| 甲酸脱氢酶基因在大肠杆菌Rosetta中的高效表达 |
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| 引用本文： | 徐娴,贾红华,何冰芳,韦萍. 甲酸脱氢酶基因在大肠杆菌Rosetta中的高效表达[J]. 食品与发酵工业, 2007, 33(5): 5-8 |
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| 作者姓名： | 徐娴 贾红华 何冰芳 韦萍 |
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| 作者单位： | 南京工业大学,制药与生命科学学院,江苏南京,210009 |
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| 基金项目： | 国家重点基础研究发展计划(973计划);国家自然科学基金 |
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| 摘 要： | 甲酸脱氢酶是氧化还原酶辅酶NAD(P)~+和NAD(P)H再生循环体系中的关键酶。实验中,用PCR方法扩增了假丝酵母Candida boidinii中甲酸脱氢酶基因(fdh),分别构建了诱导型表达载体pUC-dh和pET- fdh,以E.coli Rosetta为表达宿主,获得了能够高效表达甲酸脱氢酶的重组菌。经诱导后重组菌Rosetta/pET- fdh的FDH比酶活为0.399U/mg(蛋白),Rosetta/pUC-fdh的FDH比酶活为0.163U/mg(蛋白)。经SDS- PAGE分析,Rosetta/pET-fdh和Rosetta/pUC-fdh表达的FDH蛋白分别占菌体可溶性总蛋白的17%和10%,实现了fdh基因在Rosetta中的高效表达。研究中所构建的重组菌为氧化还原反应中的辅酶再生奠定了良好的基础。
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| 关 键 词： | 辅酶再生 甲酸脱氢酶 甲酸脱氢酶基因 高效表达 |
| 修稿时间： | 2006-11-17 |
Effective Expression of NAD-dependent Formate Dehydrogenase Gene in E.coli Rosetta |
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| Xu Xian,Jia Honghua,He Bingfang,Wei Ping. Effective Expression of NAD-dependent Formate Dehydrogenase Gene in E.coli Rosetta[J]. Food and Fermentation Industries, 2007, 33(5): 5-8 |
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| Authors: | Xu Xian Jia Honghua He Bingfang Wei Ping |
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| Affiliation: | College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology Nanjing, 210009, China |
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| Abstract: | Formate dehydrogenase(FDH)is the key enzyme in NAD(P)~+-NAD(P)H coenzyme regenera- tion system.In this article,NAD~+-dependent formate dehydrogenase gene(fdh)was amplified from genome DNA of Candida boidinii by PCR,and constructed recombinant plasmids pET-fdh and pUC-fdh which were then transformed into E.coli Rosetta respectively.The enzymatic activity of FDH in Rosetta/pET-fdh is 0.399U/rag protein,while in Rosetta/pUC-fdh is 0.163U/mg protein.SDS-PAGE showed that the expres- sion of FDH in Rosetta/pET-fdh and Rosetta/pUC-fdh is 10% and 17% of all total soluble protein respec- tively.This study laid good foundation for the coenzyme regeneration system. |
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| Keywords: | coenzyme regeneration formate dehydrogenase fdh effective expression |
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