SUMO(Small Ubiquitin-like Modifier) chip for analysis of SUMO-conjugation to a target protein

Abstract

SUMOylation, the process of conjugating small ubiquitin-like modifier (SUMO) to a target protein, is one of typical post translational modifications of proteins in eukaryotic cells and mediated by multiple enzymes. We developed a chip-based assay of SUMO conjugation by employing a domain protein of RanGAP1 as a target substrate. The relevant protein components to SUMOylation including E1 (SAE1/SAE2), E2 (Ubc9), and SUMO protein were expressed in E. coli and purified to homogeneity. To examine the selectivity of SUMO conjugation, the RanGAP1 domain with a single mutation (K526A) was also tested. Target substrate was immobilized on a surface-mo ified glass slide followed by addition of the reaction mixture for SUMOylation, and SUMO conjugation was detected using dye-labeled antibodies. For reliable and sensitive detection of on-chip SUMOylation, the reaction conditions were optimized with respect to the concentrations of protein components, the composition and the reducing state of the reaction mixture for SUMOylation, and the concentrations of dye-labeled antibodies. Under established conditions, the on-chip SUMOylation of RanGAP1 domain resulted in at least 7-fold higher fluorescence intensity than that of its mutant (K526A) irrelevant to SUMO conjugation. The on-chip SUMOylation was validated and quantified by using surface plasmon resonance spectroscopy. The chip-based assay developed here seems to provide a strategy enabling a high throughput analysis of SUMO conjugation.