Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria

The objective of this study was to introduce a simple and cheap method for purification of flagellin. So, flagellin proteins of Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Citrobacter freundii (C. freundii) and Salmonella typhi (S. typhi) were purified by a modified simple method. Bacterial cultures were precipitated by centrifugation. Precipitates were washed twice and flagellin proteins were detached by shaking vigorously (in PBS pH = 2), and then flagellin proteins were precipitated by ammonium sulfate saturation. Evaluation of purification efficiency and concentration were examined by SDS-PAGE and Bradford assay. Polyclonal antibodies were produced against S. typhimurium FRC and cross-reactivity of anti-S. typhimurium was assessed against other flagellins. Bioactivity of flagellins was evaluated by cell proliferation and IL-8 protein expression assay in HEK293 cells, and also, IL-6 and TNF-alpha genes expression in chicken cells. Results showed a single band for flagellin proteins of all bacteria on %10 SDS-PAGE, which concentration ranged from 150 to 400 mu g/mL. All flagellin proteins increased cell proliferation, and IL-8 levels were increased after treatment by flagellins and levels of IL-6 and TNF-alpha were increased after treatment with S. typhimurium FRC. All flagellin proteins showed cross-reactivity with antibodies. Findings showed that application of our method, not only reduced time and cost, but also, the purified flagellin proteins had acceptable bioactivity.