Design of FRET-based GFP probes for detection of protease inhibitors.
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.
Zhang
Bin
B
Institute of Basic Medical Sciences, Zhejiang University School of Medicine, 353 Yan'an Road, Hangzhou 310031, PR China. bzhang@visenmedical.com
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
D006629:Hirudins; D008164:Luminescent Proteins; D015335:Molecular Probes; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013917:Thrombin