葡萄糖异构酶突变体酶GIG138P和GIG138P-G247D在变铅青链霉菌中的高效表达及检测

Overexpression and Detection of the Mutated Glucose Isomerase GIG138Pand GIG138P-G247D in Streptomyces lividans

将含有G138P单点突变和G138P G2 47D双点突变的GI结构基因 ,分别克隆入E .coli 链霉菌穿梭载体pHZ 12 72 ,成功构建了穿梭表达载体pHZGI1和pHZGI2。通过原生质体的转化 ,将穿梭表达载体导入变铅青链霉菌TK5 4菌株。 30℃振荡培养 2 4h ,加入 2 μg mL硫链丝菌素诱导表达 12h。SDS PAGE电泳表明 ,两个穿梭载体在TK5 4菌株内表达出 42 5kD特异性条带。薄层扫描显示 ,突变体酶GIG138P和GIG138P G2 47D分别约占可溶性蛋白的19%和 2 2 %。Western杂交进一步证实GIG138P和GIG138P G2 47D在变铅青链霉菌TK5

The shuttle expression vectors pHZGI1 and pHZGI2 were successfully constructed by inserting structural genes of GI containing single mutated site G138P and double mutated site G138P-G247D into E.coli- Streptomyces shuttle vector pHZ-1272, respectively. Then they were transformed into S. lividans TK54 strain by protoplast transformation. SDS-PAGE indicated that two shuttle vectors in TK54 strain expressed obviously specific bands at 42.5 kD after inducted by 2μg/mL thiostrepton. Optical densitometric scan showed that the content of the mutant enzymes GIG138P and GIG138P-G247D were about 19% and 22% of dissoluble proteins, respectively. Western blotting farther proved that GIG138P and GIG138P-G247D were expressed in S.lividans TK54.