Production of a novel recombinant Drosophila melanogaster acetylcholinesterase for detection of organophosphate and carbamate insecticide residues.
A novel recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE) produced in Pichia pastoris was first reported in this study. We cloned the DmAChE cDNA by reverse transcription PCR with removal of the signal for glycosylphosphatidylinositol (GPI) anchor attachment and the endogenous signal peptide coding sequence, and inserted it into P. pastoris vector pPIC9K under control of the alcohol oxidase gene AOX1 promoter (5'AOX1). The expression cassette of AChE cDNA was then introduced into methylotrophic yeast GS115 and several recombinant strains expressing R-DmAChE were obtained. The secreted R-DmAChE showed high stability in neutral phosphate buffer at 4 degrees C, and its kinetic parameters were identical to those of the native DmAChE. The bimolecular rate constants of R-DmAChE to dichlorvos, aldicarb and carbaryl were ranging from three to six times higher than of native DmAChE. Within six insecticides, the R-DmAChE was more sensitive than EeAChE, NbAChE and HuAChE. For 10 widely used insecticides, the IC50 values to the R-DmAChE were much lower than those to AChEs commonly used in China. With the R-DmAChE-based assay, samples spiked with three concentrations of pesticides caused enzymatic activity inhibition with R.S.D. of 0-13.7%. These results suggest that the R-DmAChE can be useful for detection of organophosphate and carbamate insecticide residues.
Xu
Songci
S
Shanghai Jiao Tong University, Shanghai Institutes for Biological Sciences, Pennsylvania State University Joint Center for Life Sciences, Key Laboratory of Microbial Metabolism, Ministry of Education, Shanghai, PR China.
Wu
Aibo
A
Chen
Haode
H
Xie
Yang
Y
Xu
Yuquan
Y
Zhang
Lei
L
Li
Jie
J
Zhang
Dabing
D
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics*; D015374:Biosensing Techniques / Q000379:methods; D002219:Carbamates / Q000032:analysis*; D002800:Cholinesterase Inhibitors / Q000032:analysis; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism*; D007306:Insecticides / Q000032:analysis*; D010755:Organophosphates / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D002219:Carbamates; D002800:Cholinesterase Inhibitors; D029721:Drosophila Proteins; D007306:Insecticides; D010755:Organophosphates; D011994:Recombinant Proteins; D000110:Acetylcholinesterase