Purification and biological osteoinductive activity analysis of recombinant human bone morphogenetic protein 9 by eukaryotic expression

Abstract The present paper is aimed to explore the biological osteoinductive activity of recombinant human bone morphogenetic protein 9 (rhBMP-9) by various biological technologies. In this study, we firstly obtained hBMP-9 cDNA by PCR and inserted it into vector pcDNA4/His Max to reconstruct hBMP-9 eukaryotic expression vector pcDNA4/His Max-BMP-9. Recombinant Chinese hamster ovary (rCHO) cell line expressing high-level rhBMP-9 was reconstructed by co-transfecting the expression vectors pcDNA4/His* Max-hBMP-9 and plasmid pSV2-dhfr into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification by the methotrexate. We finally obtained a monoclonal cell line expressing the highest level protein. We purified the medium after culturing the highest-producing monoclonal by Ni-NTA His-Bind Resin columns and concentrated to by a Centricon 50 at 4 degrees C and stored at 70 degrees C until it was used. Western blot and SDS-PAGE analyses showed a specific band of about 32kD in pro-region lane and a specific band of about 50kD in pro-region complex lane. Biological activities of rhBMP-9 were tested by colorimetric determination and histochemical staining of Alkaline Phosphatase (ALP) Activity, osteocalcin and oesteopontin for C3H10 T1/2 cells, which were stimulated culture by different concentration (20, 50, 100 microg/mL) of rhBMP-9. The results showed that the rhBMP-9 could induce osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro, and were proportional to the amount. This study can provide experimental data for further tests in vivo and clinical applications.