An enzyme-based biosensor for monitoring and engineering protein stability in vivo

Significance Protein stability is central to the pathogenesis of several major human diseases and is the key to extensive research. Improved methods are needed for protein stability engineering. Here, we present a high-throughput screening strategy to stabilize proteins by linking their stabilities to the fluorescent readout of cells expressing an engineered bacterial enzyme. This strategy is generally applicable to proteins of different sources, sizes, and structural characteristics, including industrial-related enzymes and disease-related proteins. We also combined this strategy with deep mutational scanning to comprehensively understand how mutations shape the stability landscape of an important epigenetic enzyme. Our strategy expands the current toolbox of protein research and will also facilitate a variety of protein design.