High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication

Document Type : Original Article

Authors
Sobhan Faezi 1
Ahmad Reza Bahrmand 1
Mehdi Mahdavi 2
Seyed Davar Siadat 1
Iraj Nikokar 3
Soroush Sardari 4
Ian Alan Holder 5
1 Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran
2 Departments of Immunology, Pasteur Institute of Iran, Tehran, Iran
3 Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Guilan University of Medical Sciences, Guilan, Iran
4 Biotechnology Research Center, Drug Design and Bioinformatics Group, Pasteur Institute of Iran, Tehran, Iran
5 Departments of Microbiology and Surgery, College of Medicine, University of Cincinnati, and Shriners Burns Institute, Cincinnati, Ohio, USA
Abstract
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.
Keywords
Pseudomonas aeruginosa
flagellin protein
pET28a
purification
vaccine