SUMO Protease is derived from Saccharomyces cerevisiae and recombinantly expressed in Escherichia coli. It recognizes the tertiary structure of the ubiquitin-like SUMO domain and cleaves the peptide bond immediately after the Gly-Gly motif in the X‑Gly‑Gly‑X sequence at the C‑terminus of the SUMO domain. In addition to naturally SUMO-modified proteins, this enzyme is also used for cleaving recombinant SUMO fusion proteins.

The optimal pH for SUMO Protease cleavage is 8.0, and the optimal temperature is 30 °C. It maintains high enzymatic activity over a pH range of 6.0–10.0, a temperature range of 2–30 °C, and an ionic strength range of 0–400 mM NaCl. Furthermore, its cleavage activity is enhanced in the presence of 0.5–2 mM DTT as a reducing agent. Adding an appropriate concentration of DTT to the digestion system significantly improves cleavage efficiency, especially during prolonged digestion, such as overnight incubation at 4 °C.