Abstract
Fusion proteins composed of an organophosphorus hydrolase (OPH) and pHluorin, a pH-sensitive green fluorescent protein variant, were constructed as whole-cell biosensors to measure organophosphorus pesticides. pHluorin was used to detect the pH changes because of the hydrolase of paraoxon by OPH. To examine the order of fusion of OPH and pHluorin, pHluorin-OPH and OPH-pHluorin fusion proteins were constructed. In addition, a peptide linker consisting of 15 amino acid was inserted between pHluorin and OPH to reduce steric hindrance. OPH and pHluorin activities were evaluated in cells expressing the four fusion proteins. The both activities of pHluorin-OPH and pHluorin-linker-OPH were higher than that of OPH-pHluorin and OPH-linker-pHluorin. Effects of the peptide linker on the activities were slight. Therefore, pHluorin-OPH and pHluorin-linker-OPH were found to be suitable for organophosphorus pesticide measurements. Using cells expressing pHluorin-linker-OPH, 0.5μg/mL of paraoxon could be measured.
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