Biochemical study on the FRET efficiency of various fluorescent protein pairs

초록

Fluorescence Resonance Energy Transfer (FRET) occurs when two different fluorophores are positioned close enough together within 10nm that the emission spectrum of one fluorophore overlaps with the absorption spectrum of the other. In this structural arrangement, the energy from the excited state of the donor fluorophore is transferred non-radiatively to the acceptor fluorophore, which then emits light at a longer wavelength. FRET can be used to detect molecular interactions, conformational changes, and distances between molecules at the molecular level. The efficiency of energy transfer depends on the distance between the donor and acceptor molecules. We generated a series of bacterial expression vectors for His-tagged FRET pairs using different fluorescent proteins such as Cerulean, YPet and Cherry. We also generated various FRET pairs in different orders such as YPet-Cherry and Cherry-YPet. Various FRET pair proteins were expressed in BL21(DE3) E. coli strain and purified to homogeneity with nickel column chromatography. We measured fluorescence of various FRET pair proteins using BioTek Synergy HTX Multi-Mode Microplate Reader and Jasco FP-8550 spectrofluorometer. The FRET efficiency of various FRET pair proteins were analyzed for further application to in vitro protein-protein interaction studies.