EurekaMag.com logo
+ Site Statistics
References:
53,869,633
Abstracts:
29,686,251
+ Search Articles
+ Subscribe to Site Feeds
EurekaMag Most Shared ContentMost Shared
EurekaMag PDF Full Text ContentPDF Full Text
+ PDF Full Text
Request PDF Full TextRequest PDF Full Text
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter
Follow on LinkedInFollow on LinkedIn

+ Translate

Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein



Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein



Analytical Biochemistry 414(2): 239-245



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.

(PDF emailed within 0-6 h: $19.90)

Accession: 055720792

Download citation: RISBibTeXText

PMID: 21453669

DOI: 10.1016/j.ab.2011.03.031



Related references

Firefly Luciferase-Based Sequential Bioluminescence Resonance Energy Transfer (BRET)-Fluorescence Resonance Energy Transfer (FRET) Protease Assays. Methods in Molecular Biology 1461: 101-115, 2018

Detection of protein-protein interaction by bioluminescence resonance energy transfer from firefly luciferase to red fluorescent protein. Journal of Bioscience and Bioengineering 94(4): 362-364, 2005

Detection of protein-protein interaction by bioluminescence resonance energy transfer from firefly luciferase to red fluorescent protein. Journal of Bioscience & Bioengineering 94(4): 362-364, 2002

Bioanalytical systems based on bioluminescence resonance energy transfer using firefly luciferase. Combinatorial Chemistry & High Throughput Screening 18(10): 946-951, 2016

Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: applications of terbium-fluorescein and terbium-green fluorescent protein fluorescence resonance energy transfer pairs. Analytical Biochemistry 356(1): 108-116, 2006

Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase. Journal of Bioscience & Bioengineering 93(6): 537-542, 2002

Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase. Journal of Bioscience and Bioengineering 93(6): 537-542, 2005

Ratiometric fluorescent pH nanoprobes based on in situ assembling of fluorescence resonance energy transfer between fluorescent proteins. Analytical and Bioanalytical Chemistry 409(21): 5073-5080, 2017

Near infrared bioluminescence resonance energy transfer from firefly luciferase--quantum dot bionanoconjugates. Nanotechnology 25(49): 495606-495606, 2015

Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease. Analytica Chimica Acta 724: 104-110, 2012

A naphthalimiderhodamine ratiometric fluorescent probe for Hg2+ based on fluorescence resonance energy transfer. Dyes and Pigments 92(3): 909-915, 2012

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone. Photochemistry and Photobiology 92(1): 158-165, 2015

Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET). Biochemical and Biophysical Research Communications 425(3): 625-629, 2012

Dynamics of NF kB and IkBa studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNA by fluorescence resonance energy transfer. The Journal of Biological Chemistry 275(22): 035-42, 2000

Dynamics of NF kappa B and Ikappa Balpha studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNa by fluorescence resonance energy transfer. Journal of Biological Chemistry 275(22): 17035-17042, 2000