Towards an antibiotic resistance-based assay for protease characterization and engineering
(English)Manuscript (preprint) (Other academic)
Proteases attract a lot of interest, not only because of their involvement in many biological processes, but also as essential tools in biomedical research and industry. Here, we present a novel genetic method for identification of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that upon processing by a coexpressed protease confer antibiotic resistance to cells in proportion to the cleavage efficiency.
We demonstrate that cells expressing cleavable or non-cleavable reporters together with tobacco etch virus protease (TEVp), could be distinguished from each other by growth in selective media. Moreover, the growth rate proved to correlate with the substrate processing efficiency. Thus by applying competitive growth in antibiotic-containing medium, we could also show that the substrate preferred by TEVp was enriched at the expense of other less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases towards improved and/or new functionality.
proteases, flow cytometry, CAT, protein engineering, ssrA, ClpXP, tobacco etch virus, TEV, site-specific, high-throughput, selection, proteolysis, libraries
Research subject SRA - Molecular Bioscience
IdentifiersURN: urn:nbn:se:kth:diva-33548OAI: oai:DiVA.org:kth-33548DiVA: diva2:416060
QC 201105132011-05-132011-05-102011-05-16Bibliographically approved