Isolation and Purification of Flagellin Proteins

The flagellin proteins were isolated as described previously [9 (link)] with a few modifications. In brief, S. Enteritidis stock culture was inoculated into Brain Heart Infusion Broth (Sigma-Aldrich, MO) and grown for 48 hours at 37°C without shaking. The bacterium was washed two times with PBS (pH 7.4) by centrifugation at 4,000 ×g for 40 minutes. The bacterial cell pellet was treated with 3M Potassium Thiocyanate in PBS for 2 hours at room temperature with magnetic stirring. The cell suspension was ultracentrifuged at 35,000 ×g for 30 minutes. The supernatant containing the flagellin protein-enriched extract was dialyzed against PBS (pH 7.4) followed by dialysis in Milli-Q water. The protein concentration was assessed and freeze-dried using 5% sucrose as a cryoprotectant.