Preparation of Sumoylated Substrates for Biochemical Analysis
- 3k Downloads
Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via “loss of modification” analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a “gain in modification” analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.
Key wordsSUMO E2-25K in vitro sumoylation assay with recombinant enzymes protein purification
Our special thanks go to Katharina Maderböck and Neha Nigam for critical reading of the manuscript. This work was funded by the Vienna Science and Technology Fund WWTF LS05003 and FWF P18584-B12 to A.P., and EU-Rubicon, NWO-CW pionier and CBG to T.K.S.
- 16.Lin, D. Y., Huang, Y. S., Jeng, J. C., Kuo, H. Y., Chang, C. C., Chao, T. T., Ho, C. C., Chen, Y. C., Lin, T. P., Fang, H. I., Hung, C. C., Suen, C. S., Hwang, M. J., Chang, K. S., Maul, G. G., and Shih, H. M. (2006) Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol. Cell 24, 341–354.PubMedCrossRefGoogle Scholar