Isolation by multistep chromatography improves the consistency of secreted recombinant influenza neuraminidase antigens

Capsid virus-like particle display improves recombinant influenza neuraminidase antigen stability and immunogenicity in mice

“…Tag/Catcher cVLPs have previously been shown to mediate the display of large oligomeric antigens in a high-density, unidirectional format, 35 , 47 making it an ideal system to examine if multivalent viral-like display can improve the immunogenicity of soluble rNA antigens ( Figure 2 A). To implement this system, we used a multistep chromatography procedure 48 to purify a secreted rN1 chimera with a 15-residue split-protein binding Tag on the N-terminus. The isolated rN1 was enzymatically active ( Figure 2 B), showed high purity on Coomassie stained SDS-PAGE gels ( Figure 2 C) and was recognized by several N1 specific mAbs ( Figure 2 D), indicating the rN1 was a functional tetramer and retained antigenic epitopes.…”

“…Clarified culture medium was adjusted to pH 7.5 with NaOH, passed through a 0.22 μm PES membrane and the rN1 and rN2 were purified by multistep column chromatography as previously described. 48 Briefly, rN1 was initially captured using Lentil-lectin Sepharose (1 mL/50 mL media). Beads were washed with Buffer A (30 mM Tris pH7.5, 0.5 M NaCl, 1.0 mM CaCl 2 ) and proteins were eluted in Buffer A containing 0.5 M α-methyl mannose.…”