Cloning, expression and purification and antigenicity of recombinant PilS2 protein from Pseudomonas aeruginosa
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Mojgan Arefian Gazi,1 Mehdi Ghoudarzi,2 Bahareh Hajikhani,3 Mojgan Bandehpour,4 Gholamhossein Ebrahimipour,5,*
1. Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
2. Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3. Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5. Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
- Introduction: Introduction
Pseudomonas aeruginosa is an opportunistic and gram-negative bacterium that is one of the most important causes of nosocomial infections. Although many advances have been made in the treatment of Pseudomonas aeruginosa infections, infections caused by this bacterium are still one of the leading causes of death in human societies. Various pathogens such as lipopolysaccharide, pili, alginate, flagellum, and proteases are involved in the pathogenicity of this bacterium. Type IV pili as an important virulence factor in this bacterium are divided into three subtypes that according to the available reports, subtype b of the fourth type pili (PilS2) can be a significant target in therapeutic and preventive studies and of infection by this bacteria should be considered. The aim of the present study was to design, induction, and purification of this protein in a recombinant form. The production of this recombinant protein could be the prelude to the design of an effective vaccine based on a specific antibody against the PilS2 binding region, which may be used as a practical strategy to prevent Pseudomonas infection.
- Methods: Methods
Escherichia coli (E.coli) has been used as an efficient system for expressing recombinant PilS2. The constructed recombinant pET-28a-pilS2 vector into the E.coli BL21 expression system and protein was expressed as inclusion bodies following induction with IPTG. Solubilization of inclusion bodies in urea was followed by refolding of protein in nickel affinity chromatography. The refolded histidine-tagged PilS2 protein was purified and eluted from the column using imidazole and its purity was confirmed by SDS-PAGE. Western blotting was used to investigate the antigenicity of the recombinant protein.
- Results: Results
PilS2 recombinant protein was highly expressed after induction by 100 mM IPTG at 37 °C for 3.5 h in E. coli BL21 host cells. Observation of a 17 kDa protein band on SDS-PAGE gel was an indication of protein expression. Western blotting showed that the recombinant protein was properly produced and purified and formed a single band on the nitrocellulose membrane after exposure to the antibody.
- Conclusion: Conclusion
Based on the results of this study, the recombinant PilS2 protein was produced properly. This protein can be used in future studies to study immunogenicity against Pseudomonas infections.
- Keywords: Keywords
PilS2, Pseudomonas aeruginosa, Type IV pili.
