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

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.