Directed evolution improves the catalytic efficiency of TEV protease

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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Additional data beyond that provided in the Figures and Supplementary Information are available from the corresponding author upon request.

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Acknowledgements

We are grateful to Stanford, the Chan Zuckerberg Biohub, the Beckman Technology Development Seed Grant and NIH (R01 MH119353) for support of this work. FACS was performed at the MIT Koch Institute Flow Cytometry Core and at the Stanford Shared FACS Facility. W. Wang (University of Michigan) provided plasmids and advice. L. Ning (Stanford University) provided rat brain tissue. A. G. Johnson (Stanford) gave advice on TEV expression, and N. Samiylenko helped reproduce some experiments. B. Babin, J. Yim and M. Bogyo (Stanford University) provided access to their HPLC. G. Liu (MIT) built the LED box used for blue light irradiation of cells. M. Djuristic (Stanford University) assisted with electrical stimulation of neurons. M.I.S. was supported by an EMBO long-term post-doctoral fellowship (ALTF 1022-2015).

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Authors and Affiliations

  1. Departments of Genetics, Biology and Chemistry, Stanford University, Stanford, CA, USA Mateo I. Sanchez & Alice Y. Ting
  2. Chan Zuckerberg Biohub, San Francisco, CA, USA Mateo I. Sanchez & Alice Y. Ting
  1. Mateo I. Sanchez