Establishment of Fluorescence Assays for the Characterization of Site-Specific Proteases in E. coli

Recent years have seen the increasing development and application of protease-based sensors and switches in the construction of artificial signaling functions in synthetic biology. Even though they were extensively characterized in vitro, robust and quantitative assays to characterize them in vivo remains elusive. This particularly concerns assays that can provide real-time kinetic data on the apparent activity of proteases and protease-based sensors and switches in the genetically-tractable E. coli and are compatible with high-throughput screening formats. Addressing these limitations, several different protease sensor designs featuring different types of fluorescent protein reporters were developed and tested over the course of the project. Protease-cleavable degradation tags (degrons) were identified as the best way to directly modulate the fluorescent output signal through proteolytic activity in E. coli. To this end, two different assay modes were successfully established: The first assay mode is based on a conventional positive read-out where the fluorescent signal scales proportionately with the protease activity while the second mode provides enhanced quantitative data based on a ratiometric two color assay. The assay was validated with the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) while the general utility of the assay was additionally validated with the NIa protease of tobacco etch virus (TEV) and the NS3 protein of hepatitis C virus (HCV). Finally, the assay was utilized for the preliminary screening of a library of opto-switchable auto-inhibited TVMV candidates.