Fluorescence Resonance Energy Transfer-Based Assay for DNA-Binding Protein Tagged by Green Fluorescent Protein
Specific interaction between green fluorescent protein (GFP)-tagged human α- or γ-enolase<SUP>97-242</SUP> (α or γENO<SUP>97-242</SUP>) and the rhodamine-labeled DNA fragment containing the c-<I>myc</I> P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO<SUP>97-242</SUP> and the c-<I>myc</I> P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R<SUB>as</SUB>) of ENO<SUP>97-242</SUP> mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.
- Bioscience, biotechnology, and biochemistry
Bioscience, biotechnology, and biochemistry 70(8), 1921-1927, 2006-08-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry