In vitro sumoylation of recombinant proteins and subsequent purification for use in enzymatic assays

The sumoylation reaction is mechanistically similar to ubiquitination. It is ATP-dependent and in vitro can be completed in two steps using purified E1 (SAE1/SAE2), E2 (ubc9), and SUMO. Even without the inclusion of an E3 ligase, many substrates can be modified at the same lysines in vitro as in vivo. Here we describe a simplified in vitro sumoylation protocol using recombinant sumoylation substrate, E1, E2, SUMO, and an ATP-regenerating system. The modified substrate can then be repurified from the reaction mixture in a single step to be used in assays to assess the impact of sumoylation on enzymatic and/or other activities.