Recombinant antibody Fabs were transiently expressed in FreeStyleTM 293F (Invitrogen) suspension cultures by co-transfection of pVRC8400 plasmids containing expression constructs for light chain and Fab heavy chain using polyethyleneimine (Polysciences). Cell growth was harvested on day 6 post transfection. eOD-GT6 was also produced in FreeStyleTM 293F (Invitrogen) suspension cultures by transient transfection using polyethyleneimine (Polysciences) of a pHLSec plasmid containing mammalian codon-optimized eOD-GT6 with a C-terminal Avi and His6x affinity tag. Proteins were harvested from the supernatant after 96 h.
The secreted proteins were purified by using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) nickel affinity chromatography followed by size exclusion chromatography (SEC) using a Superdex 200 26/600 (Fabs) or Superdex 75 26/600 (eOD) column (GE Healthcare) in 10 mM Tris pH 8.0, 150 mM NaCl SEC buffer. Peak fractions containing Fabs or eOD-GT6 were pooled. Protein purity was analyzed by SDS-PAGE and concentrated where possible to ~10 mg/mL. BG505-SOSIP was requested from the Vaccine Research Center at the National Institute of Health (47 (link)).
The secreted proteins were purified by using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) nickel affinity chromatography followed by size exclusion chromatography (SEC) using a Superdex 200 26/600 (Fabs) or Superdex 75 26/600 (eOD) column (GE Healthcare) in 10 mM Tris pH 8.0, 150 mM NaCl SEC buffer. Peak fractions containing Fabs or eOD-GT6 were pooled. Protein purity was analyzed by SDS-PAGE and concentrated where possible to ~10 mg/mL. BG505-SOSIP was requested from the Vaccine Research Center at the National Institute of Health (47 (link)).
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