| 鸡细胞色素P450 1A5的原核表达与活性重组 |
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| 引用本文： | 安俊峰,王运浩,侯鑫.鸡细胞色素P450 1A5的原核表达与活性重组[J].生物技术通报,2012(6):94-98. |
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| 作者姓名： | 安俊峰 王运浩 侯鑫 |
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| 作者单位： | 内蒙古大学生命科学学院,呼和浩特,010021 |
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| 基金项目： | 国家重点基础研究发展计划“973”项目 |
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| 摘 要： | 旨在对鸡细胞色素P450 1A5(CYP1A5)蛋白进行体外功能研究,采用大肠杆菌系统进行CYP1A5的异源表达。以鸡的cDNA为模板,扩增出CYP1A5基因,将该基因的N端编码区进行修饰,并连接到pCW载体中构建His-CYP1A5,经IPTG诱导在大肠杆菌中表达。经CO-差示光谱检测,所获得的His-CYP1A5具有典型的P450吸收峰。该蛋白与细胞色素P450还原酶(CPR)进行体外重组,构成的重组酶系表现出乙氧基试卤灵-O-脱乙基酶活性。结果表明,所采用的表达策略可以成功产生出具有催化活性的鸡细胞色素P450 1A5(CYP1A5)蛋白。
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| 关 键 词： | 细胞色素 P4501A5(CYP1A5) 异源表达 体外重组 酶活性 |
Functional Expression of Chicken Cytochrome P450 1A5 in Escherichia coli |
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| An Junfeng
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Wang Yunhao
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Hou Xin.Functional Expression of Chicken Cytochrome P450 1A5 in Escherichia coli[J].Biotechnology Bulletin,2012(6):94-98. |
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| Authors: | An Junfeng Wang Yunhao Hou Xin |
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| Institution: | An Junfeng Wang Yunhao Hou Xin(College of Life Science,Inner Mongolia University,Hohhot 010021) |
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| Abstract: | To survey the in vitro activities of chicken cytochrome P450 1A5(CYP1A5),genetically engineered Escherichia coli was used in the present study for recombinant expression of target protein.Chicken cDNA was used as template to amplify CYP1A5 gene which was then connected to pCW vector(EcoR I/Sal I) and induced by IPTG in E.coli.Through the detection of CO-difference spectra,the expression of CYP1A5 had the typical P450 absorption peak.The expressed protein reconstituted with cytochrome P450 reductase(CPR) showed 7-ethoxyresorufin O-deethylase activity.The results suggested that the expressions used in this study can successfully produce the catalytic activity of the chicken cytochrome P450 1A5(CYP1A5) protein. |
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| Keywords: | Cytochrome P450 1A5(CYP1A5) Heterologous expression Reconstitution in vitro Enzymatic activity |
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