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.