부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.bbrc.2006.12.145 Byline: Yueh-Ying Hsu (a), Yu-Ning Liu (a), Wenyen Wang (b), Fu-Jen Kao (c), Szu-Hao Kung (a) Keywords: FRET; Enterovirus; Protease; GFP; DsRed 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. Author Affiliation: (a) Faculty of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, ROC (b) Incubation Center, National Yang-Ming University, Taipei, Taiwan, ROC (c) Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan, ROC Article History: Received 12 December 2006