| 大肠杆菌glgC基因的定点突变及高效表达 |
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| 引用本文： | 姚庆荣,郭运玲,孔华,张金文,郭安平,贺立卡,刘恩平.大肠杆菌glgC基因的定点突变及高效表达[J].热带作物学报,2007,28(1):60-64. |
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| 作者姓名： | 姚庆荣 郭运玲 孔华 张金文 郭安平 贺立卡 刘恩平 |
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| 作者单位： | 1. 中国热带农业科学院热带作物生物技术国家重点实验室,海口,571101;中国热带农业科学院热带生物技术研究所,海口,571101
2. 甘肃农业大学农学院,兰州,730070 |
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| 基金项目： | 中国热带农业科学院科研基金 |
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| 摘 要： | 通过PCR方法,从E.coliJM109基因组DNA中扩增到全长为1296bp的glgC基因,对其进行定点突变,获得第296位和336位氨基酸同时突变的glgC336基因。将2者亚克隆进pET-30a,成功构建了重组表达载体pET-C和pET-C336,转化大肠杆菌BL21(DE3),在1mmol/LIPTG诱导下进行表达。SDS-PAGE电泳分析显示,在约53kD处有1条明显的蛋白表达带,证明目的基因已得到高效表达,且重组蛋白表达量占菌体蛋白总量的77.3%。
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| 关 键 词： | glgC基因 定点突变 原核表达 |
| 修稿时间： | 2006-12-142007-02-06 |
Site-directed Mutagenesis and High-level Expression of glgC Gene from E. coli |
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| Yao Qingrong,Guo Yunling,Kong Hua,Zhang Jinwen,Guo Anping,He Lika,Liu Enping.Site-directed Mutagenesis and High-level Expression of glgC Gene from E. coli[J].Chinese Journal of Tropical Crops,2007,28(1):60-64. |
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| Authors: | Yao Qingrong Guo Yunling Kong Hua Zhang Jinwen Guo Anping He Lika Liu Enping |
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| Institution: | 1 State Key Biotechnology Laboratory for Tropical Crops, CATAS Haikou Hainan 571101 China; 2 Institute of Tropical Bioscience and Biotechnology, CATAS Haikou Hainan 571101 China; 3 College of Agronomy, Gansu Agricultural University Lanzhou Gansu 730070 China |
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| Abstract: | By using genomic DNA of E. coli JM109 as a template, glgC gene was amplified by polymerase chain reaction (PCR). The full length of this gene was 1 296 bp. The nucleotide 1 007 was changed from G to A via site-directed mutagenesis, and the amino acid was changed from glycine 336 to aspartic acid accordingly. The mutated gene named glgC336 and glgC was subcloned into prokaryotic expression vector pET-30a, and this recombinant expression plasmid was transformed into E. coli BL21(DE3) for effective expression. The SDS-PAGE analysis indicated that the recombinant protein was 53 kDa which is the same as reported. The expression level of glgC and glgC336 protein was about 77.3 % of the total protein of the induced recombinant bacteria at the presence of IPTG. |
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| Keywords: | glgC gene site-directed mutagenesis prokaryotic expression |
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