A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance
2014 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 9, no 1, 155-162 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2014. Vol. 9, no 1, 155-162 p.
Protease engineering, Protease substrate profiling, Selection system, Tobacco Etch Virus
IdentifiersURN: urn:nbn:se:kth:diva-140898DOI: 10.1002/biot.201300234ISI: 000337540100016ScopusID: 2-s2.0-84891908926OAI: oai:DiVA.org:kth-140898DiVA: diva2:693508
FunderSwedish Research Council, 2012-5236 2004-4667
QC 201402042014-02-042014-02-042015-05-21Bibliographically approved