title: Design of FRET-based GFP probes for detection of protease inhibitors.
abstract: In this study, tandem Green fluorescent protein (GFP) fusion proteins were designed to detect proteolytic activity of thrombin based on the principle of fluorescence resonance energy transfer (FRET). The thrombin-specific recognition sequence, LVPR, was strategically placed in between a cyan-emitting mutant of the green fluorescent protein and an enhanced yellow-emitting fluorescent protein to allow thrombin-specific cleavage with detectable changes of FRET signal. A 4.6-fold increase of fluorescence emission ratio was observed upon addition of thrombin. This FRET-based probe was further tested for dose-dependent effects of thrombin specific inhibitor, hirudin. Our result showed a nice correlation between fluorescence emission ratios and concentrations of hirudin with subnanomolar sensitivity. We propose that FRET-based GFP probes can be used for high-throughput screening of protease inhibitors.
authors.affiliation: Institute of Basic Medical Sciences, Zhejiang University School of Medicine, 353 Yan'an Road, Hangzhou 310031, PR China. bzhang@visenmedical.com
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
mesh_terms: D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006629:Hirudins / Q000032:analysis* / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D015335:Molecular Probes / Q000096:biosynthesis / Q000737:chemistry; D011480:Protease Inhibitors / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics
chemical_list: D006629:Hirudins; D008164:Luminescent Proteins; D015335:Molecular Probes; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013917:Thrombin