Design of FRET-based GFP probes for detection of protease inhibitors |
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Authors: | Zhang Bin |
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Institution: | Institute of Basic Medical Sciences, Zhejiang University School of Medicine, 353 Yan’an Road, Hangzhou 310031, PR China |
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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. |
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Keywords: | FRET GFP Thrombin Protease inhibitor |
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