Cell free application: Sumoylation characterization

Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport,
transcriptional regulation, apoptosis, protein stability and response to stress.

In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off to allow formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.

Cell free expression can be used to characterize sumoylation of proteins. Target proteins are expressed in a rabbit reticulocyte cell free system (supplemented with necessary addition components,). Proteins that have been modified can be analyzed by a shift in migration on polyacrylamide gels, when compared to control reactions.

The following references illustrate the use of cell free expression for this application.

Brandl, A. et al. (2012) Dynamically regulated sumoylation of HDAC2 controls p53 deacetylation and restricts apoptosis following genotoxic stress. J. Mol. Cell. Biol. (online only)

Janer, A. et al. (2010). SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7. Human. Mol. Gen. 19, 181—95.

Rytinki, M. et al. (2009) SUMOylation attenuates the function of PGC-1alpha. J. Biol. Chem. 284, 26184-93.

Klein, U. et al. (2009) RanBP2 and SENP3 function in a mitotic SUMO2/3 conjugation-deconjugation cycle on Borealin. Mol. Cell. Biol. 20, 410–18.

Seo, W. and Ziltener, H. (2009) CD43 processing and nuclear translocation of CD43 cytoplasmic tail are required for cell homeostasis. Blood, 114, 3567–77.

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Gary Kobs

Strategic Marketing Manager at Promega Corporation
Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Marketing Manager Protein Analysis.

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