【摘要】 为提高常规稀释复性方法的复性效率,以重组绿色荧光蛋白(green fluorescent protein,GFP)包涵体为模型,开发一种新的双变性-稀释复性方法。新方法选取含有精氨酸的碱性复合溶液作为第一变性剂溶解GFP包涵体,通过梯度降低变性液的酸碱度析出溶解目的蛋白,再以尿素为第二变性剂溶解析出的蛋白,随后进行稀释复性。结果显示:与常规方法比,新方法复性的绿色荧光蛋白活性收率提高至1.5～2.3倍,复性蛋白对温度、溶液酸碱度及变性剂的稳定性提高。增强型绿色荧光蛋白(enhanced GFP,EGFP)及其融合蛋白的包涵体采用新方法进行复性,均取得80%以上的活性回收率,说明新方法对GFP系列融合蛋白的包涵体具有一定的适用性。新方法从变性剂使用与包涵体结构的关系出发,突破常规操作中变性剂单次使用的局限,既保留了简便性又提高了常规稀释复性方法的效率。更多还原

【Abstract】 Target proteins overexpressed in Escherichia coli often result in the formation of inclusion bodies, which need further refolding in vitro to gain their native structures and biological functions. In order to improve the refolding rate by dilution refolding method, recombinant green fluorescent protein (GFP) inclusion bodies were used as the model to develop a novel dual denaturation-dilution refolding method in this study. This novel method used the alkaline solution containing arginine as the first denaturant to dissolve GFP inclusion bodies, and then exhibited a gradient reduction in pH of the solution to mildly form the target protein aggregates. The denatured aggregates were refolded by dilution in urea solution. Results indicated that the recovery rate of refolded GFP by this novel method was over 80%, which was 1.5 to 2.3 times higher than that of traditional methods. Moreover, the stability of refolded GFP by this novel method was highly consistent with the natural protein. Therefore, this developed method for protein refolding could be used to refold proteins, especially GFP-related proteins from inclusion bodies with high efficiency. This investigation will also provide optimal methods for protein refolding from inclusion bodies through combinatorial design and extended applications of different denaturants.更多还原