Reconstituting posttranslational modification with SUMO in vitro is an essential tool in the analysis of sumoylation. In this article, we provide detailed protocols that allow to set up and perform sumoylation reactions using a purified recombinant sumoylation machinery. The protocols include purification of the SUMO E1 enzyme His-Aos1/Uba2, untagged E2 enzyme Ubc9, untagged SUMO, and the RanBP2 E3 ligase fragment IR1 + M. Using these components, we provide step-by-step instructions to set up sumoylation reactions. Two established SUMO model substrates, His-RanGAPtail and HisYFP-Sp100, complement the described tool box; these proteins serve as positive controls in E3 ligase-independent and -dependent sumoylation reactions and are valuable instruments to adjust the reaction conditions if necessary.
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We are grateful to Nicolas Stankovic-Valentin for critical reading of the manuscript, Anja Schreieck for excellent assistance in establishing the improved version of the E1 purification procedure, Tina Lampe for cloning HisYFP-Sp100, and all lab members for sharing reagents and their experience. The group acknowledges funding from the DFG (SFB 523, GRK 1188) and the EU (NoE Rubicon).
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