title: An expression of an insect membrane-bound cytochrome P450 CYP6AA3 in the Escherichia coli in relation to insecticide resistance in a malarial vector.
abstract: This laboratory investigation was carried out at the Faculty of Sciences, Mahidol University, Thailand during October 2007 to May 2009. The objectives of this study include: the search for heterologous expression of the cytochrome P450 CYP6AA3 enzyme of the Anopheles minimus mosquitoes in relation to Malaria disease and to provide some information on molecular mechanism of insects' pyrethroid resistance. The polymerase chain reaction aided by the Pfu DNA polymerase and some specific generated primers were used to modify the CYP6AA3 gene. The PCR product was ligated with a predigested pET-3a at the NdeI and BamHI restriction sites. The modified CYP6AA3 enzyme was expressed in the Escherichia coli BL21 (DE3) pLysS in order to achieve a high amount of soluble form of its expression. The results showed that the use of the isopropyl-beta-D-thiogalactopyranoside (IPTG) and incubation together with ferric chloride and delta-aminolevulinic acid did not increase any soluble form of the CYP6AA3 enzyme. A significant amount of soluble enzyme was produced upon the replacement of the 30 N-terminal residues with a short peptide where it gave Ldelta30CYP6AA3 protein and after purification process was taken place, it yielded up to 10.64 mg 10 L(-1) or approximately 1 mg L(-1) of the homogenous Ldelta30CYP6AA3. When this purified Ldelta30CYP6AA3 protein was used in a metabolizing process with the cypermethrin, deltamethrin and permethrin substrates, it gave their apparent Km values for cypermethrin and deltamethrin of 12.5 and 23.5 microM, respectively. The heterologous expression carried out with the use of the E. coli gave a high amount of soluble CYP6AA3 enzyme of the An. minimus mosquitoes hence the modified technique being used was successfully achieved.
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Sciences, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: C
authors.initials: C
authors.lastname: Budriang
authors.forename: P
authors.initials: P
authors.lastname: Rongnoparut
authors.forename: J
authors.initials: J
authors.lastname: Yuvaniyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism / Q000469:parasitology; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017344:Genes, Insect; D006801:Humans; D007300:Insect Control; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007303:Insect Vectors / Q000235:genetics / Q000469:parasitology; D007305:Insecticide Resistance / Q000235:genetics; D007700:Kinetics; D008288:Malaria / Q000517:prevention & control / Q000635:transmission; D008969:Molecular Sequence Data; D011722:Pyrethrins / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D019476:Insect Proteins; D011722:Pyrethrins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System