The crystallized HIV-1-Env construct from strain BG505 was generated following published reports10 (link),15 (link),16 (link), using BG505 genbank accession numbers ABA61516 and DQ20845846 (link); including the “SOS” mutations (A501C, T605C), the isoleucine to proline mutation at residue 559 (I559P), and the glycan site at residue 332 (T332N); mutating the cleavage site to 6R (REKR to RRRRRR); and truncating the C terminus to residue 664 (all HIV-1 Env numbering according to the HX nomenclature). This construct is referred to as BG505 SOSIP.664 throughout this entire manuscript.
The BG505 SOSIP.664 construct was co-transfected with furin in HEK 293 GnTI−/− cells using 600 μg of BG505 SOSIP.664 and 150 μg of furin plasmid DNAs as described previously16 (link). Transfection supernatants were harvested after 7 days, and passed over either a 2G12 antibody- or VRC01 antibody-affinity column. After washing with phosphate-buffered saline (PBS), bound proteins were eluted with 3M MgCl2, 10 mM Tris pH 8.0. The eluate was concentrated to less than 5 ml with Centricon-70 and applied to a Superdex 200 column, equilibrated in 5 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% azide. The peak corresponding to trimeric HIV-1 Env was identified, pooled, concentrated and used immediately or flash-frozen in liquid nitrogen and stored at −80° C.
The BG505 SOSIP.664 construct was co-transfected with furin in HEK 293 GnTI−/− cells using 600 μg of BG505 SOSIP.664 and 150 μg of furin plasmid DNAs as described previously16 (link). Transfection supernatants were harvested after 7 days, and passed over either a 2G12 antibody- or VRC01 antibody-affinity column. After washing with phosphate-buffered saline (PBS), bound proteins were eluted with 3M MgCl2, 10 mM Tris pH 8.0. The eluate was concentrated to less than 5 ml with Centricon-70 and applied to a Superdex 200 column, equilibrated in 5 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% azide. The peak corresponding to trimeric HIV-1 Env was identified, pooled, concentrated and used immediately or flash-frozen in liquid nitrogen and stored at −80° C.
