Ni conjugated agarose bead column

The plasmids pET32a harboring PTD-RBD-VIF or other chimeric genes were transformed into E. Coli BL21 competent cells (Novagen) respectively. After the expression of proteins was induced by 1 mM isopropylthio-β-d-galactoside, the bacterial cells were lysed by sonication. The insoluble fraction was pelleted at 10,000 × g for 10 min, and the supernatant was applied to a Ni-conjugated agarose bead column (GE). After washing, the bound His fusion proteins were eluted with 500 μM Imidazole. The proteins were then suspended in PBS buffer and the concentration was measured by the Bradford method. For the in vivo tumor model experiments, the His-tag was removed first. And then the no-tagged PTD-RBD-VIF proteins was pruified by a heparin-agarose chromatography and subsequently ion exchange [51 (link)]. The proteins were then suspended in PBS buffer and the concentration was measured by the Bradford method. The purities of expressed proteins were > 95%. Moreover, the possible endotoxin had been removed from the recombinant protein with Triton X-114 as described previously and the residual endotoxin was examined with limulus amoebocyte lysate (LAL) assay (Cambrex Bio Science) by following the instruction of manufacturer [52 (link)]. The samples were then aliquoted and frozen at −80°C.

The plasmid harboring 6×His-tagged RBD was transfected into HEK293F cells using polyetherimide. Supernatants were collected at 48 h p.t. and filtered through a 0.45 μm pore-size filter. The supernatants were subsequently applied to a Ni-conjugated agarose bead column (GE Healthcare, Buckinghamshire, UK). After washing with 30 mM Imidazole (Sangon Biotech, Shanghai, China), the bound His fusion proteins were eluted with 500 mM Imidazole. Finally, the proteins were suspended in PBS buffer and the concentration was measured by the Bradford method. The purified RBD was incubated with purified exosomes at 4 °C for 4 h, followed by enriched with a Ni-conjugated agarose bead column. The bound protein was further washed with PBS buffer containing 0.25% NP40 and 50 mM NaCl for three times and eluted in protein gel loading buffer. The eluted samples were then analyzed by SDS-PAGE and detected by western blot assay.

The plasmid pET32a harboring His-tagged Nef or Nef ^E63A/F68A genes were transformed into E. coli BL21(DE3) competent cells (Novagen), respectively. After the expression of proteins was induced by 1 mM isopropylthio-b-D-galactoside, the bacterial cells were lysed by sonication. The insoluble fraction was pelleted at 12,000 × g for 15 min, and the supernatant was applied to a Ni-conjugated agarose bead column (GE). After washing, the bound His fusion proteins were eluted with 500 mM imidazole. Then the proteins were suspended in PBS buffer and the concentration was measured by the Bradford method. The samples were then aliquoted and frozen at −80°C.