Continuous Evolution of Proteases Using a T7 Bacteriophage System

The directed evolution of proteases for enhanced activity or altered specificity is a difficult process on account of cell toxicity and difficulty to select for dynamic range of function. Here, we present a T7 bacteriophage-based continuous evolution scheme for protease activity with distinct advantages over other methods, chief among them ease-of-use. Our system works by adding the protease of interest to be evolved in place of the major capsid protein of the T7 bacteriophage (T7Δ10). T7Δ10 bacteriophages will not propagate on their native E. coli host without complementation with the major capsid protein in trans. We have created a fusion between the major capsid protein and the E. coli thioredoxin gene with a protease-specific cleavage site in between which does not allow for successful bacteriophage replication in the absence of protease cleavage. We initially show a proof of concept validation of the system whereas a known tobacco etch virus protease (TEV protease) mutant reverts back to the more active wild-type TEV protease after ten passages. We also show how simple genetic drift of a self-cleavage resistant TEV protease can mutate to lead to increased enzymatic speed in twenty passages. Additionally, we have drifted TEV protease to recognize four different single mutant cleavage sites away from the wild-type cleavage site (WT:ENLYFQ/S, EΔW: WNLYFQ/S, YΔH:ENLHFQ/S, YΔE:ENLEFQ/S, and QΔH:ENLYFH/S). As four distinction populations, these quasi-species evolved for 20-30 passages on the single mutant cleavage sites so that each population showed preference for its own versus any other single mutant cleavage sites in cell lysis assays. Although most lines to some degree lyse on other mutant cleavage sites, the YΔE and EΔW lines lyse on the QΔH cleavage site the slowest and, in turn, the QΔH line lyses slowest on the YΔE and EΔW cleavage sites, suggesting some degree of orthogonality. Preliminary results suggest other proteases can be used in the same system. In summary, we have developed a novel, T7-based continuous evolution system for evolving proteases which, in addition to evolving better protease activity and unique specificity, could be used to evolve proteases to cleave therapeutically relevant targets as well as rapidly characterize the emergence of resistant proteases in response to inhibitors.