Blasticidin resistance gene

The constructs expressing JRFL-C3d and 426c-ΔGly3-C3d fusion proteins with different linkers were transiently transfected into suspension 293F cells as previously described (55 (link)). Env proteins were harvested 4 days post transfection and purified by lectin affinity chromatography (Galanthus nivalis, Vector Labs) followed by size exclusion chromatography (SEC) on a Superdex 200 16/60 or Superdex 200 10/300 GL (GE Healthcare) columns. The trimer peak was subjected to negative selection by the non-neutralizing mAb F105 to remove disordered trimers (31 (link)). The flow-through from the F105 column, containing the well-ordered trimers, was resolved by a second SEC step.
C3d-Fd proteins were produced in stably transfected Drosophila S2 cells as previously described (53 (link)). Briefly, S2 cells were cotransfected with C3d-Fd and selection vector pCoBlast, which contains the blasticidin resistance gene (Invitrogen). After blasticidin selection, stable S2 transfectants were generated that were used for protein production. Briefly, when S2 cells reached 10 M/ml in 1 L of Express Five SFM medium without FBS, 5 μM CdCl₂ was added into culture medium to induce protein expression; 3 days after the induction, the culture supernatants were harvested, clarified by centrifugation and filtration. Filtered supernatants were purified by Ni-NTA column followed by SEC.