葡萄糖异构酶突变体酶GIGl38P和GIG138P-G247D在变铅青链霉菌中的高效表达及检测

将含有G138P单点突变和G138P－G247D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-1272，成功构建了穿梭表达载体pHZGI1和pHZGI2。通过原生质体的转化，将穿梭表达载体异入变铅青链霉菌TK54菌株。30℃振荡培养24h，加入2μg/mL硫链丝菌素诱导表达12h。SDS－PAGE电泳表明，两个穿梭载体在TK54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－G247D分别约占可溶性蛋白的19％和22％。Western杂交进一步证实GIG138P和GIG138P－G247D在变铅青链霉菌TK54中获得了表达。

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

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 2 micrograms/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.