青蒿素生物合成关键酶CYP71AV1的表达纯化和晶体培养

Prokaryotic expression,purification and crystallization of CYP71AV1,key enzyme in artemisinin biosynthesis

疟疾是一种严重危害人类健康的世界性流行疾病,在青蒿素广泛应用之前,全世界每年约4亿人次感染疟疾,至少有100万人的死亡病例。因此疟疾被世界卫生组织列为世界三大死亡疾病之一。青蒿素的发现,开创了疟疾治疗新方法,全球数亿人因这种＂中国神药＂而受益。紫穗槐-4,11-二烯氧化酶是一种植物细胞色素P450蛋白（CYP71AV1）,参与催化紫穗槐-4,11-二烯生成青蒿酸的三步氧化反应,是青蒿素生物合成途径上的关键酶。作者拟通过p CW Ori（＋）-CYP71AV1重组质粒在原核表达系统中的可溶性表达及蛋白纯化,采用一氧化碳差式光谱分析和基于血红素辅基的生物活性测定,验证CYP71AV1蛋白的活性,培养该蛋白的晶体。结果表明,构建的重组基因p CW Ori（＋）-CYP71AV1在大肠杆菌中得以功能性表达,经过亲和层析和凝胶过滤层析纯化得到了毫克级、有活性的目的蛋白,筛选出了重组CYP71AV1蛋白的晶体生长条件。这些研究为解析青蒿素生物合成关键酶CYP71AV1的晶体结构,以及通过合成生物学方法批量生产青蒿素奠定了基础,同时为其他植物来源的P450蛋白的表达纯化与结晶提供参考。

Malaria is a worldwide epidemic that extensively endangers health of human beings. Before artemisinin was developed to treat with malaria,about 400 million person-time of malaria infections and at least 1 million deaths from malaria were reported in the world every year. Thus malaria has been listed as one of the world＇s three major death diseases by the WHO. The discovery of artemisinin by Chinese scientists created a novel therapy approach to treat with malaria effectively. Amorpha-4,11-diene oxidase is a plant cytochrome P450 enzymes,i. e. CYP71 AV1,which catalyzes each of the three oxidation steps from amorpha-4,11-diene to form artemisinic acid,the intermediate of artemisinin. CYP71 AV1 is the key enzyme in artemisinin biosynthesis. By constructing the prokaryotic expression vector p CWOri（＋）-CYP71 AV1,functional expression and purification of complementary CYP71 AV1 were performed. The enzyme activity was monitored by CO differential spectrum assay and the heme-based activity analysis. The preliminary crystallization condition was obtained by crystallization screening. These studies provide basis for resolving the crystal structure of CYP71 AV1 and for producing artemisinin in large scale through biosynthetic biology approach,and will provide references for over expression,purification and crystallization of other plant P450 enzymes.