• Bruce R Branchini, Justin C Rosenberg, Danielle M Ablamsky, Kelsey P Taylor, Tara L Southworth, and Samantha J Linder.
• Department of Chemistry, Connecticut College, 270 Mohegan Avenue, New London, CT 06320, USA.
• Anal. Biochem. 2011 Jul 15; 414 (2): 239-45.

AbstractWe report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41nM for caspase 3, 1.0nM for thrombin, and 58nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.Copyright © 2011 Elsevier Inc. All rights reserved.

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