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

Area of Science:

• Biochemistry
• Enzymology
• Proteomics

Background:

• Serine hydrolases are crucial enzymes involved in various biological processes.
• Accurate quantification of serine hydrolase expression is essential for understanding their function and for drug development.
• Existing methods for quantifying serine hydrolases can be limited in their universality and accuracy.

Purpose of the Study:

• To establish a universal and quantitative western blotting method for measuring recombinant serine hydrolase expression levels.
• To validate the accuracy, precision, and linearity of the developed method.
• To apply the method for assessing the expression and hydrolytic activities of carboxylesterase (CES) isozymes.

Main Methods:

• Development of a quantitative western blotting assay using Fluorophosphonate (FP)-biotin as a universal probe.
• Assessment of linearity, accuracy, and precision using known concentrations of human carboxylesterase (CES) 1, butyrylcholinesterase, and porcine liver esterases (PLE).
• Quantification of recombinant CES isozyme expression in S9 fractions and assessment of their hydrolytic activities.

Main Results:

• The FP-biotin method demonstrated excellent linearity (r² > 0.99) and accuracy (97.1-107.2% recovery) for serine hydrolase quantification.
• Porcine liver esterases (PLE) were validated as a stable calibration standard.
• Expression levels of human and cynomolgus macaque CES isozymes were successfully quantified, revealing differences in hydrolytic activities between species and isozymes.

Conclusions:

• A robust and universal quantitative western blotting method for serine hydrolases was established using FP-biotin.
• The method allows for accurate assessment of enzyme expression levels and subsequent determination of hydrolytic activities.
• This technique has broad applicability for the quantitative analysis of diverse recombinant serine hydrolases.