大肠杆菌细胞周质底物结合蛋白gsiB基因的克隆及其表达条件的优化

doi:10.3724/SP.J.1005.2010.00505

遗传 ›› 2010, Vol. 32 ›› Issue (5): 505-511.doi: 10.3724/SP.J.1005.2010.00505

大肠杆菌细胞周质底物结合蛋白gsiB基因的克隆及其表达条件的优化

王中山, 向泉桔, 王海燕, 张义正

四川大学生命科学学院四川省分子生物学和生物技术重点实验室, 成都 610064

收稿日期:2009-12-11 修回日期:2010-01-16 出版日期:2010-05-20 发布日期:2010-05-05
通讯作者: 张义正 E-mail:yizzhang@scu.edu.cn
基金资助:
国家自然科学基金项目(编号: 30871344)资助

Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli

WANG Zhong-Shan, XIANG Quan-Ju, WANG Hai-Yan, ZHANG Yi-Zheng

College of Life Sciences, Sichuan University, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Chengdu 610064, China

Received:2009-12-11 Revised:2010-01-16 Online:2010-05-20 Published:2010-05-05
Contact: ZHANG Yi-Zheng E-mail:yizzhang@scu.edu.cn

摘要/Abstract

摘要：

为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能, 对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列, 利用PCR方法扩增到该基因的编码区序列, 利用SLIC (Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中, 成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达, 通过改变培养温度和IPTG浓度等条件, 得到了能够大量表达目标蛋白的重组子。结果表明: 大肠杆菌BL21(DE3)是 gsiB基因表达的最佳宿主菌; 18℃低温诱导培养有利于gsiB基因的大量表达; 0.1 mmol/L IPTG足够诱导gsiB基因表达, 增加IPTG浓度(0.1 mmol/L~1.0 mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达, 其分子量大小与预期相符。

关键词: 大肠杆菌, 谷胱甘肽转运系统, 细胞周质底物结合蛋白, gsiB基因, 基因表达, 蛋白质印迹

Abstract:

Cloning and expression of gsiB was carried out for studying protein structure and function of glutathione transport system. The coding sequence of Escherichia coli gsiB that encodes the periplasmic solute-binding protein of a glutathione transporter was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring GFP reporter gene through the method Sequence and Ligation–Independent Cloning (SLIC). The resulting recombinant plasmid pWaldo-GFP-GsiB was transformed into different E. coli strains and the expression conditions were optimized. It was found that E. coli BL21(DE3) was the best strain for gsiB gene expression among the four strains tested. Induction at lower incubation temperature of 18°C and 0.1 mmol/L of IPTG led to higher expression of gsiB in E. coli BL21(DE3). Western blotting analysis also showed the expression of gsiB and the molecular weight of expressed protein as expected.

Key words: Escherichia coli, glutathione transporter, periplasmic solute-binding component, gsiB, gene expression, Western blotting

王中山，向泉桔，王海燕，张义正. 大肠杆菌细胞周质底物结合蛋白gsiB基因的克隆及其表达条件的优化[J]. 遗传, 2010, 32(5): 505-511.

WANG Zhong-Shan, XIANG Quan-Jie, WANG Hai-Yan, ZHANG Xi-Zheng. Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli[J]. HEREDITAS, 2010, 32(5): 505-511.

参考文献

[1] Meister A. A brief history of glutathione and a survey of its metabolism and functions. In: Dolphin ID, Poulson R, Avramovic O. Glutathione, chemical, biochemical, and medical aspects. John Wiley and Sons, 1989. New York, 1–48.

[2] Yuan L, Kaplowitz N. Glutathione in liver diseases and hepatotoxicity. Mol Aspects Med, 2009, 30(1–2): 29–41.

[3] Biswas S, Rahman I, Environmental toxicity, redox sig-naling and lung inflammation: the role of glutathione. Mol Aspects Med, 2009, 30(1–2): 60–76.

