大肠杆菌表达的重组人骨形成蛋白-7的复性

Renaturation of recombinant human bone morphogenetic protein-7

带有pBV221-hBMP-7的E.coli表达得到的rhBMP-7以不溶的包涵体形式存在,用高浓度的变性剂溶解后,经过DEAE-FF纯化,得到高纯度的目的蛋白,达95%以上。分别用尿素浓度梯度降低法、添加促复性剂及人工分子伴侣法对蛋白质进行复性,并通过不同方法对复性结果进行比较。Westernblot中辉度扫描结果显示,GSH/GSSG法样品二聚体/单体比例为79.5/20.5,尿素浓度梯度降低法二聚体/单体比例为73.6/26.4,表明GSH/GSSG法复性样品溶液上清中含较高比例的蛋白质二聚体。根据不同复性样品对NIH3T3细胞ALP活性影响大小的比较结果,氧化还原剂最有助于二聚体的形成,蛋白质活性最高。

E.coli transduced with pBV221-rhBMP-7 can express hBMP-7. For some reasons the protein exists as inclusion body and needs renaturation. After solution in 8 mol/L urea, it is purified through DEAE-FF to 95% and renaturated by several methods: lower urea gradiently, dialysis to renaturant, adorpt artificial chaperon. Among them, oxidize-reduced pair is the most efficient with dimer/monomer 79.5/20.5, and lower urea gradiently with 73.6/26.4. Comparison of the effect on the activity of NIH3T3 prove it too. This owes to the construction and character of BMP-7.