Chelating sepharose fast flow resin

Cell pellets from E. coli cultures overproducing Ycf54 proteins were re-suspended in binding buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 10 mM imidazole) containing EDTA-free protease inhibitor tablets (Roche), and the cells disrupted on ice by sonication. The lysate was clarified by centrifugation at 40 000×g for 30 min at 4°C. The soluble fraction was applied to Chelating Sepharose FastFlow resin (GE Healthcare) equilibrated with NiSO₄. The column was washed first with binding buffer, then a 50 mM imidazole wash, followed by a 100 mM imidazole wash and eluted with 500 mM imidazole. The eluted proteins were buffer exchanged into PBS and the N-terminal His₆-tag was removed by cleaving with Thrombin 80 U ml⁻¹ (GE Healthcare) at room temperature overnight. The sample was re-applied and washed through the Chelating Sepharose resin and the cleaved protein was buffer exchanged into 100 mM NaHCO₃, pH 8.3, 50 mM NaCl buffer and concentrated to 10 mg ml⁻¹ for crystallisation.
FLAG pulldown experiments were performed on solubilised thylakoid membranes prepared from cells harvested from 8L cell culture as described in [19 (link)].

Transformed E. coli BL21(DE3) cells were grown in Overnight Express autoinducing medium (Formedium) containing 100 μg/mL ampicillin for 16 h at 37 °C. Cells were harvested by centrifugation at 4000× g for 20 min and lysed using B-PER lysis reagent (Thermo Fisher Scientific, Frederick, MD, USA)). Soluble material was clarified by centrifugation of the lysate at 12,500× g for 20 min at 4 °C, followed by 0.22 μm filtration. The filtrate was applied to Q-Sepharose (Sigma Aldrich Ltd., Dublin, Ireland)) resin equilibrated with 50 mM Tris-HCl pH 7.0 as a negative ion-exchange step to remove E. coli proteins. The unbound fraction was applied to Chelating Sepharose Fast Flow resin (GE Healthcare Bio-Science AB, Uppsala, Sweden) pre-charged with 50 mM NiSO₄ and equilibrated with 150 mM NaCl and 50 mM Tris-HCl, pH 7.9. Following five column volume washes with this buffer containing 20 mM imidazole, proteins were eluted with 0.5 M imidazole and buffer was exchanged into either PBS or DMEM for further analysis. Prior to cell infection experiments, endotoxins were removed using Endotoxin removal columns (Thermo Fisher Scientific, Frederick, MD, USA) according to the manufacturer’s instructions.