Kifunensine

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.

Using the NovaCHOice transfection kit (Merck Millipore), the recombinant MUC2-C expression vector was transiently transfected into CHO-S cells growing in 800 ml flasks and FreeStyle CHO growth medium (Gibco) without serum. The extracellular recombinantly expressed protein was collected 72 h after the transfection by centrifuging the cell media at 500 × g and 6000 × g and discarding the cell pellet. To obtain the high-mannose recombinant MUC2-C the expression was done in presence of 1 μg/ml kifunensine (Toronto Research Chemicals).
The recombinant protein was purified from the supernatant by Immobilized metal affinity chromatography (IMAC) method using Ni-Sepharose® Excel (Cytiva) in a loaded gravity column (BioRad). The loaded resin was washed with 300 mM NaCl, 20 mM HEPES pH 7.4 and 40 mM imidazole, and the protein was eluted with 300 mM NaCl, 20 mM HEPES pH 7.4 and 300 mM imidazole. For the high-mannose MUC2-C the elution was dialyzed over-night against 150 mM NaCl, 20 mM MES, pH 5.5, and treated with EndoH (Roche) glycosidase at 4 °C. The products were then purified by Size Exclusion Chromatography (SEC) using an Äkta HPLC system (GE Healthcare) with a HiLoad 16/600 Superdex 200 column (GE Healthcare) in 150 mM NaCl, 20 mM HEPES, pH 7.4.