A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance.
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.
Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
Lisa
L
Sandersjöö
George
G
Kostallas
John
J
Löfblom
Patrik
P
Samuelson
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002701:Chloramphenicol / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D003470:Culture Media / Q000737:chemistry; D004352:Drug Resistance, Microbial; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D010447:Peptide Hydrolases / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017800:Potyvirus / Q000201:enzymology / Q000254:growth & development; D015202:Protein Engineering / Q000379:methods*; D059748:Proteolysis; D054852:Small Molecule Libraries; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D003470:Culture Media; D054852:Small Molecule Libraries; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D010450:Endopeptidases; D010447:Peptide Hydrolases; C099488:TEV protease
Protease engineering; Protease substrate profiling; Selection system; Tobacco Etch Virus