Protein purification and gel filtration chromatography

The human SART3 HAT domains (residues 1–691) and DUSP-UBL domain of USP15 (residues 1–226) were cloned into pET-28a (Novagen) to generate N-terminal histidine-tagged proteins. The N-domain (residues 1–333) and C-domain (residues 85–499) of human PRP31 were cloned into pET-32a (Novagen) as N-terminal histidine-tagged thioredoxin fusion proteins. All proteins were expressed in Escherichia coli Rosetta (DE3) cells with induction by 0.5 mM isopropyl-β-d-thiogalactoside at 18°C for 18 h. Proteins in 50 mM Tris–HCl (pH 8.0) and 150 mM NaCl buffer were purified with His-Trap affinity column (GE Healthcare) eluted with 25–500 mM imidazole. Affinity tags were removed with thrombin, and the resultant proteins were then further purified with a Superdex 200 26/60 gel filtration column (GE Healthcare) which had been pre-equilibrated with 50 mM Tris–HCl (pH 8.0) and 150 mM NaCl.

For the separation of protein complexes, cell lysates overexpressed with HA-SART3, Myc-USP4 and Flag-USP15 were co-fractionated by gel filtration with a Superdex 200 10/300 column (GE Healthcare) in a buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl and 1 mM EDTA. Molecular weight markers (Sigma-Aldrich) with a range of 66 000–443 000 Da were used for size calibration. Eluted fractions (0.5 ml) were collected and aliquots were subject to the SDS-PAGE and detected by western blotting.