A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe

European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences
Amira Abdel-DaimTeruko Imai

Abstract

In the present study, we established a quantitative western blotting method to measure the expression level of recombinant serine hydrolases based on their catalytic mechanism. Fluorophosphonate (FP)-biotin was selected as a universal probe to quantify their expression levels, since FP moiety irreversibly inhibits serine hydrolases through strong stoichiometric binding to active serine residue. The linearity of detection using FP-biotin was assessed on three serine hydrolases; human carboxylesterase (CES) 1, butyrylcholinesterase and porcine liver esterases (PLE). Similar response signals were obtained from the equimolar concentrations of these enzymes and excellent linearity was observed at the range of 0.4-3.4pmol/lane (r2>0.99). Accuracy and precision of the proposed method were proved using PLE with recovery of 97.1-107.2% and relative standard deviation of 5.56%. PLE was selected as a calibration standard because of its high stability and commercial availability. As an application of the developed method, we measured the expression levels of four recombinant CES isozymes from human and cynomolgus macaque in S9 fraction of HEK293 cell homogenates. The expression levels of human CES1 and CES2, and cynomolgus macaque CES1 and...Continue Reading

Citations

Feb 1, 2018·Xenobiotica; the Fate of Foreign Compounds in Biological Systems·Yasuhiro UnoTeruko Imai
Dec 15, 2019·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Kayoko OhuraTeruko Imai

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