Preparation of SUMO proteases and kinetic analysis using endogenous substrates.

SUMO proteases catalyze two reactions, deconjugation of SUMO from substrates and processing of precursor SUMO isoforms to prepare SUMO for conjugation. The SUMO protease family includes two members in yeast (Ulp1 and Ulp2) and as many as six members in human (SENP1-3, SENP5-7). SENP/Ulp proteases each contain conserved C-terminal domains that catalyze protease activity. The C-terminal protease domains exhibit unique specificities during SUMO processing and deconjugation in vitro. While there are many available reagents to assess these activities, including fusion proteins and chemically modified SUMO isoforms, our studies have indicated that the composition of substrates C-terminal to the scissile bond can substantively influence the activity of the protease. As such, we have relied extensively on assays that utilize endogenous substrates, namely wild-type SUMO precursors and SUMO conjugated substrates. In this chapter, we will describe methodological details for purification and characterization of SUMO precursors, SUMO conjugated substrates, and SUMO proteases. We will also describe methods for kinetic analysis of SUMO deconjugation and maturation using endogenous substrates.