| 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 |
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| 引用本文： | 朱国萍,张颖,徐旸,唐建国,徐冲. 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[J]. 生物工程学报, 2002, 18(3): 304-307 |
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| 作者姓名： | 朱国萍 张颖 徐旸 唐建国 徐冲 |
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| 作者单位： | 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 |
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| 基金项目： | 国家“8 6 3”高技术研究与发展计划项目 (No.130 13 0 2 0 4)~~ |
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| 摘 要： | 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。
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| 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 |
| 文章编号： | 1000-3061(2002)03-0304-04 |
| 修稿时间： | 2001-12-10 |
Overexpression and Detection of the Mutated Glucose Isomerase GIG138P and GIG138P-G247D in Streptomyces lividans |
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| ZHU Guo-Ping ZHANG Ying XU Yang TANG Jian-Guo XU Chong. Overexpression and Detection of the Mutated Glucose Isomerase GIG138P and GIG138P-G247D in Streptomyces lividans[J]. Chinese journal of biotechnology, 2002, 18(3): 304-307 |
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| Authors: | ZHU Guo-Ping ZHANG Ying XU Yang TANG Jian-Guo XU Chong |
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| Affiliation: | Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei 230026, China. |
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| Abstract: | 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. |
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| Keywords: | glucose isomerase mutant enzyme shuttle vector Streptomyces lividans overexpression |
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