银胶菊丙二烯氧化物合成酶的表达纯化及其活性分析

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。

Expression, Purification and Activity Assay of Allene Oxide Synthase from Parthenium argentatum

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.