Ff chelating sepharose resin

The N43Q mutant construct was transiently expressed in HEK 293T cells in the presence of 5 μM of the N-glycosylation inhibitor kifunensine (Toronto Research Chemicals, North York, ON, Canada). The cell media, containing the secreted HCV E1, was harvested 4 days after transfection after centrifugation at 5,000 g at 15 °C for 20 min to remove debris. The media was filtered and incubated with Ni²⁺-charged resin (FF Chelating Sepharose resin, GE Healthcare) in a shaking incubator for 90 min at 15 °C. The resin was separated from the media, washed with 15 mM Tris-HCl pH 8.0, 0.1 M NaCl, 20 mM imidazole and the bound protein was eluted with the same buffer but containing 250 mM imidazole. To remove the C-terminal His-tag and to deglycosylate the protein, the eluted protein was incubated overnight at 22 °C with recombinant TEV protease and endoglycosidase F1 produced inhouse. The supernatant was filtered and the protein purified by size-exclusion chromatography in 20 mM Tris-HCl pH 7.0 and 0.1 M NaCl on a Superdex 75 column (GE Healthcare). Fractions containing pure protein (nE1) were collected and 3-(1-Pyridino)-1-propane sulphonate (NDSB 201, Soltec Ventures Inc.) was added to a final concentration of 300 mM, to enable the protein to be concentrated to between 17 and 22 mg ml⁻¹.

DNA coding for the ectodomain of HCV E1 (residues 1–79) was synthesized with a mutation at one of the glycosylation sites (N43Q) and was cloned into the pHLsec vector (Aricescu et al., 2006 ▶ ). The construct containing a C-terminal His₆ tag (Fig. 1 ▶a) was transiently expressed in HEK293T cells in the presence of 5 µM kifunensine to limit N-glycosylation of the remaining sites (Toronto Research Chemicals, North York, Ontario, Canada). Ni²⁺-affinity purification (FF Chelating Sepharose resin, GE Healthcare) was followed by TEV protease and endoglycosidase F1 treatment before size-exclusion chromatography on a Superdex 75 column (GE Healthcare). The protein was estimated to be greater than 95% pure by SDS–PAGE (Fig. 1 ▶b). 3-(1-Pyridino)-1-propanesulfonate (NDSB 201; Soltec Ventures Inc.) was added to HCV nE1 to a final concentration of 300 mM in order to reach concentrations of between 17 and 22 mg ml⁻¹.