This method, called bioluminescence resonance energy transfer (BRET), takes advantage of a naturally occurring phenomenon, namely, the Förster resonance energy transfer between a luminescent donor and a fluorescent acceptor. BRET can be observed in the sea pansy Renilla reniformis. This organism expresses a luciferase, which emits blue light when it is purified. If the luciferase is excited in intact cells, green light occurs, because in vivo the luciferase is associated with the green fluorescent protein (GFP), which accepts the energy from the luciferase and emits green light.
The transfer efficiency depends on the degree of the spectral overlap, the relative orientation, and the distance between the donor and acceptor. BRET typically occurs in the 1-10 nm regions, which is comparable with the dimensions of biological macromolecules and makes BRET an ideal system for the study of protein-protein interaction in living cells.
BRET – the assay method
BRET is an advanced, non-destructive, cell-based assay technology that is perfectly suited for proteomics applications, including receptor research and the mapping of signal transduction pathways. The assay is based on non-radiative energy transfer between fusion proteins containing a bioluminescent luciferase and a GFP mutant.
In most applications the fused donor is Renillaluciferase (Rluc) rather than aequorin, to avoid any intrinsic affinity for Aequorea-derived GFP mutant; the acceptor is the Yellow Fluorescent Protein (YFP), to increase the spectral distinction between the two emissions. When the donor and acceptor are in close proximity, the energy resulting from catalytic degradation of the coelenterazine derivative substrate is transferred from the luciferase to the YFP, which will then emit fluorescence at its characteristic wavelength.
To demonstrate the clear discrimination between positive and negative control of the BRET assay technology, the luminescence and fluorescence signals of the BRET^2™demo kit (Perkin Elmer Life Sciences) were quantified on the microplate reader POLARstar OPTIMA (BMG LABTECH, Fig.1), allowing the monitoring of the kinetic curves and the calculation of the BRET ratio. The POLARstar OPTIMA´s internal reagent injectors for 384-well plate format combined with high-end simultaneous dual emission detection offer a unique advantage for fast kinetic assays where simultaneous emission detection at two wavelengths is required.
The BRET²™demo kit applies the cell-permeable and non-toxic coelenterazine derivative substrate DeepBlueC™ (DBC) and a mutant of the Green Fluorescent Protein (GFP²) as acceptor. These compounds show improved spectral resolution and sensitivity over earlier variants.
Fig 1: The POLARstar OPTIMA is perfectly suited for monitoring BRET assays due to its simultaneous dual emission detection system, which allows collecting 50 kinetic data per second, and its internal reagent injectors for 384-well plate format.
The BRET² kit was performed as described in the kit instructions. The reaction was measured in a white 384-well plate at two channels in simultaneous dual emission detection mode with the highest possible resolution of 0.02 s for every data point. Four sets of samples were run in triplicate, a blank (non-transfected cells), a positive control (Rluc-GFP²), a negative control (Rluc + GFP²), and a buffer control (BRET² assay buffer). Readings were started immediately after the automated injection of the luciferase substrate DBC.

The kinetic curves of the negative control are shown in Fig.2 for both channels. The low values of the 515 nm channel indicate that no resonance energy transfer occurred. Whereas the positive control shows reduced values at the 410 nm and elevated values at the 515 nm channel due to the BRET effect.
Fig 2: Resonance energy transfer is obvious for the positive control. No BRET occurs for the negative control.
The calculated BRET ratio indicates the occurrence of protein-protein interaction in vivo. This type of detection eliminates data variability caused by fluctuations in light output which can be found with variations e.g. in assay volume, cell types, number of cells per well and/or signal decay across the plate. In Fig.3 the blank corrected BRET² ratios for both, negative and positive control, are shown and were determined as:

The signal for negative and positive control here reveals a value of around 0.06 and 3.3 respectively, which leads to a factor of around 50 and a clear discrimination between these controls.

BRET - a new method for assaying protein-protein interactions in living cells

Category: BRET

The signal for negative and positive control here reveals a value of around 0.06 and 3.3 respectively, which leads to a factor of around 50 and a clear discrimination between these controls.
Fig 3: Ratio of negative and positive control.
The high factor between these controls is caused by the artificial fusion construct of the positive control (Rluc-GFP²) resulting in an extremely high BRET. Real assay samples will presumably result in lower ratios. Nevertheless, the large spectral resolution between donor and emission peaks in BRET² (115 nm) greatly improves the signal to background ratio over traditionally used BRET and FRET technologies that typically have only a ~50 nm spectral resolution.³
Advantages of BRET over FRET
The BRET technique is related to an existing method for monitoring biomolecular interactions and conformational changes, fluorescence resonance energy transfer (FRET). In FRET, the luminescent donor is replaced by a second fluorophore, which emission spectrum overlaps with the excitation spectrum of the acceptor fluorophore. By using two spectral mutants of GFP, it is possible to genetically attach donor and acceptor fluorophores to proteins, which allows the study of protein interactions in native organisms under physiological conditions.
The main disadvantages of FRET, as opposed to BRET, are the consequences of the required excitation of the donor with an external light source. BRET assays show no photo bleaching or photoisomerization of the donor protein, no photodamage to cells, and no light scattering or autofluorescence from cells or microplates, which can be caused by incident excitation light. In addition one main advantage of BRET over FRET is the lack of emission arising from direct excitation of the acceptor.
This reduction in background should permit detection of interacting proteins at much lower concentrations than it is possible for FRET. However, BRET requires the addition of a cofactor and for some applications, e.g. determining the compartmentalization and functional organization of living cells, the GFP-based FRET method is superior to BRET due to the much higher light output.
BRET applications
The BRET technology was first described in 1999 from Xu and colleagues¹ and has been used successfully for a wide range assay types including protein-protein interactions (e.g. interaction of cardian clock proteins¹), GPCR functional assays⁴ (incl. orphan receptors), receptor oligomerization², and protease activity assays in living cells². BRET has been further used for Ca^{2 +} detection. By fusing GFP directly to the luminescent jellyfish luciferase aequorin, which metabolizes coelenterazine in response to binding free calcium ions, a sensor was produced, that reports calcium ion flux by increases in GFP fluorescence.⁵
Conclusion
BRET is a new energy transfer based technique that offers the ability to directly study complex protein-protein interactions in living cells. There is no need for an excitation light source. Therefore photosensitive tissue can be used for BRET, and problems associated with FRET-based assays such as photobleaching, autofluorescence and direct excitation of the acceptor are eliminated. This powerful technology has been applied in a range of interesting applications in academia and drug discovery. Its homogeneous nature and the development of sensitive plate readers, which offers injection features, have made high-throughput screening using BRET in live cells possible.
References

