昆虫学报2018，Vol.61Issue(1)：48-58,11.DOI:10.16380/j.kcxb.2018.01.06
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)
摘要
Abstract
[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.关键词
中华按蚊/羧酸酯酶/代谢抗性/异源表达/AsAe7结晶Key words
Anopheles sinensis/carboxylesterase/metabolic resistance/heterologous expression/AsAe7 crystallization分类
生物科学引用本文复制引用
张甜甜,苗娅,许柏英,陈斌..中华按蚊α-羧酸酯酶AsAe7的异源表达、纯化与结晶[J].昆虫学报,2018,61(1):48-58,11.基金项目
重庆市基础与前沿研究计划项目(cstc2015jcyjBX0142,cstc2016jcyjA0375) （cstc2015jcyjBX0142,cstc2016jcyjA0375）
重庆市教委科学技术研究项目(KJ1500326) （KJ1500326）
重庆师范大学博士启动项目(15XLB009) （15XLB009）
“两江学者”计划专项经费 （）
国家自然科学基金项目(31672363,31372265) （31672363,31372265）
国际原子能机构CRP项目(18268) （18268）
国家科技基础性工作专项重点项目(2015FY210300) （2015FY210300）
