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:: Volume 5, Issue 1 (Int J Mol Cell Med 2016) ::
Int J Mol Cell Med 2016, 5(1): 37-48 Back to browse issues page
High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication
Sobhan Faezi1, Ahmad Reza Bahrmand 2, Mehdi Mahdavi3, Seyed Davar Siadat1, Iraj Nikokar4, Soroush Sardari5, Ian Alan Holder6
1- Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran.
2- Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran. , Padideh79@yahoo.com
3- Departments of Immunology, Pasteur Institute of Iran, Tehran, Iran.
4- Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Guilan University of Medical Sciences, Guilan, Iran.
5- Biotechnology Research Center, Drug Design and Bioinformatics Group, Pasteur Institute of Iran, Tehran, Iran.
6- Departments of Microbiology and Surgery, College of Medicine, University of Cincinnati, and Shriners Burns Institute, Cincinnati, Ohio, USA.
Abstract:   (4705 Views)

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
Full-Text [PDF 373 kb]   (2249 Downloads)    
Type of Study: Original Article | Subject: Medical Pharmacology
Received: 2015/12/23 | Accepted: 2016/01/25 | Published: 2016/02/22
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Faezi S, Bahrmand A R, Mahdavi M, Siadat S D, Nikokar I, Sardari S et al . High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication. Int J Mol Cell Med. 2016; 5 (1) :37-48
URL: http://ijmcmed.org/article-1-437-en.html


Volume 5, Issue 1 (Int J Mol Cell Med 2016) Back to browse issues page
International Journal of Molecular and Cellular Medicine (IJMCM) International Journal of Molecular and Cellular Medicine (IJMCM)
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