Expression and purification of recombinant proteins
His-tagged proteins were expressed in E. coli strain Rosetta2(DE3). Cells were cultivated in LB medium at 37oC to an optical density between 0.6 and 0.8. After induction with 1 mM of isopropyl-β-D-thiogalactopyranosid (IPTG) the cells were grown for additional 3 hours at 30 oC. Collected cells were resuspended in washing buffer (20 mM HEPES, 300 mM NaCl, 40 mM imidazole, 2 mM DTT, pH 8.0). Cells were disrupted by sonification and after centrifugation, the supernatant was either loaded onto a nickel-nitrilotriacetic acid column (GE Healthcare) or incubated with nickel-nitrilotriacetic acid resin (Qiagen). After washing with washing buffer the fusion constructs were eluted with elution buffer (20 mM HEPES, 300 mM NaCl, 250 mM imidazole, 2 mM DTT, pH 8.0). Subsequently, the imidazole was removed from the buffer using PD-10 columns (GE Healthcare). The protein was kept on ice and used directly or was stored at -20°C until usage.
GST-Get3b was expressed as described above with the following modifications. Collected bacterial cells were resuspended in 1x PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) before sonication. Proteins were purified using glutathione-sepharose (GE Healthcare) and eluted with 10 mM reduced glutathione in 1xPBS buffer. The buffer was exchanged to 1xPBS using PD-10 columns (GE Healthcare).