Binding of SGTA to Rpn13 selectively modulates protein quality control

Leznicki, Pawel; Korac-Prlic, Jelena; Kliza, Katarzyna; Husnjak, Koraljka; Nyathi, Yvonne; Dikic, Ivan; High, Stephen

Escherichia coli strain BL21 transformed with the pGEX-4T1 plasmid encoding a GST-tagged protein of interest was grown to OD600=0.5; expression was induced with 0.5 mM IPTG followed by overnight incubation at 16°C. Cells were harvested, lysed by sonication in lysis buffer [20 mM Tris-HCl pH 7.5, 10 mM EDTA, 5 mM EGTA, 150 mM NaCl, 0.5% (v/v) Triton X-100] and insoluble material pelleted by centrifugation (20 min, 4°C, 10,000×g). Pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) were added to the supernatant, incubated for 1 h at 4°C and extensively washed with the lysis buffer. Bound GST fusion proteins were either stored on the resin at 4°C or released by using thrombin (GE Healthcare) at 24°C in 1× cleavage buffer (20 mM Tris-HCl, pH 8.4, 150 mM NaCl, 2.5 mM CaCl2, 1 mM DTT) overnight. Beads were then pelleted by centrifugation and thrombin was inactivated with PMSF. The interaction between purified components was followed by incubating one Glutathione-Sepharose-bound protein with a potential binding partner that had been released from its GST tag through thrombin cleavage. The His-tagged Trx-SGTA fusion protein and a K160E/R164E mutant version were expressed and purified as previously described (Leznicki et al., 2011). Binding reactions were performed in 1× incubation buffer [20 mM Tris-HCl pH 8.4, 150 mM NaCl, 2.5 mM CaCl2, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mM DTT] for 4 h at 4°C, beads washed extensively with incubation buffer, SDS sample buffer added and samples resolved by SDS-PAGE followed by western blotting.