State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China.
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China
Proc Natl Acad Sci U S A. 2021 Mar 30;118(13). doi: 10.1073/pnas.2101618118.
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 (CysG) 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.
蛋白质稳定性影响蛋白质的生理功能，也是许多蛋白质工程任务中所期望的特性，但由于缺乏直接监测细胞内蛋白质稳定性的方法，提高蛋白质稳定性具有挑战性。在这里，我们报告了一种体内稳定性生物传感器，其中感兴趣的蛋白质（POI）插入到微生物酶（CysG）中，该酶催化内源性荧光化合物的形成，从而将 POI 稳定性与简单的荧光读数相关联。我们证明了该生物传感器在定向进化中的实用性，以获得两种 POI 的稳定、不易聚集的变体（包括人胰岛淀粉样多肽的非淀粉样变体）。除了工程应用之外，我们还利用我们的生物传感器进行了深度突变扫描，以实验描绘组蛋白 H3K4 甲基转移酶催化结构域中所有残基的稳定性相关贡献，从而揭示了其具有科学信息的稳定性图谱。因此，我们这种用于体内监测各种蛋白质稳定性的高可及性方法将促进基础研究和应用蛋白质工程的努力。
