showed that the rhBMP-2 refolded at pH 74 urea gradient dialysis had higher biological activity for MG-63 cell proliferation and differentiation than that refolded at pH 9 0 since pH 7 4 is closer to the conditions in vivo leading to the formation of Miners through the interchain disulfide bond Moreover the biological activity for MG-63 was promoted with the increase of rhBMP-2 concentration in the cell culture medium This work may be important for the in vitro production and biomedical application of rhBMP-2 protein (C) 2010 Elsevier Inc All rights reserved