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.

Tobacco Etch Virus protease: A shortcut across biotechnologies

“…In particular, Asp148 mutated to positively charged residues Arg or Lys was preferentially found in TEVp variants with specificity for Glu in P1 position, while selection for His on P1 favoured mutations with small amino acids (Ala, Ser or Cys) or Pro (Yi et al, 2013). More recently, a novel TEVp mutant selected by directed evolution, which had Asn171 mutated in Asp, showed higher tolerance for Thr and Pro in position P6 (Carrico et al, 2016).…”

Enhancement of malate production through engineering of the periplasmic rTCA pathway in Escherichia coli

“…To separate the periplasmic and cytoplasmic fractions of cellular proteins, osmotic shock was used (Carrico, Strobel, Atreya, Clark, & Francis, 2016). Phosphoenolpyruvate carboxykinase and malate dehydrogenase activity were determined by measurement of A340 to monitor NADH oxidation, as described (Xu, Liu, & Chen, 2012;Yang, Lubeck, & Lubeck, 2015).…”

Engineering Proteases for Mass Spectrometry‐Based Post Translational Modification Analyses

“…When using a surrogate substrate, the evolved protease must be subsequently validated to ensure that the enzyme also improves activity on the desired substrate. Nevertheless, a number of earlier directed evolution efforts have been reported albeit sporadic and mostly limited to “model viral proteases.” Reviewed here are the most recent advances and most generalized approaches in directed evolution of proteases in the author's opinion.…”