文献类型：期刊文献

英文题名：An enzyme-based biosensor for monitoring and engineering protein stability in vivo

作者：Ren, Chang[1];Wen, Xin[1];Mencius, Jun[1];Quan, Shu[1]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2021

卷号：118

期号：13

外文期刊名：PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000637394200090)】；

基金：We thank James C. A. Bardwell (University ofMichigan) for helpful advice, Yong Chen (Chinese Academy of Sciences) for sharing plasmids and helpful suggestions on the study of MLL3SET, and Zixiao Xue and Yongxin Zheng (East China University of Science and Technology) for experimental support and helpful discussions. This work was supported by National Natural Science Foundation of China Grants 31870054, 31670802, and 31661143021 (to S.Q.), the Fundamental Research Funds for the Central Universities (Grant 22221818014 to S.Q.), and the Research Program of State Key Laboratory of Bioreactor Engineering (to S.Q.).

语种：英文

外文关键词：biosensor; protein stability; protein engineering; deep mutational scanning

摘要：Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysGA) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.