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

作　　者：朱国萍[1] 张颖[1] 徐旸 唐建国 徐冲[1] 

机构地区：[1]中国科学技术大学生命科学学院分子生物学与细胞生物学系,合肥230026

出　　处：《生物工程学报》2002年第3期304-307,共4页Chinese Journal of Biotechnology

基　　金：国家"8 6 3"高技术研究与发展计划项目 (No.130 13 0 2 0 4)~~

摘　　要：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TK5The 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.

关 键 词：葡萄糖异构酶 突变体酶 穿梭载体 变铅青链霉菌 高效表达 GIG138P GIG138P-G247D 

分 类 号：Q786[生物学—分子生物学]