[4] Kannan R, Yi JR, Tang D, Li Y, Zlokovic BV, Kaplowitz N. Evidence for the existence of a sodium-dependent glu-tathione (GSH) transporter. Expression of bovine brain capillary mRNA and size fractions in Xenopus laevis oo-cytes and dissociation from γ-glutamyltranspeptidase and facilitative GSH transporters. J Biol Chem, 1996, 271(16): 9754–9758.

[5] Jamai A, Martinoia E, Delrot S. Characterization of glu-tathione uptake in broad bean leaf protoplasts. Plant Physiol, 1996, 111(4): 1145–1152.

[6] Li ZS, Szczypka M, Lu YP, Thiele DJ, Rea PA. The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump. J Biol Chem, 1996, 271(11): 6509–6517.

[7] Bourbouloux A, Chakladar A, Delrot S, Bachhawat AH, Shahi P. Hgt1p, a high affinity glutathione transporter from yeast Saccharomyces cerevisiae. J Biol Chem, 2000, 275(18): 13259–13265.

[8] Fahey R, Brown W, Adams W, Worsham M. Occurrence of glutathione in bacteria. J Bacteriol, 1978, 133(3): 1126–1129.

[9] Suzuki H, Kumagai H, Tochikura T. Isolation, genetic mapping, and characterization of Escherichia coli K-12 mutants lacking γ-glutamyltranspeptidase. J Bacteriol, 1987, 169(9): 3926–3931.

[10] Owens RA, Hartman PE. Export of glutathione by some widely used Salmonella typhimurium and Escherichia coli strains. J Bacteriol, 1986, 168(1): 109–114.

[11] Suzuki H, Hashimoto W, Kumagai H. Escherichia coli K-12 can utilize an exogenous γ-glutamyl peptide as an amino acid source, for which γ-glutamyltranspeptidase is essential. J Bacteriol, 1993, 175(18): 6038–6040. [12] Suzuki H, Kamatani S, Kim ES, Kumagai H. Aminopep-tidases A, B, and N and dipeptidase D are the four cys-teinylglycinases of Escherichia coli K-12. J Bacteriol, 2001, 183(4): 1489–1490.

[13] Hideyuki S, Takashi K, Shunsuke I, Akiko O, Hidehiko K. The yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a novel glutathione importer with an ATP-binding cassette. J Bacteriol, 2005, 187(17): 5861–5867. [14] Keseler IM, Bonavides-Martínez C, Collado-Vides J, Gama-Castro S, Gunsalus RP, Johnson DA, Krum-menacker M, Nolan LM, Paley S, Paulsen IT, Peralta-Gil M, Santos-Zavaleta A, Shearer AG, Karp PD. EcoCyc: A comprehensive view of Escherichia coli Biology. Nucleic Acids Res, 2009, 37: 464–470.

[15] Geoffrey SW, Blake MS, Joel B, Thomas CT. Rapid pro-tein-folding assay using green fluorescent protein. Nature Biotechnol, 1999, 17: 691–695.

[16] 白帆, 刘珣, 张义正. 一种新的基因克隆方法SLIC的可靠性分析. 四川大学学报(自然科学版), 2008, 45(增刊): 33–36.

[17] Mamie Z L, Stephen JE. Harnessing homologous recom-bination in vitro to generate recombinant DNA via SLIC. Nat Methods, 2007, 3(4): 251–256.

[18] Kay T. Overview of bacterial expression systems for het-erologous protein production: from molecular and bio-chemical fundamentals to commercial systems. Appl Mi-crobiol Biotechnol, 2006, 72: 211–222.

[19] David D, Mirjam L, Edmund K, Dirk JS, Jan WG. Opti-mization of membrane protein overexpression and purifi-cation using GFP fusions. Nat Mothods, 2006, 3(4): 303–313.

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大肠杆菌细胞周质底物结合蛋白gsiB基因的克隆及其表达条件的优化

Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli

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