Superdex200 prep grade 16 600 column

A human codon optimized HCV immunogen was inserted into the donor vector between the cytomegalovirus promoter and bovine growth hormone poly‐A sequence by In‐Fusion cloning to generate the shuttle vector. The shuttle vector was cloned into the ChAdOx1 destination vector, using ThermoFisher’s LR gateway cloning method, and linearized using PmeI restriction and transfected into T‐REx‐293 cells (ThermoFisherScientific) to generate ChAdOx1 vaccines. For MVA vaccines, the HCV immunogen was cloned at the F11 locus of the pMVA‐shuttle vector and the recombinant MVA generated by recombining the pMVA‐shuttle vector and WT MVA. HCV viral vector vaccines were manufactured by the Viral Vector Core Facility (Jenner Institute, University of Oxford, Oxford, UK).
Recombinant proteins were expressed using the Freestyle 293‐F cells stable transfected cell clone, purified from the culture supernatants using cobalt‐charged TALON metal affinity resin (Clontech) by the C‐terminal polyhistidine tag followed by size‐exclusion chromatography on a Superdex200 prep grade 16/600 column (GE Healthcare), using the ÄKTA pure fast protein liquid chromatography system (GE Healthcare) equilibrated in PBS (pH 6.8). Fractions containing the HMW species were pooled and concentrated through an Amicon® 30‐kDa molecular‐weight cut‐off ultracentrifugal device (Merck).

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).