Expression and purification of the PRMT5-MEP50 complex

Full-length human PRMT5 (GenBank accession number NM_001039619.2) and MEP50 (GenBank accession number NM_024102.2) were cloned into the baculovirus transfer vector pFB-LIC-Bse. Baculovirus expression was performed as previously described (59). After coexpression of PRMT5 and MEP50, the Sf9 cells were harvested and the pellets were stored at −80°C until use. Frozen insect cell pellets of PRMT5-MEP50 were resuspended in lysis buffer [50 mM Hepes (pH 7.5), 250 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM tris(2-carboxyethyl)phosphine–HCl (TCEP), 0.1% Triton X-100, 1× protease inhibitors (Roche)]. The cells were lysed by sonication for 5 min and clarified by centrifugation. The supernatant was applied to 2 ml of nickel beads (Qiagen) and incubated for 2 hours. Nickel beads were washed three times with wash buffer [50 mM Hepes (pH 7.5), 250 mM NaCl, 10% glycerol, 30 mM imidazole, 0.5 mM TCEP] and eluted in 5 ml of elution buffer [50 mM Hepes (pH 7.5), 250 mM NaCl, 10% glycerol, 250 mM imidazole, 0.5 mM TCEP]. The eluted protein was run on SDS-PAGE and applied to a HiPrep 26/60 Sephacryl S-300 column that had been equilibrated with gel filtration buffer [25 mM Hepes (pH 7.5), 150 mM NaCl, 10% glycerol, 2 mM DTT]. The fractions containing PRMT5-MEP50 complex were pooled, concentrated, and frozen at −80°C.