Optimized HCMV gB Protein Expression

The postfusion gB plasmid encodes residues 32–692 of HCMV gB (strain AD169) with an artificial N-terminal signal sequence and a C-terminal HRV3C protease cleavage site, 8×HisTag, and a TwinStrep tag. The plasmid was transiently transfected into FreeStyle 293-F cells (Gibco) using polyethylenimine for expression. To enhance solubility, we combined published successful engineering strategies of gB expression and introduced the following substitutions: Y155G, I156H, Y157R, Y206H, S238N, W240T, L241T, Y242H and C246S [17 (link), 46 (link)]. Cells transfected with the gB construct were treated with 5 μM kifunensin three hours post-transfection to ensure uniform high-mannose glycosylation. Cell supernatants were harvested six days after transfection. The postfusion gB protein was purified using Strep-Tactin resin (IBA Lifesciences) before being run over a Superose6 10/300 column (GE Healthcare) in 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN₃.