Production and Purification of SUMO-UBC9 and SUMO-RANGAP1^CTD

SUMOylation is a post-translational modification catalyzed by a multi-step enzymatic cascade. To gain structural biology insights into the last step of this process, where SUMO is transferred from a SUMO~UBC9 molecule onto a substrate, stable complexes with SUMO covalently linked to UBC9, the substrate, or both are essential. Here, building on previously published approaches and our experience, we describe detailed protocols for the generation of a simple stable mimetic of human SUMO~UBC9, as well as a stably SUMOylated version of a model substrate, the C-terminal domain of RANGAP1. Both of these molecules can be used to reconstitute noncovalent complexes with a fragment of a SUMO E3 ligase for biophysical and structural analysis.