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.

中文翻译：

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定向进化提高了 TEV 蛋白酶的催化效率。

烟草蚀刻病毒蛋白酶 (TEV) 是生物技术中使用最广泛的蛋白酶之一，因为它具有出色的序列特异性。然而，一个限制是它的催化速度慢。我们开发了一个通用的基于酵母的平台，用于蛋白酶催化特性的定向进化。蛋白酶活性是通过膜锚定转录因子的蛋白水解释放读出的，我们使用光感 LOV 域临时调节对 TEV 裂解底物的访问。通过逐渐减少曝光时间，我们在多轮选择中丰富了更快的 TEV 变体。我们的 TEV-S153N 突变体 (uTEV1Δ) 在加入钙积分器 FLARE 后，将信号/背景比提高了 27 倍，并能够以 60 秒的时间分辨率记录培养物中的神经元活动。