GFP-β-DGNLS was purified from bacteria as His6-tagged proteins using nickel affinity chromatography under native conditions. Briefly, BL21 (DE3) bacteria were transformed to express the fusion protein, grown to OD600 = 0.6 and induced overnight with 1 mM IPTG. The bacteria was centrifuged at 14,000 rpm and the pellets resuspended in His-lysis buffer (8 M Urea, 0.1 M NaH2PO4, 0.01 M Tris and 1 M NaCl) containing 3 mg/ml lysozyme and lysed on ice for 30 min in the presence of 1 unit/ml DNase1 and Complete EDTA-free protease inhibitors (Roche Applied Science). The lysate was centrifuged at 11,000 x g for 45 min at 4°C and the soluble fraction incubated with 4 ml of pre-equilibrated Ni-NTA bead slurry (Qiagen) for 1 h at 4°C. The beads were washed and the protein was eluted in His-Lysis buffer containing 200 mM imidazole, followed by dialysis against PBS. The protein was further purified by gel filtration using a HiPrep 26/60 Sephacryl S-200 high resolution column attached to an ÄKTA Purifier system (GE Healthcare) and concentrated using Amicon centrifugal concentration devices (Millipore Corporation, Billerica, MA, USA). Protein concentration was estimated by absorbance measurement at 280 nm and the theoretical molar extinction coefficient.
GST-ezrin (mouse), GST-IMPα, GST-IMPβ and GST alone were purified from bacteria under native conditions, as described previously [21] (link).
GST-ezrin (mouse), GST-IMPα, GST-IMPβ and GST alone were purified from bacteria under native conditions, as described previously [21] (link).