Technologies

Cut-N-Glow is the first fully, biological in vivo protease mapping tool that emits fluorescence. Our assay is easily tailored via standard cloning techniques to detect different proteases or to map protease specificity. No chemical synthesis is required therefore there is no need for co-factors or co-substrates. Additionally, this assay only requires two reagents and both are proteins that can be easily obtained following over-expression of E. coli. In addition to the advantage of emitting a fluorescent signal in the presence of proteases, Cut-N-Glow, as its name implies, is designed with a conditional distortion to convert self-assembling GFP proteins into a site specific protease switch. Our approach to the distortion is reversible through proteolysis by constraining the N and C termini of GFP 11 with a protease-sensitive tether, eglin C. The feature distinguishing our system from other GFP reporters is that there is a gain of fluorescence, rather than a loss of fluorescence in proteolysis both in vitro and in vivo.

Proteases occur naturally in all organisms and are valuable tools in medical diagnostics serving as initiators of cell signaling, as regulators of immune responses, and as agents of infectious disease. Therefore, mapping proteases in parasitic diseases and bacteria as well as assayable proteases associated with cancer could lead to the identification of shared structural similarities validating potential drug targets. Our strategy utilized split proteins in a conditionally inactive form with the aid of a conformational distortion maintained by a cleavable tether. We applied this method to convert split GFP into a latent fluorophore that can be activated by site-specific proteolysis. The chimeric GFP served as substrate for representative enzymes from the three major protease classes: serine, cysteine, and aspartic acid.