title: Molecular cloning and recombinant expression of cytochrome P450 CYP6B6 from Helicoverpa armigera in Escherichia coli.
abstract: The cytochrome P450 s play a significant role in the detoxification of plant allelochemicals and synthetic insecticides in Lepidoptera. In the cotton bollworm Helicoverpa armigera, 2-tridecanone and quercetin can induce P450-dependent monooxygenase activity increased, to further the characterization of P450, the CYP6B6 of cotton bollworm (H. armigera) was cloned, sequenced and expressed in pMAL-p2x vector and expressed in Escherichia coli. The deduced amino acid sequences of cytochrome P450 in the midgut and fat body of H. armigera showed 98.23 and 97.84 % similarity with CYP6B6, respectively. According to nomenclature of P450 s, the P450 genes we got belong to CYP6B. Purification of recombinant protein based on the affinity of MBP for maltose was achieved by Mal-Tag magnetic beads. The purified protein was used to raise polyclonal antibody according to classical procedure. SDS-PAGE and Western blot results indicated that MBP-CYP6B6 had been successfully expressed. The ethoxycoumarin-O-deethylase activity of the purified recombinant protein was 36.5 ± 8.12 pmol of 7-hydroxycoumarin/min/mg protein, which showed the fusion MBP-CYP6B6 had the ability to o-deethylase of 7-ethoxycoumarin.
authors.affiliation: College of Life Science and Technology, Xinjiang University, Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Urumqi, 830046, China.
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Liu
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Xuetao
authors.initials: X
authors.lastname: Zhang
authors.forename: Gao
authors.initials: G
authors.lastname: Xiwu
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D004926:Escherichia coli; D019476:Insect Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007305:Insecticide Resistance; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D019476:Insect Proteins; D011994:Recombinant Proteins; C017299:7-ethoxycoumarin; D001189:Aryl Hydrocarbon Hydroxylases