中华按蚊α-羧酸酯酶AsAe7的异源表达、纯化与结晶

Heterologous expression, purification and crystallization of α-carboxylesterase AsAe7 from Anopheles sinensis(Diptera:Culicidae)

【目的】羧酸酯酶(carboxylesterase,COE)是昆虫体内一类重要的解毒酶,与昆虫的抗药性相关。本研究旨在对中华按蚊Anopheles sinensis羧酸酯酶Ae7(Asae7)进行初步晶体学研究,为解析As Ae7的空间结构及探讨其分子功能奠定基础。【方法】首先对Asae7进行生物信息学分析,然后进行分子克隆,并利用原核表达系统在体外对Asae7进行重组表达;结合Ni-NTA金属螯合层析和葡聚糖凝胶层析方法纯化融合表达蛋白;通过葡聚糖凝胶层析和化学交联结果分析As Ae7的聚合状态;采用坐滴气相扩散法对As Ae7进行结晶筛选。【结果】生物信息学分析表明,As Ae7是亲水性蛋白,分子量为61.053 k D,无跨膜区和信号肽;3D结构预测结果分析显示,As Ae7采取的是α/β-水解酶超家族折叠模式。多序列比对结果表明,在不同昆虫中Ae7蛋白具有高度保守性。分子克隆得到中华按蚊As Ae7的编码基因Asae7序列,大小为1 626 bp。成功构建重组质粒p ET28aAsae7;在大肠杆菌Escherichia coli中表达的融合蛋白As Ae7主要分布在上清中。通过镍柱亲和层析和凝胶过滤层析纯化出了高纯度且稳定的目的蛋白;通过凝胶过滤层析和化学交联获得纯化的As Ae7主要呈单体状态;同时通过晶体筛选获得了As Ae7的晶体。【结论】运用结晶学的方法初步获得了As Ae7的晶体,为后续解析As Ae7的晶体结构以及在原子分辨率水平上直观阐释As Ae7参与代谢抗性的分子机制奠定了基础。

【Aim】 Carboxylesterase is one kind of the important detoxifying enzymes in insects, and is associated with insecticide resistance. The aim of this study is to explore the structure and the function of the carboxylesterase Ae7 from Anopheles sinensis （AsAe7） based on its preliminary crystallography. 【Methods】 The Asae7 gene was bioinformatically analyzed, cloned, and expressed in prokaryotic expression system. The recombinant protein was purified with nickel chelate affinity chromatography and gel filtration chromatography. The polymerization of AsAe7 was detected by gel filtration chromatography and chemical crosslinking analysis, and the crystal screening was performed by sitting drop vapor diffusion technique. 【Results】 Bioinformatic analysis revealed that AsAe7 is a hydrophilic protein of 61.053 kD without transmembrane regions and signal peptide. The 3D structural prediction showed that AsAe7 adopts an α/β-hydrolase superfamily fold. Multiple sequence alignment result demonstrated that Ae7 proteins are highly conserved in different insects. The 1 626 bp coding sequence of Asae7 was cloned, and the recombinant plasmid pET28a-Asae7 was constructed correctly. The SDS-PAGE analysis illustrated that the fusion protein AsAe7 expressed in Escherichia coli mainly existed in the supernatant. The highly purified and stable protein was then obtained with two-step affinity chromatography. Gel filtration chromatography and chemical crosslinking analysis showed that AsAe7 mainly exists as monomer in vitro. Finally, protein crystals of AsAe7 were obtained by crystal screening. 【Conclusion】 Crystals of the recombinant AsAe7 have been obtained by crystallography, which lays the foundation for illustrating the crystal structure of AsAe7 and the molecular mechanisms of AsAe7-mediated metabolic resistance at atomic resolution.