重组人骨形成蛋白-2在大肠杆菌中的表达、纯化和复性(英文)

Expression, purification and refolding of recombinant human bone morphogenetic protein-2 in Escherichia coli

目的:通过对大肠杆菌表达重组人骨形成蛋白-2的复性研究,得到高活性的重组人骨形成蛋白-2。方法:在大肠杆菌中通过温度诱导表达重组人骨形成蛋白-2经过Triton X-100清洗之后,又通过DEAE离子交换层析纯化包涵体,包涵体在8 mol/L尿素变性溶解,在氧化-还原(还原型和氧化型谷胱甘肽)复性系统中,通过简单的稀释复性,通过肝素亲和层析一步纯化法纯化重组人骨形成蛋白-2 ,最后通过诱导C2C12细胞产生碱性磷酸酶检测重组人骨形成蛋白-2活性。结果:温度诱导表达重组人骨形成蛋白-2是以包涵体单体的形式存在,经过几步纯化后,得到高纯度的包涵体。重组人骨形成蛋白-2在不同氧化-还原剂比例,和不同小分子化学辅助剂浓度中复性,得到复性的效率也不同。亲和层析纯化后,本实验得到重组人骨形成蛋白-2的生物学活性比商业化的重组人骨形成蛋白-2更高。结论:重组人骨形成蛋白-2属于转化因子-β家族,此复性方法可能应用于此家族的其他成员同时得到成本低、产量高的重组人骨形成蛋白-2 ,可能为临床应用创造了良好的条件。

AIM： To get high biological activity of recombinant human bone morphogenetic protein- 2 （ rhBMP - 2） expressed in Escherichia coli by the methods of refolding. METHODS ： The rhBMP - 2,expressed in Escherichia coil, was washed by Triton X- 100 and further purified by DEAE chro, atography.The inclusion bodies were resolved in 8 mol/L urea, and were refolded and dimerized in the redox systems （reduced and oxidized glutathione）. Finally, a one - step purification procedure based on the heparin affinity chromatography was implemented. The biological activity of purified rhBMP - 2 were tested by induction of the alkaline phosphatase activity in C2C12 cells. RESULTS： The rhBMP- 2 was expressed in Escherichia coli in a non - active aggregated form of inclusion bodies using a temperature - inducible expression system. The high- purified rhBMP - 2 was obtained in the form of inclusion bodies by several purification courses. The rhBMP- 2 was refolded and dimerizcd in the redox systems （ reduced and oxidized glutathione） and a one - step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP - 2 dimers and monomers. The purified rhBMP - 2 dimers showed the higher biological activity than the commercial rhBMP - 2. CONCLUSIONS： The method achieved the refolding of rhBMP - 2 would be applied to the whole TGF - β superfamily because the BMP - 2 belongs to the superfamily. Meanwhile, the inexpensive,high yield rhBMP - 2 is suitable for clinic application.