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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 p...

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Autores principales: Faezi, Sobhan, Bahrmand, Ahmad Reza, Mahdavi, Mehdi, Siadat, Seyed Davar, Nikokar, Iraj, Sardari, Soroush, Holder, Ian Alan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Babol University of Medical Sciences 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916782/
https://www.ncbi.nlm.nih.gov/pubmed/27386437
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author Faezi, Sobhan
Bahrmand, Ahmad Reza
Mahdavi, Mehdi
Siadat, Seyed Davar
Nikokar, Iraj
Sardari, Soroush
Holder, Ian Alan
author_facet Faezi, Sobhan
Bahrmand, Ahmad Reza
Mahdavi, Mehdi
Siadat, Seyed Davar
Nikokar, Iraj
Sardari, Soroush
Holder, Ian Alan
author_sort Faezi, Sobhan
collection PubMed
description 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.
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spelling pubmed-49167822016-07-06 High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication Faezi, Sobhan Bahrmand, Ahmad Reza Mahdavi, Mehdi Siadat, Seyed Davar Nikokar, Iraj Sardari, Soroush Holder, Ian Alan Int J Mol Cell Med Original Article 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. Babol University of Medical Sciences 2016 /pmc/articles/PMC4916782/ /pubmed/27386437 Text en This is an Open Access article distributed under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Faezi, Sobhan
Bahrmand, Ahmad Reza
Mahdavi, Mehdi
Siadat, Seyed Davar
Nikokar, Iraj
Sardari, Soroush
Holder, Ian Alan
High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication
title High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication
title_full High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication
title_fullStr High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication
title_full_unstemmed High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication
title_short High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication
title_sort high yield overexpression, refolding, purification and characterization of pseudomonas aeruginosa type b-flagellin: an improved method without sonication
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916782/
https://www.ncbi.nlm.nih.gov/pubmed/27386437
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