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 Vol 9: Versatile Recombinant SUMOylation System for the Production of SUMO-Modified Protein.


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This article is from PLoS ONE, volume 9.AbstractPosttranslational modification by small ubiquitin-like modifiers SUMO is being associated with a growing number of regulatory functions in diverse cellular processes. The biochemical investigation into the underlying molecular mechanisms, however, has been lagging behind due to the difficulty to generate sufficient amounts of recombinant SUMOylated proteins. Here, we present two newly designed two-component vector systems for the expression and purification of SUMO-modified target proteins in Escherichia coli. One system consists of a vector for SUMO conjugation, expressing human SUMO-activating SAE1-SAE2 and conjugating Ubc9 enzymes together with His6-tagged SUMO1, 2 or 3, that can be combined with commonly used expression constructs for any gene of interest. To facilitate SUMOylation of targets normally requiring a SUMO-E3 ligase for efficient modification, a second system is designed to express the target protein as a fusion with the human SUMO-conjugating enzyme Ubc9, thus compensating the absence of a potential SUMO ligase. We demonstrate the proficiency of these systems by SUMOylation of two DNA repair proteins, the thymine DNA glycosylase TDG and XRCC1, and describe purification schemes for SUMOylated proteins in native and active form. This SUMO toolbox facilitates -in-cell- and -in-extract- production and purification of recombinant SUMO-modified target proteins for functional and structural analysis.



Autor: Weber, Alain R.; Schuermann, David; Schar, Primo

Fuente: https://archive.org/







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