Isolation and purification of cross-linked and native HCMV gB with fusion inhibitor

Minor variations on the following protocol were used to prepare lots of prefusion HCMV gB used for structure determination. Human foreskin fibroblast cells in roller bottle culture in Dulbecco’s modified Eagle’s medium with 15% heat-inactivated fetal bovine serum were inoculated with HCMV strain Towne at a multiplicity of infection of 0.01 (based on titration in MRC-5 cells) and incubated at 37°C for 12 days, when nearly complete cytopathic effect was observed. The remaining attached cells were scraped from the roller bottles, and the cell culture medium was harvested, frozen and thawed, sonicated, and clarified by low-speed centrifugation. WAY-174865 (18, 19) was added to 1 mg/liter before the harvest was concentrated by tangential flow filtration with a 300-kDa cutoff membrane, and the fusion inhibitor was maintained at this concentration or higher throughout all subsequent steps. The concentrated virus was frozen, thawed, and pelleted twice through 15% sucrose in phosphate-buffered saline (PBS) onto 40% iodixanol cushions. The concentrated virus was cross-linked with 0.2 mM bis(sulfosuccinimidyl) glutarate-d0 (Thermo Fisher Scientific) at room temperature for 1 hour before quenching with 5 mM tris. The purified and cross-linked virus was lysed with 2% n-dodecyl β-d-maltoside (DDM) in the presence of WAY-174865 (2 mg/liter). The solubilized material was clarified by high-speed centrifugation, concentrated by ultrafiltration with an Ultracel-15 Amicon 100-kDa membrane, and incubated overnight at 4°C with a FAb of mAb SM5-1 (21) that had been fused at the C terminus to a histidine tag and a Strep-tag (LakePharma). The FAb-bound protein was purified by affinity chromatography with a HiTrap TALON cobalt column (GE Healthcare) with imidazole elution and addition of EDTA to 10 mM following elution. After concentration with a 100-kDa ultrafiltration membrane, FAb-bound gB was further purified by size exclusion chromatography with a Superose 6 10/300 GL (GE Healthcare) column in PBS (pH 7.4), 0.1% DDM, and WAY-174865 (2 mg/liter). Pooled fractions were concentrated by ultrafiltration as before, and the FAb-bound gB was further purified by affinity chromatography with a StrepTrap HP Purification column (GE Healthcare) in the same buffer, with elution by 2.5 mM desthiobiotin. The purified gB-FAb complex was concentrated by ultrafiltration as above and dialyzed against PBS, 0.1% DDM, 1 mM EDTA, and WAY-174865 (2 mg/liter) before being used for cryo-EM grid preparations.