增强型绿荧光蛋白突变体mut4EGFP在大肠杆菌中的表达与纯化  

The Expression and Purification of Enhanced Green Fluorescent Protein mutant mut4EGFP in E. coli

作　　者：牛传双[1] 方六荣[1] 肖少波[1] 张辉[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院动物病毒室,武汉430070

出　　处：《华中农业大学学报》2004年第5期489-491,共3页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目 (3 9970 5 5 9)资助

摘　　要：增强型绿荧光蛋白突变体mut4EGFP(EGFP/V16 3A/S175G)是一种发光能力更强、更稳定的新型绿色荧光蛋白。为了获得大量高纯度的蛋白 ,将mut4EGFP基因插入原核表达载体 pTWIN1中 ,使之与几丁质结合域 (CBD) 内含肽 (intein)融合 ,获得原核表达质粒 pTWIN M 4。将 pTWIN M 4转化大肠杆菌BL2 1(DE3)plysS ,IPTG诱导后进行SDS PAGE电泳分析 ,结果显示 ,CBD intein mut4EGFP融合蛋白在BL2 1(DE3) plysS中获得高效表达并以可溶性蛋白的形式存在。进一步采用几丁质柱纯化系统对表达产物进行纯化 ,得到了高纯度的mut4EGFP。mut4EGFP, a novel enhanced green fluorescent protei nmutant (EGFP/V163A/S175G), is more bright and stable than EGFP. In orde r to obtain the purified protein, the DNA fragment encoding mut4EGFP was inserte d into prokaryotic expression vector pTWIN1, resulting in the expression plasmid pTWIN-M4. pTWIN-M4 was further transformed into BL21(DE3)plysS, induced by IP TG and expression was analyzed using SDS-PAGE. The results showed that mut4EGF P was expressed and existed in soluble form. The expressed products were further purified using Chitin Beadsand mg-grade recombinant proteins were obta ined.

关 键 词：绿荧光蛋白 突变体mut4EGFP 表达 纯化 

分 类 号：Q786[生物学—分子生物学]