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| 银胶菊丙二烯氧化物合成酶的表达纯化及其活性分析 | |
| 引用本文： | 刘云华,周秋虎,张灵芝,常振战. 银胶菊丙二烯氧化物合成酶的表达纯化及其活性分析[J]. 生物技术通报, 2012, 0(8): 76-82 |
| 作者姓名： | 刘云华 周秋虎 张灵芝 常振战 |
| 作者单位： | 1. 黑龙江生态工程职业学院生物技术系,哈尔滨150025;北京大学基础医学院生物物理系,北京100191 2. 北京大学基础医学院生物物理系,北京,100191 3. 北京大学药学院天然药物学系,北京,100191 |
| 基金项目： | 国家自然科学基金资助项目，北京市自然科学基金资助项目 |
| 摘 要： | 植物来源细胞色素P450的表达和结晶都十分困难,成为成功解析该类蛋白晶体结构的瓶颈。探索了使用大肠杆菌表达系统表达、纯化银胶菊丙二烯氧化物合成酶(allene oxide synthase,AOS)的方法。氨基酸序列比对分析显示,银胶菊AOS缺失N-末端的膜锚定序列和信号肽序列,推测其在大肠杆菌中应表达为水溶性蛋白质。试验中纯化得到的银胶菊AOS是一个分子量为54.5 kD的八聚体分子,均一性好,不含去垢剂,达到毫克量级,适合下一步的晶体培养研究;还原一氧化碳差示光谱显示该酶在450 nm处有特征吸收峰,酶活性测定得到该酶的米氏常数为45.3μmol/L,显示该重组AOS具有很高的酶活性。 |
| 关 键 词： | 银胶菊(Parthenium argentatum) 丙二烯氧化物合成酶 细胞色素P450 表达纯化 酶活性分析 |
Expression, Purification and Activity Assay of Allene Oxide Synthase from Parthenium argentatum |
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| Liu Yunhua , Zhou Qiuhu , Zhang Lingzhi , Chang Zhenzhan. Expression, Purification and Activity Assay of Allene Oxide Synthase from Parthenium argentatum[J]. Biotechnology Bulletin, 2012, 0(8): 76-82 | |
| Authors: | Liu Yunhua Zhou Qiuhu Zhang Lingzhi Chang Zhenzhan |
| Affiliation: | 1Department of Biotechnology,Heilongjiang Vocational Institute of Ecological Engineering,Harbin 150025;2Department of Biophysics,School of Basic Medical Sciences,Peking University,Beijing 100191;3Department of Natural Medicines, School of Pharmaceutical Sciences,Peking University,Beijing 100191) |
| Abstract: | Expression and crystallization of Cytochrome P450 from plants is difficult,and becomes the bottleneck of structural study for plant P450s.It was researched the expression and purification of AOS from guayule using E.coli expression system.In order to realize expression and purification of the AOS,its amino acid sequence was aligned with that of other CYP74A enzymes.Results showed that the AOS was the absence of the amino-terminal membrane anchor and the putative transit sequence.It could be expressed in E.coli as water-soluble form.Experimental purified recombinant AOS was an active octamer of molecular mass of 54.5 kD,it was milligram quantities of homogeneous,detergent-free AOS protein which would be used for crystallographic study.The reduced-CO difference spectrum of recombinant AOS had a characteristic band at 450 nm.Enzymatic activity assay was performed.Value of Km was 45.3 μmol/L,that is,the purified AOS exhibited high activity. |
| Keywords: | Guayule(Parthenium argentatum) Allene oxide synthase Cytochrome P450 Expression and purification Enzyme activity assay |
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