SUMOylation is a dynamic and reversible post-translational modification that is widely present in eukaryotes and plays crucial biological roles.SUMO proteases are responsible for two key functions:deSUMOylation of substrate proteins(isopeptidase activity)and maturation of precursor SUMO molecules(endopeptidase activity),which are indispensable for SUMOylation cycle and the functional regulation of substrate proteins.Recently,a novel SUMO protease belonging to the Desi family has been identified in both mice and humans.Through homologous sequence analysis,eight members of the Desi family have been identified in Arabidopsis thaliana.Among these,only Desi3A has been functionally characterized,with demonstrated isopeptidase activity directly related its functions.However,whether Desi3A possesses endopeptidase activity and the enzymatice properties of the remaining seven Desi proteins remain unclear.Investigating the functional roles of Desi proteins through enzymatic activity analysis represents a promising research strategy.Therefore,we selected Desi3A as a representative candidate.The full-length CDS of Desi3A was cloned into the pGEX-6P-1 prokaryotic expression vector,and subsequently analyzed using an in vitro reaction system after induction,expression and purification.CBB staining and Western blotting assays confirmed the successful expression of GST-Desi3A in BL21(DE3)cells.However,the purified GST-Desi3A failed to cleave preSUMO1-EBS after incubation for 1 h,3 h,or 5 h,whereas the positive control GST-ESD4 could almost completely cleaved preSUMO1-EBS within 1 h.These results suggest that Desi3A may lack endopeptidase activity toward precursor SUMO molecules,or its enzymatic activity is very low and requires extended incubation time or optimized conditions for detection.This study provides a methodological reference for the prokaryotic expression and purification of Desi family proteins and lays the groundwork for further investigation into their enzymatic activities and biological functions.