Cut-N-Glow™ is the first fully biological in vivo protease mapping tool that emits fluorescence. This assay is easily tailored via standard cloning techniques to detect specific proteases or protease inhibitors or to map protease substrate preference in vitro or in vivo. Chemical synthesis is not required and 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.
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. This strategy utilizes split proteins in a conditionally inactive form with the aid of a conformational distortion maintained by a cleavable tether. This method is applied to convert split GFP into a latent fluorophore that can be activated by site-specific proteolysis. The chimeric GFP serves as a substrate for representative enzymes from the three major protease classes: serine, cysteine, and aspartic acid.
Factor X, or prothrombinase, is a serine endopeptidase of the coagulation cascade. Factor X is activated into factor Xa, which functions to cleave prothrombin to yield thrombin. Thrombin acts as a serine protease that catalyzes the conversion of soluble fibrinogen into insoluble strands of fibrin, in addition to catalyzing many other coagulation-related reactions.
This assay kit is specific for Factor Xa activity and contains sufficient reagents for one 96-well plate (96 tests)
The Cut-N-Glow™ approach involves the introduction of a structural distortion into one of the complementary fragments (GFP 11), through the use of a conditionally stable tether, which serves to constrain the N and C termini of GFP11 closely in space, thereby diminishing the mutual affinity of the two fragments and blocking protein self-assembly until the tether is cleaved. The distortion can be reversed upon proteolysis of the tether, resulting in fragment assembly with GFP 1-10, generating reconstituted, functional GFP.
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