A protease substrate profiling method that links site‐specific proteolysis with antibiotic resistance

Abstract

Proteases are involved in many biological processes and have become important tools in biomedical research and industry. Technologies for engineering and characterization of, for example, proteolytic activity and specificity are essential in protease research. Here, we present a novel method for assessment of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that, upon processing by a co-expressed protease, confer antibiotic resistance to bacteria in proportion to the cleavage efficiency. We have demonstrated that cells co-expressing cleavable reporters together with tobacco etch virus protease (TEVp) could be discriminated from cells with non-cleavable reporters by growth in selective media. Importantly, the resistance to antibiotics proved to correlate with the substrate processing efficiency. Thus, by applying competitive growth of a mock library in antibiotic-containing medium, we could show that the substrate preferred by TEVp was enriched relative to less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases and protease recognition sequences towards improved or even new functionality.

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

“…This potential has encouraged several labs to invent screens and selections for evolving proteases for new substrate selectivity. The screens have used fluorescence (Kostallas and Samuelson, 2010;Varadarajan et al, 2008;Yi et al, 2013), growth (Kohler, 2003), or colorimetric changes (Renicke et al, 2013;Sellamuthu et al, 2008) to detect new proteases in vitro, and the selections have used antibiotic resistance (Sandersjoo et al, 2014;Verhoeven et al, 2012), translation (Dickinson et al, 2014), or non-toxicity (O'Loughlin et al, 2006) to identify microorganisms expressing proteases with new sequence specificity. The screens offer greater versatility than selections, but they typically cost more and take longer for the investigation of the large numbers of mutants.…”

YESS 2.0, a Tunable Platform for Enzyme Evolution, Yields Highly Active TEV Protease Variants

“…Protease variants with the desired phenotypes are isolated from a large pool, so a high-throughput protease screening system should ideally exhibit a high operational range (sensitivity over a large variation in input), and a high dynamic range (signal-to-noise ratio). Also, it should avoid laborious design–build–test cycles that slow progress. ,− …”

Novel export and import pathways in S. cerevisiae identified by an engineered SUMO system

“…fluorescence or bacterial growth) (Sandersjöö et al 2014;Kostallas et al 2011;Kostallas & Samuelson 2010). In contrast to our selection system, these studies did not provide any fruitful application of the system and did not offer the possibility to select against protein cleavage.…”