Bacterial expression of TEV cleavable fluorescent protein pairs for in vitro FRET efficiency study

초록

Fluorescence (Forster) Resonance Energy Transfer (FRET) is a mechanism by which energy is transferred non-radiatively from an excited donor molecule to an acceptor molecule. FRET is dependent on the distance between the donor and acceptor, and can be used to study molecular interactions and distances in the range of 1 to 10 nanometers. We generated bacterial expression vectors for various fluorescent protein pairs and individual fluorescent proteins to study the biochemistry of FRET efficiency between two fluorescent proteins. A bacterial expression vector for the EBFP-mScarlet fusion protein was generated with a linker sequence between the two fluorescent proteins. The linker sequence includes 12 amino acids for flexibility and the ENLYFQG sequence for TEV protease digestion. Expression of the EBFP-mScarlet fusion protein in an E. coli strain, BL21(DE3) was observed by SDS-PAGE. The optimal ratio between the fusion protein and TEV protease was determined for further FRET study. Various spectroscopic parameters such as absorbance and fluorescence were monitored using a UV/VIS spectrophotometer, spectrofluorometer, fluorescence microplate reader, and UV transilluminator. FRET was observed with the crude extract of the EBFP-mScarlet fusion protein. The addition of TEV protease eliminated FRET signals. The cleavage of the EBFP-mScarlet fusion protein into two individual fluorescent proteins (EBFP and mScarlet) was observed by SDS-PAGE. These results suggest that FRET efficiency between two proteins can be studied in vitro using crude extracts and purified proteins.