Sensitive Assay of Protease Activity on a Micro/Nanofluidics Preconcentrator Fused with the Fluorescence Resonance Energy Transfer Detection Technique

2014 ◽  
Vol 86 (6) ◽  
pp. 3216-3221 ◽  
Author(s):  
Chen Wang ◽  
Jun Ouyang ◽  
Yun-Yi Wang ◽  
De-Kai Ye ◽  
Xing-Hua Xia
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Protease Activity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Detection Technique ◽  
Sensitive Assay ◽  
Fluorescence Resonance

scholarly journals Fluorescence Resonance Energy Transfer-Based Assay for Characterization of Hepatitis C Virus NS3-4A Protease Activity in Live Cells

2008 ◽  
Vol 53 (2) ◽  
pp. 728-734 ◽  
Author(s):  
Rosario Sabariegos ◽  
Fernando Picazo ◽  
Beatriz Domingo ◽  
Sandra Franco ◽  
Miguel-Angel Martinez ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescence Resonance Energy Transfer ◽  
Protease Activity ◽  
Cleavage Site ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance

ABSTRACT The NS3/4A protease from hepatitis C virus (HCV) plays a key role in viral replication. We report a system for monitoring the activity of this enzyme in single living mammalian cells. We constructed a fluorescence resonance energy transfer (FRET) probe that consists of an enhanced cyan fluorescent protein-citrine fusion, with a cleavage site for HCV NS3/4A protease embedded within the linker between them. Expression of the biosensor in mammalian cells resulted in a FRET signal, and cotransfection with the NS3/4A expression vector produced a significant reduction in FRET, indicating that the cleavage site was processed. Western blot and spectrofluorimetry analysis confirmed the physical cleavage of the fusion probe by the NS3/4A protease. As the level of FRET decay was a function of the protease activity, the system allowed testing of NS3/4A protease variants with different catalytic efficiencies. This FRET probe could be adapted for high-throughput screening of new HCV NS3/4 protease inhibitors.

A quantum-dot based protein module for in vivo monitoring of protease activity through fluorescence resonance energy transfer

2011 ◽  
Vol 47 (18) ◽  
pp. 5259 ◽  
Author(s):  
Payal Biswas ◽  
Lakshmi N. Cella ◽  
Seung Hyun Kang ◽  
Ashok Mulchandani ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Fluorescence Resonance Energy Transfer ◽  
Protease Activity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
In Vivo Monitoring ◽  
Fluorescence Resonance
Sign in / Sign up

Export Citation Format

Share Document