Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage

Biosensors & Bioelectronics
H DacresS C Trowell

Abstract

In this study, a representative FRET system (CFP donor and YFP acceptor) is compared with the BRET(2) system (Renilla luciferase donor, green fluorescent protein(2) (GFP(2)) acceptor and coelenterazine 400a substrate). Cleavage of a thrombin-protease-sensitive peptide sequence inserted between the donor and acceptor proteins was detected by the RET signal. Complete cleavage by thrombin changed the BRET(2) signal by a factor of 28.9+/-0.2 (R.S.D. (relative standard deviation), n=3) and the FRET signal by a factor of 3.2+/-0.1 (R.S.D., n=3). The BRET(2) technique was 50 times more sensitive than the FRET technique for monitoring thrombin concentrations. Detection limits (blank signal+3sigma(b), where sigma(b)=the standard deviation (S.D.) of the blank signal) were calculated to be 3.05 and 0.22nM thrombin for FRET and BRET(2), respectively. This direct comparison suggests that the BRET(2) technique is more suitable than FRET for use in proximity assays such as protease cleavage assays or protein-protein interaction assays.

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Citations

Jan 2, 2015·Biomicrofluidics·Nam Cao Hoai LeStephen C Trowell
Apr 1, 2009·Expert Opinion on Drug Discovery·Hideto Hoshino
Dec 4, 2015·Nature Communications·Matthew B RobersKeith V Wood
Mar 18, 2009·Biochemical and Biophysical Research Communications·Young-Pil KimHak-Sung Kim
Mar 6, 2012·Analytical Biochemistry·Helen DacresStephen C Trowell
May 11, 2017·The Analyst·Irvine Lian Hao Ong, Kun-Lin Yang
Jul 3, 2021·International Journal of Molecular Sciences·Emmiliisa VuorinenHarri Härmä
Oct 13, 2010·Bioconjugate Chemistry·Bruce R BranchiniJustin C Rosenberg

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