PERRC: Protease Engineering with Reactant Residence Time Control.

Proteases with engineered specificity hold great potential for targeted therapeutics, protein circuit construction, and biotechnology applications. However, many proteases exhibit broad substrate specificity, limiting their use in such applications. Engineering protease specificity remains challenging because evolving a protease to recognize a new substrate, without counterselecting against its native substrate, often results in high residual activity on the original substrate. To address this, we developed Protease Engineering with Reactant Residence Time Control (PERRC), a platform that exploits the correlation between endoplasmic reticulum (ER) retention sequence strength and ER residence time. PERRC allows precise control over the stringency of protease evolution by adjusting counterselection to selection substrate ratios. Using PERRC, we evolved an orthogonal tobacco etch virus protease variant, TEVESNp, that selectively cleaves a substrate (ENLYFES) that differs by only one amino acid from its parent sequence (ENLYFQS). TEVESNp exhibits a remarkable 65-fold preference for the evolved substrate, marking the first example of an engineered orthogonal protease driven by such a slight difference in substrate recognition. Furthermore, TEVESNp functions as a competent protease for constructing orthogonal protein circuits in bacteria, and molecular dynamics simulations analysis reveals subtle yet functionally significant active site rearrangements. PERRC is a modular dual-substrate display system that facilitates precise engineering of protease specificity.