Promega Corporation

A Novel Protease Assay Using Engineered Luciferase Scientific Poster

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Abstract

Frank Fan, Braeden Butler, Ken Lewis, Susan Frackman, Fen Huang and Keith Wood
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711

We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the original enzyme termini were joined by a peptide containing a protease site of interest. Using caspase 3 as our initial example, we showed that a circularly permutated luciferase with the corresponding cleavage site (DEVD) had low luminescence activity in the absence of protease. Upon treatment with caspase 3, the activity increased up to >3000 fold in a dosage dependent manner. The luminescence activity correlated with the amount of peptide cleavage as determined by SDS-PAGE, and was blocked by caspase 3 inhibitors. Based on these results, we developed a caspase 3 assay that detects as low as 0.5pg of caspase 3 and has a dynamic range of over four orders of magnitudes. We further demonstrated the broad applicability of this protease assay using other proteases and their cognate sites, including caspase 8, enterokinase, tobacco etch virus (TEV) protease, prostate-specific antigen (PSA) protease, human rhinovirus 3C (PreScission) protease and SARS-CoV 3CL proteases. For all experiments, the mutant luciferases were easily generated by in vitro transcription/translation and used directly. Compared with other protease assay methods, this assay has the following advantages: bioluminescent readout which allows high sensitivity, wide dynamic range and easy quantitation; rapid generation of substrate using molecular cloning and expression; the protease cleavage site within a protein context, and potential for cell-based protease assay.

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  • Part# PS018
  • Printed in USA.

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