Prepared by the method of different refolding rhBMP.docVIP

 PAGE \* MERGEFORMAT 16 Prepared by the method of different refolding rhBMP Author: Zhao Wei-Qin, Chen Sumin, Wang Tao, Zhang Xiaonan, Wang Li [Keywords:] recombinant human bone morphogenetic protein-2 mature peptide;, high-density fermentation;, protein purification;, protein refolding Comparison of different refolding methods in inducing ectopic bone formation by rhBMP2m [Abstract] AIM: To refold recombinant human bone morphogenetic protein2 mature peptide (rhBMP2m) by different methods, and compare the activity of inducing the formation of ectopic bone. METHODS: The engineered strain, which could express rhBMP2m 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 ionexchange chromatography. The purified rhBMP2m was refolded by three different methods: ① rhBMP2m was dialyzed with pH 4.8 buffer for refolding. ② rhBMP2m was refolded by means of dilution method in pH 9.0 buffer. ③ rhBMP2m 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 rhBMP2m was 98% after ionexchange chromatography. ① The resulting rhBMP2m 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 rhBMP2m was planted into mouse muscles, it induced ectopic cartilage after 2 weeks and trabecular bone after 4 weeks. ② rhBMP2m was soluble after refolding with pH 9.0 buffer. The refolded rhBMP2m seemed dissolvable by macroscopic observation, but lost over 90% after filtrating sterilization.After 1 mg of this lyophilized rhBMP2m was planted into muscles, cartilage appeared after 1 week, bone formed after 2 weeks, and trabecular bone was seen after 3 weeks. ③ After dialysis, rhBMP2m was precipitated. When 1 mg of insoluble rhBMP2m was planted into muscles, it induced tr