›› 2018, Vol. 61 ›› Issue (1): 48-58.doi: 10.16380/j.kcxb.2018.01.006

• RESEARCH PAPERS • Previous Articles     Next Articles

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

ZHANG Tian-Tian, MIAO Ya, XU Bo-Ying*, CHEN Bin*   

  1. (Chongqing Key Laboratory of Vector Insects, Institute of Entomology and Molecular Biology, Chongqing Normal University, Chongqing 401331, China)
  • Online:2018-01-20 Published:2018-01-20

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.

Key words: Anopheles sinensis, carboxylesterase, metabolic resistance, heterologous expression, AsAe7 crystallization