Ni nta bead slurry

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).

Solubilized PfFNT (35 ml from yeast expression, or 2 ml cell-free reaction) was incubated overnight with 333 μl Ni-NTA bead slurry (Qiagen) using a rotating mixer at 4°C. The slurry loaded on columns and was washed with 20 volumes of 20 mM imidazole-containing buffer of 20 mM TRIS, pH 8.0, plus 200 mM NaCl, 10% (v/v) glycerol and 0.05% Brij 78, 0.17% n-decyl-β-D-maltopyranoside (DM), 0.05% n-dodecyl-β-D-maltopyranoside (DDM), 1.2% n-octyl-β-D-glucoside (β-OG), or 0.27% LDAO as indicated. PfFNT was eluted in fractions of 5 column volumes with increasing imidazole concentrations of 80–500 mM. Fractions with the highest PfFNT content were pooled and subjected to size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare) in the purification buffer without imidazole with a constant flowrate of 0.35 ml min^-1, 1.2 MPa at 4°C.