Recombinant Expression and Purification of USP2a

Human USP2a (residues 258-605) was expressed in the Escherichia coli BL21 (DE3, Invitrogen). Cells were grown in LB medium containing 100 μg/ml ampicillin at 37 °C and induced with 0.5 mM IPTG at OD₆₀₀ of 0.7-0.9 and cultured for additional 5 h at 37 °C. Cells were harvested by centrifugation and frozen at -20 °C. USP2a purification was carried out according to optimized protocol (Renatus et al., 2006 (link)). In brief, cells from 6 liters culture were resuspended in 300 ml of lysis buffer (10 mM Tris/HCl pH=8.0, 1 mM MgCl₂, 5 mM β-mercapthoetanol, 10 μM PMSF) and ruptured by sonication. After centrifugation supernatant was loaded on a Chelating Sepharose Fast Flow (GE Healthcare) charged with nickel ions. The column was washed with lysis buffer and protein was eluted with lysis buffer supplemented with 250 mM imidazole. Fractions containing USP2a were combined and further purified on Q-Sepharose Fast Flow (GE Healthcare) column. USP2 protein was in the flow-through fraction.
The last purification step consisted of size exclusion chromatography on HiLoad 16/60 Superdex 75 prep grade (GE Healthcare) in PBS pH=7.4 containing 5 mM DTT. USP2a was stored for further experiments as 0.01 mM protein stock with 10% glycerol at -80 °C.