Preprint Article Version 1 NOT YET PEER-REVIEWED

Molecular Cloning and Expression Analysis of COX II from Sitophilus zeamais, a Potential Action Site of AITC

  1. Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling 712100, China
  2. Research Center of Biopesticide Technology and Engineering, Yangling 712100, China
Version 1 : Received: 24 August 2016 / Approved: 25 August 2016 / Online: 25 August 2016 (10:11:13 CEST)

How to cite: Hou, C.; Wang, J.; Wu, H.; Liu, J.; Ma, Z.; Feng, J.; Zhang, X. Molecular Cloning and Expression Analysis of COX II from Sitophilus zeamais, a Potential Action Site of AITC. Preprints 2016, 2016080203 (doi: 10.20944/preprints201608.0203.v1). Hou, C.; Wang, J.; Wu, H.; Liu, J.; Ma, Z.; Feng, J.; Zhang, X. Molecular Cloning and Expression Analysis of COX II from Sitophilus zeamais, a Potential Action Site of AITC. Preprints 2016, 2016080203 (doi: 10.20944/preprints201608.0203.v1).

Abstract

COX II containing a dual core CuA active site is one of the three core subunits of mitochondrial Cco, which plays a significant role in the physiological process. In this report, the full-length cDNA of COXⅡ gene was cloned from Sitophilus zeamais, which had an ORF of 684 bp encoding 227 amino acids residues. The predicted COXⅡ protein had a molecular mass of 26.2 kDa with pI value of 6.37, and multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXⅡ had high sequence identity 78.51% with the COXⅡ of other insect species, especially similarity to sitophilus oryzae. This gene was subcloned into the prokaryotic expression vector pET-32a, and induced by IPTG in E.coli Transetta (DE3) expression system. Finally the COXⅡ with 6-His tag was purified using affinity chromatography with Ni2+-NTA agarose. WB showed the recombinant COXⅡ was about 44 kD, and the concentration of fusion protein was 50μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXⅡ could catalyze the oxidation of substrate Cytc, and influenced by AITC. It was found that AITC could form a hydrophobic region with COXⅡ protein via molecular docking, besides, a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results will provide valuable information for elucidating the role of COXⅡ in Sitophilus zeamais responses to AITC, meanwhile, it will helpful to carry out a point mutation in AITC binding sites for the future research.

Subject Areas

Sitophilus zeamais; COXⅡ; Soluble proteins; Enzyme activity; AITC

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