skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

Abstract

An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP{sup 2})-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A{sup pro}) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A{sup pro} from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research.

Authors:
 [1];  [1];  [2];  [3];  [4]
  1. Faculty of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan (China)
  2. Incubation Center, National Yang-Ming University, Taipei, Taiwan (China)
  3. Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan (China)
  4. Faculty of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan (China). E-mail: szkung@ym.edu.tw
Publication Date:
OSTI Identifier:
20979806
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 353; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2006.12.145; PII: S0006-291X(06)02782-3; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ENERGY TRANSFER; FLUORESCENCE; IN VIVO; PEPTIDES; SUBSTRATES; VIRUSES

Citation Formats

Hsu, Y.-Y., Liu, Y.-N., Wang Wenyen, Kao, Fu-Jen, and Kung, S.-H. In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2006.12.145.
Hsu, Y.-Y., Liu, Y.-N., Wang Wenyen, Kao, Fu-Jen, & Kung, S.-H. In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair. United States. doi:10.1016/j.bbrc.2006.12.145.
Hsu, Y.-Y., Liu, Y.-N., Wang Wenyen, Kao, Fu-Jen, and Kung, S.-H. Fri . "In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair". United States. doi:10.1016/j.bbrc.2006.12.145.
@article{osti_20979806,
title = {In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair},
author = {Hsu, Y.-Y. and Liu, Y.-N. and Wang Wenyen and Kao, Fu-Jen and Kung, S.-H.},
abstractNote = {An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP{sup 2})-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A{sup pro}) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A{sup pro} from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research.},
doi = {10.1016/j.bbrc.2006.12.145},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 353,
place = {United States},
year = {Fri Feb 23 00:00:00 EST 2007},
month = {Fri Feb 23 00:00:00 EST 2007}
}