Soluble E2 proteins were produced via transient transfection of Freestyle 293-F cells. Plasmids pE2RBD, pE2RBDA7, pE2Δ123, and pE2Δ123A7 were transfected using 293Fectin (Invitrogen) according to the manufacturer’s instructions. Soluble glycoproteins secreted into the culture supernatants were affinity purified using cobalt-charged TALON metal affinity resin (Clontech) and then subjected to size exclusion chromatography on a Superdex 200 prep-grade 16/600 column (GE Healthcare) using the ÄKTA pure fast protein liquid chromatography (FPLC) system (GE Healthcare) equilibrated in PBS (pH 6.8). Monomeric species, as previously defined (19 (link)), were pooled and concentrated through a centrifugal filter with a nominal molecular weight cutoff (30 kDa; Amicon).
Anti-S MAb 7C12 was kindly provided by ARTES Biotechnology (Langenfeld, Germany). Synthetic MAbs specific to epitopes located within E2 (HCV1, AR3C, 2A12, and HC84.27) were expressed and purified from 293-F cells as previously described (53 (link)). E2 MAb24 and MAb44 were produced from mouse hybridoma cell lines as previously described (17 (link)). E2 MAb AR1A was kindly provided by Mansun Law (Scripps Research, CA).
Anti-S MAb 7C12 was kindly provided by ARTES Biotechnology (Langenfeld, Germany). Synthetic MAbs specific to epitopes located within E2 (HCV1, AR3C, 2A12, and HC84.27) were expressed and purified from 293-F cells as previously described (53 (link)). E2 MAb24 and MAb44 were produced from mouse hybridoma cell lines as previously described (17 (link)). E2 MAb AR1A was kindly provided by Mansun Law (Scripps Research, CA).
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