Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage.
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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Food Futures Flagship, CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia.
Dumancic
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Horne
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Trowell
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D015374:Biosensing Techniques / Q000379:methods*; D003199:Computer Systems; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D010447:Peptide Hydrolases / Q000032:analysis / Q000737:chemistry*; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000032:analysis / Q000737:chemistry*
D010447:Peptide Hydrolases; D013917:Thrombin