In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system

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...

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Bibliographic Details
Published in:PloS one 2013-03, Vol.8 (3), p.e59710
Main Authors: Kim, Jeong Hee, Lee, Min Jun, Hwang, Inhwan, Hwang, Hyun Jin
Format: Article
Language:English
Subjects:
Animals
Apoptosis
Binding sites
Biochemistry
Biology
CHO Cells
Cleavage
Cloning
Cricetinae
Cricetulus
Cytosol
Cytosol - metabolism
Dentistry
Detection equipment
Drug Delivery Systems
Drug discovery
Engineering
Fluorescence
Fluorescent Dyes - pharmacology
Hepacivirus - enzymology
Hepatitis
Hepatitis C virus
Inhibitory Concentration 50
Intracellular
Localization
Molecular biology
Molecular Imaging - methods
Peptide Hydrolases - chemistry
Peptides
Plasmids
Plasmids - metabolism
Protease
Proteases
Protein Structure, Tertiary
Protein Transport
Proteinase
Proteins
R&D
Research & development
Target detection
Tool life
Translocation
Viral infections
Viral Nonstructural Proteins - chemistry
Viruses
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Description
Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0059710