期刊
ANALYTICAL BIOCHEMISTRY
卷 414, 期 2, 页码 239-245出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.03.031
关键词
Alexa Fluor; Bioluminescence; BRET; Caspase; Factor Xa; Firefly; FRET; Luciferase; Protease; mKate; Near-infrared; Red fluorescent protein; Thrombin
资金
- National Science Foundation [MCB0842831]
- Air Force Office of Scientific Research [FA9550-10-1-0174]
- Hans & Ella McCollum '21 Vahlteich Endowment
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 (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. 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. (C) 2011 Elsevier Inc. All rights reserved.
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