Abstract
We describe a simple, sensitive and noninvasive assay that uses nontoxic, reengineered anthrax toxin-beta-lactamase fusion proteins with altered protease cleavage specificity to visualize specific cell-surface proteolytic activity in single living cells. The assay could be used to specifically image endogenous cell-surface furin, urokinase plasminogen activator and metalloprotease activity. We have adapted the assay for fluorescence microscopy, flow cytometry and fluorescent plate reader formats, and it is amenable for automation and high-throughput analysis.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, N.I.H., Intramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Antigens, Bacterial / metabolism*
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Automation
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Bacterial Proteins / metabolism*
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Bacterial Toxins / metabolism*
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Biological Assay / methods*
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Cell Membrane / metabolism*
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Cell Membrane / ultrastructure
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Cells, Cultured / metabolism*
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Cells, Cultured / ultrastructure
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Flow Cytometry
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Furin / metabolism
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Metalloproteases / metabolism
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Microscopy, Fluorescence
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Receptors, Cell Surface / metabolism
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Receptors, Urokinase Plasminogen Activator
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Recombinant Fusion Proteins / metabolism
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Substrate Specificity
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beta-Lactamases / metabolism
Substances
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Antigens, Bacterial
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Bacterial Proteins
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Bacterial Toxins
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Receptors, Cell Surface
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Receptors, Urokinase Plasminogen Activator
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Recombinant Fusion Proteins
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anthrax toxin
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Metalloproteases
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Furin
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beta-Lactamases