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dc.contributor.authorPage, Richard
dc.contributor.authorPaththamperuma, Chathura
dc.description.abstractTobacco etch virus (TEV) protease is a widely used protease for fusion tag cleavage. Despite its widespread usage, an assay to quickly and easily quantify its activity in laboratory settings is still lacking. Thus, researchers may encounter inefficient cleavage of the desired fusion proteins due to poor activity of a given TEV protease preparation. Here, we describe the development and implementation of a fluorescence dequenching-based assay to quantify TEV protease activity and assess kinetic parameters. The peptide substrate used in this assay consists of a C-terminal TAMRA fluorophore, an N-terminal fluorescein fluorophore, and the canonical TEV protease recognition sequence. The assay is based on a reduction of fluorescence quenching of fluorescein upon cleavage by TEV protease. The substrate peptide was studied spectroscopically to assess feasibility and to propose a plausible mechanism of the assay. The assay was optimized and applied to obtain rapid assessments of TEV protease activity in purified samples and crude lysate extracts. The kinetic data obtained from improved TEV protease variants were compared with a traditional SDS-PAGE assay. Finally, the assay was applied to determine the optimum pH for TEV protease. Further, the study found that the assay is a rapid and simple approach to quantify TEV protease activity. The findings of the assay on crude lysate extracts, activity assay of TEV protease variants, and assessment of optimum pH for TEV protease reactions demonstrate the robust utility of the assay.en_US
dc.rightsAttribution-NonCommercial-ShareAlike 3.0 United States*
dc.titleRaw Data for: Fluorescence Dequenching Assay for the Activity of TEV Protease (YABIO_11495)en_US
dc.typeJournal Articleen_US

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  • Page, Richard
    Dr. Richard Page - Associate Professor, Department of Chemistry and Biochemistry

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Attribution-NonCommercial-ShareAlike 3.0 United States
Except where otherwise noted, this item's license is described as Attribution-NonCommercial-ShareAlike 3.0 United States