Simultaneous selection and counter-selection for the directed evolution of proteases in E. coli using a cytoplasmic...

With the goal of generating new enzymes that can cleave custom sequences, this article describes a selection strategy for evolving proteases with desirable characteristics. Positive selection and counter-selection are combined to select for and against specified cleavage sequences simultaneously. Cleavage of the positive selection sequence permits E. coli growth, and cleavage of the counter-selection sequence slows growth. Growth occurs when cleavage of the positive selection sequence releases β-lactamase into the periplasm where it can confer antibiotic resistance. The counter-selection traps β-lactamase in the cytoplasm, preventing antibiotic resistance and growth. Thus, proteases with a preference for the positive selection sequence relative to the counter-selection sequence grow more rapidly. This system was used to select a tobacco etch virus (TEV) protease mutant with new substrate compatibility. Biotechnol. Bioeng. 2016;113: 1187-1193. © 2015 Wiley Periodicals, Inc.