SIV Env SOSIP.664 Trimer Purification

SOSIP.664 Env trimer modifications were incorporated into the SIV_mac239 Env coding sequence, as described previously (32 (link)). Amino acid substitutions were incorporated by using a QuikChange site-directed mutagenesis kit (Agilent Technologies, USA), according to the manufacturer’s instructions. All the mutations were confirmed by DNA sequence analysis (Eton Bioscience, San Diego, CA). Soluble recombinant Env trimers were expressed in HEK293F cells as described elsewhere (32 (link)). Briefly, plasmids encoding the SIV_mac239 SOSIP.664 trimer and its V2 variants were cotransfected with a furin expression construct into HEK293F cells at a 3:1 ratio using PEI-MAX 4000 transfection reagent (Polysciences, Inc.). The secreted trimer proteins were purified from cell supernatants after 5 days using agarose-bound Galanthus nivalis lectin (GNL) (Vector Labs) columns as described previously (32 (link)). Affinity-purified proteins were separated by size exclusion chromatography using Superdex 200 10/300 GL columns (GE Healthcare) in phosphate-buffered saline (PBS). The purified trimers were stored at −80°C until use.