PA strain PAO1 was injected into 2000 mL of tryptic soy broth medium (Sigma-Aldrich, St. Louis, MO) and incubated overnight at 37°C in a shaking incubator (New Brunswick Scientific, Edison, NJ) to a stationary phase (10
12 CFU/mL). Bacteria were collected by centrifugation at 7000
g at 4°C for 20 minutes and resuspended in 50 mM sodium phosphate buffer (pH 7.0). The bacterial suspension was blended to remove the flagellin from the cells. The homogenate was centrifuged at 12,000
g at 4°C for 20 minutes to remove the cells from the supernatant. The supernatant was then mixed with ammonium sulfate in 5% increments. The differentially saturated supernatants were centrifuged at 12,000
g at 4°C for 30 minutes to remove the insoluble materials from solution. The 15% and 20% insoluble fractions that contained the greatest amount of flagellin with the least amount of contaminants, were dissolved in 50 mM sodium phosphate buffer (pH 8.0) and dialyzed against the same buffer.
28 The flagellin-containing sample was applied to diethylaminoethyl (DEAE)-Sephadex A-50 (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The column was washed with 10× 50 mM sodium phosphate buffer (pH 8.0) and then eluted with 6× 0.6 mM NaCl and 50 mM sodium phosphate buffer (pH 8.0). The protein-containing fractions were collected, concentrated on filters (Amicon Centriplus YM-3; Millipore, Bedford, MA),
14 and applied to 1 mL prepacked gel affinity columns (Detoxi-Gel Affinity Pak; Pierce, Rockford, IL). The amount of LPS was determined with a quantitative limulus amebocyte lysate kit (QCL-1000; BioWhittaker, Walkersville, MD). The amount of LPS in the flagellin samples after the two steps of chromatography was 2.7 endotoxin units (EU)/mg protein, comparable to that in the LPS-free flagellin preparation reported by Rudner et al.
29 Identity of flagellin was confirmed with immunoblot analysis with rabbit anti-PA flagellum B antiserum, kindly provided by Dan Wozniak (Wake Forest University, Winston-Salem, NC).