Generation of bacterial expression system for fluorescent fusion proteins with TEV cleavage site

초록

Fluorescent proteins can transfer energies internally through FRET (Fluorescence Resonance Energy Transfer) if two fluorophores are positioned within 10 nm of each other. We developed a bacterial expression system for fluorescent fusion proteins incorporating a TEV cleavage site between the two fluorescent proteins to study FRET efficiency among various fluorescent proteins. The bacterial expression vector used for this study was based on the pET expression system with a lac promoter. This vector was engineered to express two open reading frames (ORFs) in tandem, featuring an N-terminal His-tag and a TEV cleavage site between the two proteins. This expression system facilitated the expression of the EBFP2-mCherry fusion protein in the BL21(DE3) strain. EBFP2, mCherry, and the EBFP2-mCherry fusion proteins were expressed in E. coli and purified using a nickel column. The purified EBFP2-mCherry fusion protein was successfully cleaved into EBFP2 and mCherry proteins by TEV protease. FRET efficiency and various fluorescence parameters were analyzed for EBFP2, mCherry, and the EBFP2-mCherry fusion proteins before and after TEV cleavage.