小柯机器人

定向进化提高烟草蚀纹病毒蛋白酶催化效率
2019-12-10 15:33

美国斯坦福大学Alice Y Ting研究组利用定向进化提高了烟草蚀纹病毒(TEV)蛋白酶的催化效率。该研究于2019年12月9日在线发表于国际一流学术期刊《自然—方法学》。

研究人员开发了基于蛋白酶的平台,用于蛋白酶催化特性的定向进化。蛋白酶的活性是通过膜锚定的转录因子的蛋白水解释放作为出口,研究人员利用光敏LOV结构域短暂调节对TEV裂解底物的接触。通过逐渐减少曝光时间,研究人员在多轮选择中丰富了TEV的更快变体。当研究人员将TEV-S153N突变体(uTEV1Δ)整合到钙整合蛋白FLARE中后,其信号/背景比提高了27倍,并能够以60 s的时间分辨率记录培养物中的神经元活性。鉴于TEV在生物技术中的广泛应用,研究人员进化出的TEV突变体和用于产生它们的定向进化平台都有利于广泛的应用。

据介绍,TEV蛋白酶由于其特殊的序列特异性,是生物技术中使用最广泛的蛋白酶之一。然而,其局限性在于其缓慢的催化速率。

附:英文原文

Title: Directed evolution improves the catalytic efficiency of TEV protease

Author: Mateo I Sanchez, Alice Y Ting

Issue&Volume: 2019-12-09

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.

DOI: 10.1038/s41592-019-0665-7

Source: https://www.nature.com/articles/s41592-019-0665-7

Nature Methods:《自然—方法学》,创刊于2004年。隶属于施普林格·自然出版集团,最新IF:28.467
官方网址:https://www.nature.com/nmeth/
投稿链接:https://mts-nmeth.nature.com/cgi-bin/main.plex


本期文章:《自然—方法学》:Online/在线发表

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