title: Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein.
abstract: We 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.
authors: Department of Chemistry, Connecticut College, 270 Mohegan Avenue, New London, CT 06320, USA.
authors: Bruce R
authors: BR
authors: Branchini
authors: Justin C
authors: JC
authors: Rosenberg
authors: Danielle M
authors: DM
authors: Ablamsky
authors: Kelsey P
authors: KP
authors: Taylor
authors: Tara L
authors: TL
authors: Southworth
authors: Samantha J
authors: SJ
authors: Linder
mesh_terms: D053148:Caspase 3 / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D015951:Factor Xa / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D049409:Luciferases, Firefly / Q000032:analysis* / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D019265:Spectroscopy, Near-Infrared; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D053148:Caspase 3; D015951:Factor Xa; D005456:Fluorescent Dyes; D049409:Luciferases, Firefly; D008164:Luminescent Proteins; D010447:Peptide Hydrolases; D011993:Recombinant Fusion Proteins; D013917:Thrombin; D000097573:Red Fluorescent Protein