模块化和综合活动报告器加强了酵母ER的生化研究

Samantha G Martinusen, Ethan W Slaton, Sage E Nelson, Marian A Pulgar, Julia T Besu, Cassidy F Simas, Carl A Denard
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引用次数: 0

摘要

酵母内质网封存和筛选(YESS)系统是一个通用平台,在研究翻译后修饰酶(PTM-酶)方面非常有用。研究人员可以利用该系统分析和设计 PTM 酶的活性和底物特异性,并发现抗抑制剂的酶突变体。在这项研究中,我们将 YESS 的功能元件转移到整合质粒上,从而扩展了 YESS 的功能。YESS 整合系统能以各种配置产生统一的蛋白质表达和蛋白酶活性,允许在两个独立位点整合活性报告物,并能在整合质粒和中心质粒之间拆分该系统。我们从方差系数、信噪比和折叠活化等方面描述了这些整合报告器与两种病毒蛋白酶--烟草蚀刻病毒(TEVp)和3-糜蛋白酶样蛋白酶(3CLpro)--的特性。总之,我们为基于染色体的研究提供了一个模块化框架,可以对酵母中的 PTM 酶进行严格的高通量检测。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Modular and integrative activity reporters enhance biochemical studies in the yeast ER
The yeast endoplasmic reticulum sequestration and screening (YESS) system is a generalizable platform that has become highly useful to investigate post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.
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