Fig. 1. Characterization of soluble monomeric and polymeric flagellin.

(A₁) Coomassie Blue staining of recombinant monomeric flagellin purified with Nickel affinity column. Lane 1: pre-stained SDS broad range protein marker, lane 2: band of monomeric flagellin, 5 μg (mFliC). (A₂) SDS-poyacrylamide gel electrophoresis followed by Western blot of purified mFliC, 1 μg protein: flagellin band is probed with rabbit anti-flagellin antibody and goat anti-mouse IgG HRP. (A₃)Silver stain of monomeric flagellin. (B₁) Coomassie Blue staining of purified polymeric flagellin (pFlag). Lane 1: pre-stained SDS broad range protein marker (Precision Plus, Bio-Rad, Richmond, CA), lane 2: band of pFlag after ammonium sulfate precipitation, 5 μg. (B₂) Band of pFlag, monomerized after heating at 65°C, 2 μg of protein. (B₃) Western blot of purified pFlag: flagellin band is probed with mouse anti-flagellin polyclonal antibody and goat anti-mouse IgG HRP. Lane 1: 2 μg protein, lane 2: 1 μg protein loaded (B₄) Silver stain of polymeric flagellin. (C) Electron microscopy of purified pFlag. (D) A RAW264.7 cell-based assay was used to determine the bioactivity of mFliC and pFlag. TLR5 bioactivity was expressed as shown by TNF-α production by TLR5 positive cells, subtracting that of TLR5 negative cells stimulated by the flagellins. The experiment was performed in triplicate samples in serial 10-fold dilutions per sample. EC₅₀ (concentration which produces 50% of maximal activity). To confirm that the adjuvanticity of our preparations was attributed to the activation of TLR5 receptor by flagellin and not to residual endotoxin lipopolysaccharide (LPS), we tested for LPS levels and found that they were negligible (≤0.125 EU/ml).