The first challenge in the construction of a new protease-based signaling cascade was the selection of appropriate proteases. The candidate proteases should recognize defined target cleavage sequences, preferably as long as possible; they should be active in mammalian cells, but not toxic to them and inducible, ideally through the reconstitution of split protein fragments. Most importantly, a large number of proteases with similar properties but with different specificities should be available to allow for modular construction of signaling pathways and logic functions and these proteases should be orthogonal to each other, meaning they should have specific cleavage sites not recognized by the other proteases in the system.

We found that the tobacco etch virus protease (TEVp) was the only protease described in the literature to match our criteria.