Directed evolution improves the catalytic efficiency of TEV protease
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Updated: Jan 2, 2026

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Mateo I Sanchez1,2, Alice Y Ting3,4
1Departments of Genetics, Biology and Chemistry, Stanford University, Stanford, CA, USA.
Abstract:
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 TEV's 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 TEV-S153N mutant (uTEV1Δ), 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-s 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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