SUMOylation detection using fluorescence resonance energy transfer(FRET)

Abstract

The primary goal of this research is to examine feasibility of the application of FRET detection method in SUMOylation detection. SUMOylation is one of recently discovered post-translational modification processes, which is adopted by eukaryotic cells to control various important cellular functions. This study is based on the fact that SUMO1(G) as SUMOylation modifier, after SUMOylation would covalently attach to it’s SUMOylation substrates. Human RanGAP1 domain containing SUMOylation motif was used as a model. ECFP fusion human SUMO1(G) was used as modifier and EYFP fusion human RanGAP1 domain as its substrate. Since the constant attachment between this two proteins，in short proximity, fluorescence resonance energy transfer(FRET) might occur between donor ECFP and acceptor EYFP, result in increased fluorescence intensity at EYFP emission region under 400nm ECFP excitation that normally not occur in negative control. As result, FRET occurrence was confirmed by obvious changes in increased fluorescence emission in the emission region of EYFP(510nm~530nm) after background reducing process by membrane filtration. The result enables further application of FRET method in the study of in vivo SUMOylation dynamics, or in combination with high throughput SUMO target screening system.