title: In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.
abstract: This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s). The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University, Seoul, Korea. jhkimh@khu.ac.kr
authors.forename: Jeong Hee
authors.initials: JH
authors.lastname: Kim
authors.forename: Min Jun
authors.initials: MJ
authors.lastname: Lee
authors.forename: Inhwan
authors.initials: I
authors.lastname: Hwang
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Hwang
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D016503:Drug Delivery Systems; D005456:Fluorescent Dyes / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology*; D020128:Inhibitory Concentration 50; D057054:Molecular Imaging / Q000379:methods*; D010447:Peptide Hydrolases / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D017361:Viral Nonstructural Proteins; D010447:Peptide Hydrolases
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