Xu Y, Piston DW, Johnson CH. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 1999;96:151-6.
Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M. Detection of b₂-adrenergic receptors dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 2000; 97:3684-9.
Mahajan NP, Linder K, Berry G, Gordon GW, Heim R, Herman B. Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nat Biotechnol 1998; 16:547-52.
Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M., Jockers R. Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 2002; 277:21522-8.
Baubet V, Le Mouellic H, Campbell AK, Lucas-Meunier E, Fossier P, Brûlet P. Chimeric green fluorescent protein-aequorin as bioluminescent Ca^{2 +} reporters at the single-cell level. Proc Natl Acad Sci USA 2000; 97:7260-5

Fig 3: Ratio of negative and positive control.
The high factor between these controls is caused by the artificial fusion construct of the positive control (Rluc-GFP²) resulting in an extremely high BRET. Real assay samples will presumably result in lower ratios. Nevertheless, the large spectral resolution between donor and emission peaks in BRET² (115 nm) greatly improves the signal to background ratio over traditionally used BRET and FRET technologies that typically have only a ~50 nm spectral resolution.³
Advantages of BRET over FRET
The BRET technique is related to an existing method for monitoring biomolecular interactions and conformational changes, fluorescence resonance energy transfer (FRET). In FRET, the luminescent donor is replaced by a second fluorophore, which emission spectrum overlaps with the excitation spectrum of the acceptor fluorophore. By using two spectral mutants of GFP, it is possible to genetically attach donor and acceptor fluorophores to proteins, which allows the study of protein interactions in native organisms under physiological conditions.
The main disadvantages of FRET, as opposed to BRET, are the consequences of the required excitation of the donor with an external light source. BRET assays show no photo bleaching or photoisomerization of the donor protein, no photodamage to cells, and no light scattering or autofluorescence from cells or microplates, which can be caused by incident excitation light. In addition one main advantage of BRET over FRET is the lack of emission arising from direct excitation of the acceptor.
This reduction in background should permit detection of interacting proteins at much lower concentrations than it is possible for FRET. However, BRET requires the addition of a cofactor and for some applications, e.g. determining the compartmentalization and functional organization of living cells, the GFP-based FRET method is superior to BRET due to the much higher light output.
BRET applications
The BRET technology was first described in 1999 from Xu and colleagues¹ and has been used successfully for a wide range assay types including protein-protein interactions (e.g. interaction of cardian clock proteins¹), GPCR functional assays⁴ (incl. orphan receptors), receptor oligomerization², and protease activity assays in living cells². BRET has been further used for Ca^{2 +} detection. By fusing GFP directly to the luminescent jellyfish luciferase aequorin, which metabolizes coelenterazine in response to binding free calcium ions, a sensor was produced, that reports calcium ion flux by increases in GFP fluorescence.⁵
Conclusion
BRET is a new energy transfer based technique that offers the ability to directly study complex protein-protein interactions in living cells. There is no need for an excitation light source. Therefore photosensitive tissue can be used for BRET, and problems associated with FRET-based assays such as photobleaching, autofluorescence and direct excitation of the acceptor are eliminated. This powerful technology has been applied in a range of interesting applications in academia and drug discovery. Its homogeneous nature and the development of sensitive plate readers, which offers injection features, have made high-throughput screening using BRET in live cells possible.
References

Xu Y, Piston DW, Johnson CH. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 1999;96:151-6.
Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M. Detection of b₂-adrenergic receptors dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 2000; 97:3684-9.
Mahajan NP, Linder K, Berry G, Gordon GW, Heim R, Herman B. Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nat Biotechnol 1998; 16:547-52.
Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M., Jockers R. Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 2002; 277:21522-8.
Baubet V, Le Mouellic H, Campbell AK, Lucas-Meunier E, Fossier P, Brûlet P. Chimeric green fluorescent protein-aequorin as bioluminescent Ca^{2 +} reporters at the single-cell level. Proc Natl Acad Sci USA 2000; 97:7260-5

Pharmaceutical info

Monday, January 9, 2012