Molecular cloning and expression analysis of cytochrome c oxidase subunit II from Sitophilus zeamais.
Cytochrome c oxidase subunit II (COX II) containing a dual core CuA active site is one of the core subunits of mitochondrial Cytochrome c oxidase (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 open reading frame (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. multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXII had high sequence identity with the COXII of other insect species. The gene was subcloned into the expression vector pET-32a, and induced by isopropyl β-d-thiogalactopyranoside (IPTG) in E. coli Transetta (DE3) expression system. Finally the recombinant COXII with 6-His tag was purified using affinity chromatography with Ni(2+)-NTA agarose. Western Blotting (WB) showed the recombinant protein was about 44 kD, and the concentration of fusion protein was 50 μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXII could catalyze the oxidation of substrate Cytochrome C (Cyt c), and influenced by allyl isothiocyanate (AITC). By using molecular docking method, It was found that a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results suggested that tag-free COXII was functional and one of the action sites of AITC, which will be helpful to carry out a point mutation in binding sites for the future research.
Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling, Shaanxi Province, 712100, China.
Chang-Liang
CL
Hou
Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling, Shaanxi Province, 712100, China.
Jing-Bo
JB
Wang
Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling, Shaanxi Province, 712100, China; Research Center of Biopesticide Technology and Engineering, Yangling, Shaanxi Province, 712100, China. Electronic address: wgf20102010@nwsuaf.edu.cn.
Hua
H
Wu
Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling, Shaanxi Province, 712100, China.
Jia-Yu
JY
Liu
Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling, Shaanxi Province, 712100, China; Research Center of Biopesticide Technology and Engineering, Yangling, Shaanxi Province, 712100, China.
Zhi-Qing
ZQ
Ma
Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling, Shaanxi Province, 712100, China; Research Center of Biopesticide Technology and Engineering, Yangling, Shaanxi Province, 712100, China.
Jun-Tao
JT
Feng
Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling, Shaanxi Province, 712100, China; Research Center of Biopesticide Technology and Engineering, Yangling, Shaanxi Province, 712100, China.
Xing
X
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017879:Isothiocyanates / Q000378:metabolism / Q000494:pharmacology; D062105:Molecular Docking Simulation; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D048889:Weevils / Q000201:enzymology / Q000235:genetics*
D019476:Insect Proteins; D017879:Isothiocyanates; D011994:Recombinant Proteins; D045304:Cytochromes c; C004471:allyl isothiocyanate; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV
Allyl isothiocyanate; Cytochrome c oxidase subunit II; Enzyme activity; Sitophilus zeamais; Soluble proteins