Design of FRET-based GFP probes for detection of protease inhibitors

Biochemical and Biophysical Research Communications
Bin Zhang

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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Citations

Dec 10, 2008·The Journal of Biological Chemistry·Nancy W KaruriJean E Schwarzbauer
Sep 4, 2008·Photochemistry and Photobiology·Lorenzo AlbertazziFabio Beltram
Apr 6, 2006·Microbial Cell Factories·Rosa María FerrazAntonio Villaverde
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Feb 26, 2019·Journal of Experimental Botany·Álvaro Daniel Fernández-FernándezSimon Stael
Jan 5, 2019·Chembiochem : a European Journal of Chemical Biology·Bao-Qing ZhaoKai-Hong Zhao
Sep 30, 2010·The Analyst·Katharina WelserJonathan W Aylott
Dec 1, 2017·Methods and Applications in Fluorescence·Alexander S GoryashchenkoAlexander P Savitsky

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