Proteases are enzymes that break down proteins by cleaving peptide bonds. They have significant potential for applications in proteome editing, targeted therapeutics, and building protein circuits. However, most proteases have broad substrate specificity, making it crucial to ensure they are specific to the substrate of interest when developing these methods. Unintended cleavage of incorrect substrates can lead to off-target effects, which limit their potential applications. To address this challenge, we developed a platform for Protease Engineering with Reactant Residence Time Control (PERRC). Unlike previous methods, PERRC allows for the adjustment of counterselection substrate (CS) and selection substrate (SS) ratios. Since proteases often retain activity on their native substrate, PERRC strengthens selection pressure by adjusting these ratios, promoting the evolution of proteases with greater specificity. Using this platform, we evolved a variant of the Tobacco Etch Virus protease (TEVp) that exhibits a 65-fold preference for a new substrate over its native substrate, despite a single amino acid difference. In future work, we aim to apply the PERRC platform to other disease-relevant proteases to develop more specific variants for protease-based therapeutics and other biotechnological applications.
