Easy and efficient purification of flagellin from salmonella typhimurium atcc 14028 without necessity of ultracentrifugation
Mohammad-hosein Khani kangarshahi
,1,* Masoumeh bagheri
,2 Fattah sotoodehnejad nematalahi
1. Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran
2. Department of genomics and engineering genetics, Razi Vaccine and Serum Research Institute, Agricultural Research, Educa
3. Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran
Gram negative bacteria's flagellin has many applications in diagnosis, treatment and vaccines. flic, the major protein of flagellin, is an agonist of tlr5 that can activate innate immune system. in this way, flagellin may be a potential vaccine adjuvant by immune system induction. isolation and purification of native flagellin had been challenged by low efficacy and ultracentrifugation (<50000 xg) requirement in previous reports. since ultracentrifuge is a prerequisite to isolate native flic from gram negative bacteria, this study was conducted to eliminate ultracentrifugation step because of its limitation in some labs.
Salmonella typhimurium atcc 14028 was cultured in broth medium and precipitated only by after 18 hours. by dissolving bacterial pellets in pbs (ph =2), bacterial flagellin was depolymerized in monomeric units. then, bacterial residuals were separated (in pellet form) a common centrifugation 10000 xg and then the supernatant containing monomeric flic was treated with saturated ammonium sulphate solution in order to aggregate and concentrate the flic monomers. salt residual was eliminated by dialyzing (ph= 7.4). in order to evaluate protein purity, a sds-page was conducted and using bradford and lowry assays, protein concentration was determined.
Sds-page results showed a single band in 50 kd without any extra band impurity. the protein concentration was 1 mg/ml in ph= 7.4 in most of experiments.
By this method, flagellin of salmonella typhimurium can be extracted and purified without any need to ultracentrifugation. on the other hand, saving time and cost can be the other advantages over previous methods.
Flagellin, flic, salmonella typhimurium, ultracentrifuge