Tobacco etch virus protease (TEV) is one of the most widely-used proteases in biotechnology because of its exquisite sequence-specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEVs cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our S153N mutant (uTEV1delta), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-second temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed evolution platform used to generate them, could be beneficial across a wide range of applications.
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