title: Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.
abstract: Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.
authors.affiliation: Comparative Genomics Centre, School of Pharmacy and Molecular Sciences, James Cook University, DB 21, James Cook Drive, Townsville, QLD 4811, Australia.
authors.forename: Samuel P
authors.initials: SP
authors.lastname: Askin
authors.forename: Isabelle
authors.initials: I
authors.lastname: Morin
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Schaeffer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D005470:Fluorometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D019272:Leukocyte Elastase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; C061910:tus protein, Bacteria; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases; D019272:Leukocyte Elastase; D014357:Trypsin; D053148:Caspase 3