Enhanced Detection of In Vivo SUMO Conjugation by Ubc9 Fusion-Dependent Sumoylation (UFDS)
- 3k Downloads
The bottleneck in studying protein sumoylation—the conjugation of the small ubiquitin-like modifier (SUMO)—is the detection of the low level of in vivo sumoylated proteins. The Ubc9 fusion-directed sumoylation (UFDS) system strongly enhances the in vivo sumoylation of a substrate protein at its specific sumoylation site. UFDS utilizes an expression plasmid for the protein of interest fused to the SUMO-conjugating enzyme Ubc9. When expressed in HEK293, COS-7, HeLa, or CHO cells, the fused target protein is conjugated with endogenous or coexpressed SUMO at its native sumoylation sites. This sumoylation requires neither SUMO ligase nor any extracellular stimulation and is easily detectable by fusion protein- or Ubc9-specific Western blotting with commercially available antibodies.
Key words:SUMO Ubc9 sumoylation Ubc9 fusion-directed sumoylation UFDS in vivo sumoylation
The author would like to thank Matthias Gaestel for critical reading of the manuscript, and Astrid Jakobs for preparing the figures shown for Ubc9 fusion-directed sumoylation. This work was supported by the Medical School Hannover, Institute of Physiological Chemistry.