Purification of p19 Protein Constructs

The PFO-based endosome-disrupting agent (C225.2/PFO^T490A,L491V) was prepared as previously described (17 (link)). The p19, p19-E6, p19-E18 clones and SUMO-E18 were expressed from the pE-SUMO vector (LifeSensors) in Rosetta 2 (DE3) Escherichia coli (Novagen) and purified by Talon metal affinity chromatography (Clontech) following previously described methods (17 (link)). Following cleavage of the SUMO tag, the p19 constructs were purified by anion exchange chromatography (AEX) and size-exclusion chromatography (SEC). AEX was performed using a HiTrap Q HP anion exchange column (GE Healthcare Life Sciences) with an increasing salt gradient (10–500 mM NaCl) in 20 mM Bis–Tris, pH 6.5. SEC was performed using a HiLoad 16/600 Superdex 75 pg column (GE Healthcare Life Sciences) in PBS. Analytical SEC was performed using a Superdex 75 10/300 GL column (GE Healthcare Life Sciences) or Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) in PBS. Detailed methods for the expression and purification of p19 are provided in Supplementary Methods.