BG505 SOSIP.664 trimers were recombinantly expressed in HEK293T, 293F or 293S (GnTI−/−) cells, and 2G12-affinity purified13 (link)21 (link)27 (link)48 (link). The JR-FL SOSIP.664 trimers were produced in 293T cells and PGT145-affinity purified49 (link). The supernatant was flown over Sepharose resin cross-linked to 2G12 or PGT145 IgG, then washed with 20 mM Tris (pH 8), 500 mM NaCl and eluted with 3 M MgCl2. The eluted trimers were dialyzed into 20 mM Tris (pH 8), 500 mM NaCl. BG505 SOSIP.664 trimers were further purified through a HiLoad 16/600 Superdex 200 SEC column (GE Healthcare) for structural and biophysical studies.
3BC315 and 3BC176 were expressed in HEK293F cells as Fabs or IgGs, where the HC and LC genes were co-transfected at a ratio of 2:1. The IgGs were purified in a single step, through a 5-ml protein A column (GE Healthcare). Fabs were purified in three steps. First, the collected media was loaded onto a 5-ml Lambda Select column (GE Healthcare), followed by cation exchange chromatography using a Mono S column (GE Healthcare). Fractions corresponding to the proper HC–LC dimer paring were then SEC-purified through a Superdex 200 column (GE Healthcare).
3BC315 and 3BC176 were expressed in HEK293F cells as Fabs or IgGs, where the HC and LC genes were co-transfected at a ratio of 2:1. The IgGs were purified in a single step, through a 5-ml protein A column (GE Healthcare). Fabs were purified in three steps. First, the collected media was loaded onto a 5-ml Lambda Select column (GE Healthcare), followed by cation exchange chromatography using a Mono S column (GE Healthcare). Fractions corresponding to the proper HC–LC dimer paring were then SEC-purified through a Superdex 200 column (GE Healthcare).