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Your Position :Home->Past Journals Catalog->2014年51 No.6

Expression, purification and crystallization ofHelicoverpa armigera CYP337B3
Author of the article:WANG Lu-Lu1, 2** WANG Jing-Min2, 3 FU Sheng2*** WANG Ying2***
Author's Workplace:1. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300222; 2. Tianjin International Joint Academyof Biotechnology and Medicine, Tianjin 300457, China; 3. NanKai University, Tianjin 300071, China
Key Words:Helicoverpa armigera, CYP337B3, protein crystallography
Abstract:[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.
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