定点突变改善红色荧光指示蛋白玉米尿卟啉原Ⅲ甲基化酶的水溶性研究

Improved the Solubility of Maize Uroporphyrinogen Ⅲ Methyltransferase as the Red Fluorescent Indicator by Site-directed Mutagenesis

摘要:

尿卟啉原Ⅲ甲基化酶是一种新型的红色荧光指示蛋白，但是，在大肠杆菌重组表达的SUMT水溶性相对较低，限制了它的应用范围，而且对于结合在蛋白的色素组分尚不清楚。利用定点突变产生玉米尿卟啉原Ⅲ甲基化酶L88R/L89G双突变体和L166A突变体，两种突变体分别在大肠杆菌中重组表达，Ni-NTA一步纯化。紫外可见光谱扫描和质谱分析确定从纯化的L88R/L89G双突变体蛋白分离的色素组分。L88R/L89G双突变体在大肠杆菌细胞内有酶活，而L166A突变体胞内酶活丧失。结合蛋白的主要组分为三甲基化咕啉。纯化的双突变体蛋白水溶性增加，为提高它作为荧光指示蛋白检测外源融合蛋白的水溶性打下基础。

Abstract:

S-adenosylmethionine-dependent uroporphyrinogen Ⅲ methyltransferase (SUMT) is a novel red fluorescence indicator. However, the production of SUMT in Escherichia coli is restricted by its relatively low solubility, and little is known about the red fluorescent materials that are associate with SUMT. Two individual SUMT mutations, L166A and L88R/L89G double mutant were produced by site-directed mutagenesis. Both mutants were overexpressed in E. coli and purified by Ni-NTA chromatography. The reddish mixtures isolated from the purified L88R/L89G double mutant were analyzed by UV-visible spectra scanning and mass analysis(MS). The L88R/L89G double mutant has enzymatic activity in vivo, whereas L166A mutant loses the activity. Trimethylpyrrocorphin is identified as the main constituent in the isolated pigments. The purified L88R/L89G mutant increases protein solubility, which is applied potentially as the fluorescent indicator denoting the solubility of protein fusion partner.