Directed evolution for obtaining improved variants of TEV protease for biotechnological applications

Abstract

Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence-specificity. A limitation of TEV is its slow catalytic rate, which limits product generation and therefore signal output. Provided is a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is determined via proteolytic release of a membrane-anchored transcription factor, and access to TEV's cleavage site is temporally regulated using a photosensory LOV domain. By gradually decreasing light exposure time, faster variants of TEV were selected over multiple rounds of selection. The mutant TEV proteases and the directed evolution platform are useful in a wide range of biotechnology applications, such as FLARE and SPARK tools.

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