A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins

Abstract

We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.

Figure 1

In vivo BRET in E. coli cells expressing the RLUC–EYFP fusion protein. (A) Diagrams of the expression cassettes of pT7/Eyfp, pT7/Rluc, and pT7/Rluc•Eyfp. P_T7, T7 promoter region; Ter, transcription terminator region. The intergenic linker sequence of 11 amino acid residues between Rluc and Eyfp in the Rluc•Eyfp fusion construct is shown below the pT7/Rluc•Eyfp diagram. (B) Luminescence (lum.) or fluorescence (fluor.) emission spectra from transformed strains expressing the proteins RLUC, EYFP, or the fusion protein RLUC•EYFP. Luminescence reactions were initiated by the addition of 1 μM coelenterazine. All spectra were normalized.

Figure 2

In vivo BRET assay for protein interaction in E. coli cells. (A) Diagrams of the gene fusion expression cassettes of pT7/Eyfp•kaiB, pT7/Rluc•kaiB, and pT7/Eyfp•kaiA. (B) Luminescence emission spectra from the transformed strains expressing unfused RLUC and EYFP, fusion proteins RLUC•KaiB and EYFP•KaiB, and fusion proteins RLUC•KaiB and EYFP•KaiA. Luminescence reactions were initiated by the addition of 1 μM coelenterazine.

Figure 3

KaiB–KaiB association in vitro revealed by BRET. Extracts containing fusion proteins EYFP•KaiB were mixed in equal proportions with those containing RLUC•KaiB. Bioluminescence emission profiles were measured at the indicated times after the extracts were combined and incubated at room temperature. Fresh coelenterazine (1 μM) was added at the beginning of the reaction at each time point.

Figure 4

Imaging and quantification of BRET luminescence from transformed E. coli strains. (A) Bioluminescence visualized through interference bandpass filters transmitting light of 480 ± 5 nm (left side) or 530 ± 5 nm (right side). Row 1 is the strain producing only RLUC; row 2 is the strain expressing the RLUC•EYFP fusion protein; row 3 is the strain expressing the fusion proteins RLUC•KaiB and EYFP•KaiA; and row 4 is the strain producing the fusion proteins RLUC•KaiB and EYFP•KaiB. Luminescence reactions were initiated by the addition of 3 μM coelenterazine. (B) Quantification of luminescence intensity in relative light units at 480 nm and 530 nm of the cultures imaged in A. (C) Calculation of the 530 nm/480 nm ratios for the data of B. For B and C, strains are labeled as in A: (1) RLUC, (2) RLUC•EYFP, (3) RLUC•KaiB and EYFP•KaiA, and (4) RLUC•KaiB and EYFP•KaiB.