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

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 0.4–3.4 pmol/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 CES2 were 2.51 ± 0.1, 1.63 ± 0.17, 0.79 ± 0.09 and 1.37 ± 0.13 pmol/5 μg S9 protein, respectively. Based on these determinations, their hydrolytic activities were accurately assessed. Cynomolgus CESs showed lower hydrolysis activities for p-nitrophenyl esters than human CESs. The hydrolase activities of CES2 isozymes were higher than CES1 in both species. Three to five folds faster hydrolysis for p-nitrophenyl butyrate than p-nitrophenyl acetate was observed in all CES isozymes except of cynomolgus CES1 that showed nearly same hydrolysis for both substrates. The provided method could be widely used for universal quantitative analysis of recombinant serine hydrolases.