Process for Determining Enzyme Activity in a Cell by Activity-based Reporter Gene Technology (AbRGT)

Abstract

Methods and materials for specific imaging of active enzyme in a live or intact cell are disclosed. The enzyme of interest tagged to reporter protein (donor) is exogenously expressed in a cell. The conversion of proenzyme to active enzyme (containing reporter protein) is achieved upon applying an appropriate stimulus to the target cells. The activated enzyme is labelled with an activity-based probe carrying a fluorophore (acceptor). The covalent labelling of active enzyme by the activity-based probe creates a FRET pair which provides the opportunity to exquisitely image the function of an “active enzyme†. This method is used for specific imaging of the function of active caspase-3,-7,-8,-9 and cathepsin-B and also for profiling of inhibitors of caspases and cathepsin B.