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bioRxiv · 10.1101/811570

Directed evolution improves the catalytic efficiency of TEV protease

Sanchez Lopez, M. I.·Ting, A. Y.
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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 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 (uTEV1{Delta}), 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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Sanchez Lopez, M. I., Ting, A. Y.. 2019-10-21. Directed evolution improves the catalytic efficiency of TEV protease. https://doi.org/10.1101/811570

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