利用绿色荧光蛋白建立包涵体变-复性新方法  

Development of A Novel Refolding Method Using Inclusion Bodies from Green Fluorescent Protein

作　　者：张颋[1] 冯延叶[1] 沈亚领[1] 金维荣[2] 杨忠[1] 王菊芳[3] 王小宁[1,3] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]生物芯片上海国家工程研究中心,上海201203 [3]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《食品科学》2010年第5期141-146,共6页Food Science

基　　金：国家"863"计划项目(2007AA021702);国家"973"计划项目(2007CB512402);广州市科技计划项目(2007J1-C0131)

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。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.

关 键 词：大肠杆菌 包涵体 变性 复性 绿色荧光蛋白 

分 类 号：Q78[生物学—分子生物学]