Creation of A Multi-Color Sensing System for Protease Activities.

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We designed a dual-protease biosensor for two different protease activities using doubly inhibited split-GFP, which was fully activated only when two proteases were present at the same time. In the system, N- and C-terminal fragments of split-GFP of split-luciferase were cyclized via the distinct protease substrate sequences, respectively. The simultaneous addition of two proteases resulted in cleavage of both substrate linkers, leading to the functional recovery of split-GFP and split-luciferase. These results clearly demonstrate the successful construction of the dual-protease biosensor for two different caspase activities by using split-GFP and split-luciferase.