棉铃虫细胞色素P450 337B3（CYP337B3）的表达、纯化及结晶
Expression, purification and crystallization ofHelicoverpa armigera CYP337B3
王璐璐1, 2** 王静敏2,3 傅 晟2*** 汪 颖2***
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DOI：10.7679/j.issn.2095-1353.2014.183
作者单位：1. 天津科技大学生物工程学院，天津 300222；2. 天津国际生物医药联合研究院，天津 300457；3. 南开大学，天津 300071
中文关键词：棉铃虫，CYP337B3，蛋白质晶体学
英文关键词：Helicoverpa armigera, CYP337B3, protein crystallography
中文摘要：
【目的】 以抗性棉铃虫Helicoverpa armigera体内发现的一种基因重组导致的嵌合型P450酶CYP337B3作为研究对象，通过基因克隆、体外重组表达、蛋白质结晶等技术手段，获得CYP337B3（△23）的晶体结构，为深入了解棉铃虫P450酶CYP337B3的结构与功能奠定基础。【方法】 对CYP337B3编码基因进行了密码子优化，通过基因重组，将其转化至BL21(DE3)感受态细胞中进行原核表达尝试，通过亲和层析及RESOURCETM Q离子交换层析对CYP337B3（△23）进行体外纯化，利用气相悬滴结晶方法对CYP337B3（△23）进行结晶条件的筛选及X射线晶体衍射。【结果】 通过密码子优化，实现了CYP337B3（△23）在大肠杆菌原核表达系统内的大量表达，经过体外纯化及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测，CYP337B3（△23）蛋白纯度可达到95%左右，我们对CYP337B3（△23）进行了蛋白结晶条件的筛选，最终获得了CYP337B3（△23）的结晶条件和蛋白晶体。【结论】 本文利用气相悬滴结晶方法获得了CYP337B3（△23）的蛋白晶体，为今后CYP337B3的三维结构解析、功能研究以及最终阐述该种新型的棉铃虫抗药机制奠定了良好的基础。
英文摘要：[Objectives] CYP337B3 is a unique P450 enzyme that arose from unequal crossing-over between two parental P450 genes. In order to conduct further research on this enzyme, we expressed and purified it to obtain the CYP337B3 (△23) crystal. [Methods] The gene of CYP337B3 (△23) was optimized and cloned into pET-28aexpression vector. The protein was successfully expressed in Escherichia coli BL21 (DE3) and purified using Ni-NTA affinity chromatography and RESOURCETM Q. The hanging drop vapor diffusion method was used for crystallization after which preliminary X-ray crystallographic analysis was carried out. [Results] CYP337B3 (△23) fused with an N-terminal His-tag was expressed in E.coli BL21 (DE3). The purified CYP337B3 (△23) was calculated to be > 95% pure based on SDS-PAGE analysis. We also performed mass spectrum analysis to confirm the correct expression of the target protein. Furthermore, initial crystals were obtained by the hanging-drop vapor-diffusion process. [Conclusions] After optimization of the crystallization process, rod-like crystals were obtained using 1, 6-Hexanediol as precipitant. Structure determination is underway. CYP337B3 provides a potential target for anti-Helicoverpa armigera drug development.