Related Experiment Video
Updated: Jun 22, 2026
Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly
Published on: February 9, 2015
Cloning, expression, purification, and characterization of recombinant flagellin isolated from Pseudomonas aeruginosa
Gholamreza Goudarzi1, Morteza Sattari, Mehryar Habibi Roudkenar
1Department of Bacteriology, Medical Sciences School, Tarbiat Modares University, Tehran, Iran.
Abstract:
Pseudomonas aeruginosa as an opportunistic pathogen causes lethal infections in immunocompromised individuals. This bacterium possesses a polar flagellum made up of flagellin subunits. Flagella have important roles in motility, chemotaxis, and establishment of P. aeruginosa in acute phase of infections. Isolation, cloning, and expression of flagellin were aimed at in this study. Flagellin gene (fliC) of P. aeruginosa strain 8821M was isolated by PCR and cloned into a pET expression vector. The recombinant flagellin (46 kDa) was overexpressed as inclusion bodies (IBs). IBs were solubilized in guanidine hydrochloride (GuHCl) followed by affinity-purification and renatured using Ni(2+)-Sepharose resin. Recombinant flagellins reacted with the serum from a rabbit previously immunized with native flagellin. In addition, polyclonal antiserum raised against the recombinant flagellin was shown to significantly inhibit the cell motility of P. aeruginosa strain 8821M in vitro.
Insights
Researchers isolated and expressed Pseudomonas aeruginosa flagellin, a key component of its motility structure. The resulting recombinant flagellin successfully elicited an immune response and inhibited bacterial movement.
Area of Science:
- Microbiology and Immunology
- Bacterial Pathogenesis
- Protein Expression and Purification
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections, particularly in immunocompromised individuals.
- The bacterium utilizes a polar flagellum, composed of flagellin subunits, for critical functions like motility and chemotaxis, aiding in infection establishment.
Purpose of the Study:
- To isolate, clone, and express the flagellin gene (fliC) from P. aeruginosa strain 8821M.
- To characterize the recombinant flagellin and assess its immunogenicity and functional impact on bacterial motility.
Main Methods:
- Isolation of the flagellin gene (fliC) using Polymerase Chain Reaction (PCR).
- Cloning into a pET expression vector for overexpression as inclusion bodies (IBs).
- Solubilization of IBs, affinity purification, and renaturation of recombinant flagellin.
Main Results:
- Successfully overexpressed recombinant flagellin (46 kDa) as inclusion bodies.
- Purified recombinant flagellin reacted with antiserum against native flagellin.
- Polyclonal antiserum against recombinant flagellin significantly inhibited P. aeruginosa motility in vitro.
Conclusions:
- The study successfully produced and purified recombinant P. aeruginosa flagellin.
- The recombinant flagellin is immunogenic and can be used to generate specific antibodies.
- Antibodies against recombinant flagellin effectively inhibit bacterial motility, suggesting potential therapeutic applications.
More Related Videos
11:31Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa
Published on: June 30, 2016
22:10Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit
Published on: June 28, 2013