title: Simultaneous selection and counter-selection for the directed evolution of proteases in E. coli using a cytoplasmic anchoring strategy.
abstract: 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.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California, 94720. z.carrico@gmail.com.
authors.forename: Zachary M
authors.initials: ZM
authors.lastname: Carrico
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Strobel
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California.
authors.forename: Meera E
authors.initials: ME
authors.lastname: Atreya
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Clark
authors.affiliation: Department of Chemistry, University of California, Berkeley, California, 94720.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Francis
mesh_terms: D003593:Cytoplasm / Q000201:enzymology; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D012641:Selection, Genetic / Q000235:genetics*
chemical_list: D010447:Peptide Hydrolases
keywords: TEV protease; beta-lactamase; cytoplasmic anchoring; directed evolution; protease; selection