Preparation of flagellin

Pst DC3000 was grown on King’s B agar plate containing 100 μg/mL rifampicin at 28 °C, then inoculated into King’s B liquid medium containing rifampicin and incubated overnight at 28 °C with shaking at 150 rpm. A portion of this culture was inoculated into ≈40 volumes of fresh King’s B liquid medium containing rifampicin and incubated overnight at 28 °C with shaking at 150 rpm. The bacterial pellet was collected by centrifugation at 3000 × g for 10 min and washed three times with 20 mM Tris-HCl (pH 8.0). To shear the flagella, the cell suspension was intensively blended for 2 min at 8000 rpm using a Polytron PT 10/35 homogenizer, and then centrifuged at 5000 × g for 10 min to pellet the cells³¹. The resulting supernatant was centrifuged at 100,000 × g for 1 h to pellet the flagella, and the resulting pellet then was suspended in 0.1 M glycine-HCl buffer (pH 2.0) for dissociation³². The flagellin protein was separated by a second round of centrifugation at 100,000 × g for 1 h, and the pH of the resulting supernatant (flagellin solution) was adjusted to 7.0 with NaOH.