Engineering Sensitive Acetylcholinesterase for Detection of Organophosphate and Carbamate Insecticides

Design of acetylcholinesterases for biosensor applications

“…More sensitive AChE variants and sophisticated multianalyte detection systems are critical issues when it comes to turning AChE biosensors into marketable products. (Ordentlich et al, 1996) Hu F295A 1800 65 830 (Ordentlich et al, 1996) Hu F295L 800 264 940 (Ordentlich et al, 1996) Hu F295L/ F297V 117 340 310 (Ordentlich et al, 1996) Hu E405A 1.9 0.09 1.5 (Ordentlich et al, 1996) Hu G121A 0.085 0.007 (Ordentlich et al, 1998) (Villatte et al, 1998) Dm B08 220 (Montesinos et al, 2000) WT Rb 80 52 0.07 (Pleiss et al, 1999) Rb F295L 550 120 5.6 (Pleiss et al, 1999) WT: wild type; Hu: human AChE, amino acid numbering corresponds to human AChE; HuBChE: human butyrylcholinesterase; Dm: Drosophila melanogaster AChE, amino acid numbering corresponds to DmAChE; Rb: rat brain AChE, amino acid numbering corresponds to RbAChE.…”

“…It has been suggested that DmE107Y and DmY412A may be part of the peripheral anionic site. Moreover, the substitution of tyrosine by phenylalanine at position 408 led to a mutant which was more sensitive to 12 out of 19 tested insecticides than the wild type (Villatte et al, 1998).…”

“…AChEs from Torpedo californica (Radic et al, 1992), Drosophila melanogaster (Chaabihi et al, 1994;Estrada-Mondaca et al, 1998) and rat brain could be actively expressed in satisfactory amounts in a baculovirus-insect cell system ( Table 2). The baculovirus system has severe limitations with respect to the production of larger amounts of protein, but seems to be very suitable for producing mutant recombinant AChEs.…”

“…Two approaches are usually applied for the purification of the enzymes: ion exchange chromatography (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998) and affinity chromatography (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998;Villatte et al, 1998;Villatte et al, 2000) based on the chemical modification of the support with reversible AChE inhibitors (Massoulie and Bon, 1976;Ralston et al, 1983) or on the addition of a His-tag to the enzyme (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998;Hussein et al, 1999;Hussein et al, 2000).…”

“…Estrada-Mondaca and Fournier combined affinity chromatography (using procainamide as ligand) with anionic-exchange chromatography for the purification of Drosophila melanogaster AChE (DmAChE) after expression in baculovirus-infected cells (Estrada-Mondaca and Fournier, 1998). The use of a diethylaminoethyl (DEAE)-Sepharose column after affinity purification allowed the separation of AChE from proteases that are co-extracted with the AChE.…”