Fig. 1

(A) Schematic diagram of adenovirus Fiber-Knob (FK)-based vaccine construct. The construct includes the knob part of the protein, the final (22nd ) shaft repeat of the adenoviral fiber and a 6-His tag at the N-terminal required for protein purification. (B)E. coli-expressed Ad5-FK (~ 22 KDa) on denaturing SDS-PAGE: FK protein was efficiently produced as a soluble protein with molecular weight of approximately 22 KDa. The purification process of the protein involved several steps including two consecutive IMAC steps using Ni-NTA columns (lanes 2–4 and 6), an intermediate step of ammonium sulfate precipitation (lane 5) and a final purification step using ion exchange chromatography (lane 7). (C) Native and denatured Ad5-FK on Coomassie Blue-stained SDS-PAGE gel. Purified FK without β-mercaptoethanol treatment (WO) was found to self-assemble into a trimer form of ~ 55 KDa, while no trimerization was detected in the β-mercaptoethanol-treated sample (W). (D) Western blot of native and denatured Ad5-FK using anti-His tag antibody. The trimerization pattern in the FK sample not treated with β-mercaptoethanol (WO), as observed on SDS-PAGE, was also confirmed by Western blotting