Ti 90

Harvested cell culture supernatants were applied to a 10% sucrose cushion in TAN buffer pH 8.0 (50 mM triethanolamine, 10 mM NaCl). Ultracentrifugation was carried out for 2 h at 50,000 rpm at 4 °C (Beckmann Ti 90) as described previously [26 (link),40 (link)]. The resulting pellets containing the viral particles were resuspended in TAN buffer pH 8.0, supplemented with 0.1% bovine serum albumin. Quantification of E by a four-layer ELISA was carried out as described above.

The HIV-1 CANC protein was purified as previously described [58 (link),61 (link),63 (link)]. Briefly, the HIV-1 CANC protein was expressed in Escherichia coli (E.coli) BL21 (DE3) and following cell lysis, polyethyleneimine to a final concentration of 0.15% (w/v) was added to the cell lysate and the nucleic acids were removed by ultracentrifugation (Beckman (Brea, CA, USA), TI 90, 55,000 rpm, 3 h, 4 °C). The HIV-1 CANC protein was purified by a combination of ion-exchange chromatography using HiPrep™SP FF 16/10 column (GE Healthcare, Chicago, IL, USA) and gel-filtration chromatography using HiLoad™26/600 Superdex™ column. The HIV-1 CANC protein was concentrated to 4 mg/mL and stored at −80 °C.