high yield overexpression, refolding, purification and characterization of pseudomonas aeruginosa type b-flagellin: an improved method without sonication

pseudomonas aeruginosa as an opportunistic pathogen is a significant cause of acute and chronic infections in patients with compromised defenses. this bacterium is motile via a single polar flagellum made of polymerized flagellin subunits differentiated into two major serotypes: a and b. flagellin plays an important role as a virulence factor in the adhesion, colonization and invasion of p. aeruginosa into host epithelial cells. to develop a functional vaccine that can be used in practical application to prevent and treat infection, type b-flagellin was produced as recombinant protein. in this work, the flic gene was introduced into a pet28a vector and expressed in escherichia coli bl21 (de3). the expressed recombinant protein was purified by a modified method without sonication using a histrap affinity column. the functional activities of produced flagellin were confirmed by elisa, western blot analysis, motility inhibition assay and opsonophagocytosis test. the purification process of the type b-flagellin was lead to a high yield. the produced recombinant type b-flagellin showed high biological activity in all of these standard assays. in conclusions, this report provides the new protocol to efficiently obtain the type b-flagellin with high biological activity and immunogenicity. this immunogen can be introduced as an adjuvant or vaccine in the future study.