Figure 1.

Characterization of Fc-based recombinant gp350 proteins. (A) Diagrams for design of Fc-based recombinant gp350 proteins. The extracellular domains (D-I, D-II, and D-III), transmembrane domains, and cytoplasmic tail (TD/CT) are shown in gray. The 6-histidine tag is shown as a hatched box, the mouse IgG2a Fc domain is shown orange, and the glycine–serine (G₄S)₃ linker is colored green. (B) Western blot of secreted recombinant gp350 constructs under reducing (+β-ME) and non-reducing conditions (−β-ME). gp350-ECD₁₂₃-6His was detected by an anti-His tag antibody, and proteins fused with Fc domain were detected with an anti-mouse Fc antibody. For each fusion protein, the same volume (15 µl) of supernatants from identical passage 1 baculovirus stocks was loaded onto the gels under reducing and non-reducing conditions. Samples were resolved on 10% SDS-PAGE. (C) Detection of the indicated recombinant gp350 forms by conformation-dependent anti-gp350 mAb72A1 antibody in the supernatant of bacmid-transfected Sf9 cells using dot blot. Cell culture supernatants from mock-transfected cells were included as negative controls. (D) SDS-PAGE analysis of SEC-purified forms of recombinant gp350. Proteins from the peak fractions were resolved on 10% gel under reducing condition and stained with Coomassie brilliant blue.