Recombinant γ-secretase substrate allows for detection of protease activity directly in cells. (a) Sb4 γ-secretase substrate. Schematic of the truncated Sb4 substrate from the amyloid precursor protein that has an engineered MBP tag as well as Avitag for purification and biotinylation, respectively. A thrombin cleavage site between the MBP tag and avitag allows for the removal of MBP by thrombin treatment following substrate purification. (b) LC-MS analysis identified Sb4 at the expected size and determined that greater than 90% of purified Sb4 is shown to be biotinylated. (c) Development of an exo-cell assay. Utilization of the Sb4 substrate in conjunction with a small amount of CHAPSO detergent allows for real-time examination of γ-secretase activity directly from cells using ECL or homogenous time-resolved fluorescence (HTRF) detection methods in 96-well format.
Shelton et al. Molecular Neurodegeneration 2009 4:22 doi:10.1186/1750-1326-4-22