Firefly Luciferase-Based Sequential Bioluminescence Resonance Energy Transfer (BRET)-Fluorescence Resonance Energy Transfer (FRET) Protease Assays

  • Bruce BranchiniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1461)


We describe 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 catalyzes yellow-green (560 nm 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 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. This method successfully employs an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence to assay physiologically important protease activities and should be generally applicable to the measurement of any endoprotease lacking accessible cysteine residues.

Key words

Bioluminescence BRET Caspase Factor Xa Firefly FRET Luciferase Protease mKate Thrombin 



Alexa Fluor 680 C2-maleimide


BRET-FRET fusion protein, a fusion protein consisting of an N-terminus hexa-His tagged mKate S158A variant joined to PpyWT-TS through the decapeptide linker GSAP4P3P2P1GSG where P4P3P2P1 is DEVD (C3), LVPR (Th), IEGR (Xa), or GSGS (GS)


The BRET-FRET substrates corresponding to the BFFP-proteins labeled with AF680


Bioluminescence resonance energy transfer


Fluorescence resonance energy transfer


Charge-coupled device


Tandem HPLC-electrospray ionization mass spectrometry


D-firefly luciferin


Photinus pyralis luciferase (E. C.



PBSA buffer

20 mM sodium phosphate buffer (pH 7.2) containing 150 mM NaCl, 5 mM EDTA and 0.8 M ammonium sulfate


Recombinant P hotinus pyralis luciferase containing the additional N-terminal peptide GPLGS


PpyWT containing the mutations T214A, A215L, I232A, F295L, and E354K


Red fluorescent protein (mKate S158A variant)


Sequential BRET-FRET.



I gratefully acknowledge the financial support provided by the National Science Foundation (MCB 1410390), the Air Force Office of Scientific Research (FA9550-14-1-0100) and the Hans & Ella McCollum’21 Vahlteich Endowment. The technical contributions of Justin C. Rosenberg, Danielle M. Fontaine, Kelsey P. Taylor, Tara L. Southworth, and Samantha J. Linder were essential to the development of the SRET substrates.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of ChemistryConnecticut CollegeNew LondonUSA

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