The sequence encoding amino acid residues (aa) 384–661 of HCV genotype 1a strain H77c (GenBank accession no. AF009606), plus an N-terminal 6-histidine tag, was inserted into the pAcGP67-A transfer vector (BD Biosciences). Sf9 cells were co-transfected with the transfer vector and Baculogold™ linearized baculovirus DNA, to make recombinant baculovirus (rBac) expressing the ectodomain of E2 as a secreted, soluble protein. The rBac E2
661 was cloned by limiting dilution, amplified, and used to infect Hi Five™ cells at a multiplicity of infection (moi) of 1. After 3 days’ incubation the supernatant medium was harvested, filtered through a 0.22 µm membrane, and dialyzed first against 20 mM sodium phosphate pH 6.8, 150 mM NaCl and subsequently against 20 mM sodium phosphate pH 8.0, 300 mM NaCl. Ultrapure imidazole was added to a concentration of 20 mM, and the supernatant loaded onto a
HisTrap HP Ni Sepharose column using an
Äkta Purifier (GE Healthcare). E2
661 was eluted with a gradient of 40–200 mM imidazole in 20 mM sodium phosphate pH 8.0, 300 mM NaCl, to separate monomeric molecules from dimers and multimers. Monomeric E2
661 was purified to homogeneity by size exclusion chromatography (SEC) on a
Superdex 200 Increase 10/300 GL column (GE Healthcare).
Cowton V.M., Owsianka A.M., Fadda V., Ortega-Prieto A.M., Cole S.J., Potter J.A., Skelton J.K., Jeffrey N., Di Lorenzo C., Dorner M., Taylor G.L, & Patel A.H. (2021). Development of a structural epitope mimic: an idiotypic approach to HCV vaccine design. NPJ Vaccines, 6, 7.