Comparison of different refolding methods in inducing ectopic bone formation by rhBMPª²2m

    ZHAO Weiª²Qin, CHEN Suª²Min, WANG Tao, ZHANG Xiaoª²Nan, WANG Li

    Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To refold recombinant human bone morphogenetic proteinª²2 mature peptide (rhBMPª²2m) by different methods, and compare the activity of inducing the formation of ectopic bone. METHODS: The engineered strain, which could express rhBMPª²2m at a high level, was fermented in high density, and induced by temperature. The bacterial cells were collected and lyzed. The gained inclusion bodies were purified by ionª²exchange chromatography. The purified rhBMPª²2m was refolded by three different methods: ¢Ù rhBMPª²2m was dialyzed with pH 4.8 buffer for refolding. ¢Ú rhBMPª²2m was refolded by means of dilution method in pH 9.0 buffer. ¢Û rhBMPª²2m was dialysed in pH 6.5 buffer to refold. It (1 mg) was planted into mouse muscles to observe the formation of bone without any carrier. RESULTS: The purity of the acquired rhBMPª²2m was 98% after ionª²exchange chromatography. ¢Ù The resulting rhBMPª²2m was soluble after refolding with pH 4.8 buffer and had no any loss after passing through sterilizing filter membrane. When 1 mg of the lyophilized rhBMPª²2m was planted into mouse muscles, it induced ectopic cartilage after 2 weeks and trabecular bone after 4 weeks. ¢Ú rhBMPª²2m was soluble after refolding with pH 9.0 buffer. The refolded rhBMPª²2m seemed dissolvable by macroscopic observation, but lost over 90% after filtrating sterilization.After 1 mg of this lyophilized rhBMPª²2m was planted into muscles, cartilage appeared after 1 week, bone formed after 2 weeks, and trabecular bone was seen after 3 weeks. ¢Û After dialysis, rhBMPª²2m was precipitated. When 1 mg of insoluble rhBMPª²2m was planted into muscles, it induced trabecular bone in 2 weeks and myeloid cells after 4 weeks. CONCLUSION: Compared with acid and alkaline condition, rhBMPª²2m refolded in neutral condition has the best activity of inducing the formation of bone, but rhBMPª²2m refolded in acid condition has better dissolubility. ......