Small ubiquitin-related modifier (SUMO) research requires large amounts of SUMO-activating enzyme (SUMO-E1) for in vitro SUMOylation assays. In this study the expression and purification of a hexahistidine-tagged mouse Aos1-Uba2-fusion protein (His6-mAU) in a baculovirus-insect cell system revealed that approximately 1.0 mg of highly-purified His6-mAU was obtained from 100 mL of Sf9 cell culture. The recombinant protein had SUMO-E1 activity in multiple in vitro SUMOylation assays.