Protease engineering: Approaches, tools, and emerging trends

Engineered proteases with bespoke substrate specificities and activities can empower broad and innovative applications in biomedicine, mass spectrometry-based proteomics, and chemical and synthetic biology. This review provides an authoritative, topical, and detailed description and discussion of the directed evolution and high-throughput strategies designed to engineer the substrate specificity of proteases in E. coli, yeast, phage, and cell-free systems. Second, we discuss emerging protease engineering strategies that complement directed evolution, including antibody-protease fusions that enable proximity catalysis, and protease substrate specificity switching driven by exogenous protein-protein interactions. Lastly, we discuss principles for engineering split and autoinhibited proteases, which are key signal-processing modules in protein circuits. Overall, readers will gain a valuable understanding of the latest advances in protease engineering, focusing on methodologies and strategies that enable precise control of protease activity and specificity.