Recombinant Env expression.

One milligram of plasmid DNA per 1 liter of cells was diluted in Dulbecco modified Eagle medium (DMEM) and mixed with polyethyleneimine (PEI). PEI-DNA mixtures were added to cells for 4 h. 293F (Invitrogen) cells were subsequently washed and diluted to 1.25 million cells/ml in Freestyle293 medium (Invitrogen). Five days after transfection, the cell culture medium was centrifuged and filtered through a 0.8-μm-pore-size filter to eliminate cells. The cell culture was concentrated with a 10-kDa-molecular-mass-cutoff Vivaflow 50. The concentrated cell culture supernatant was rotated with Galanthus nivalis lectin beads (Vistar Labs) overnight at 4°C. The beads were pelleted by centrifugation the next day and washed twice with morpholineethanesulfonic acid (MES) buffer. The protein was eluted with methyl-α-pyranoside, buffer exchanged into PBS, and stored at −80°C.