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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification
Published on: November 16, 2016
Expression, purification, and refolding of biologically active Acinetobacter baumannii OmpA from Escherichia coli
Michael J McConnell1, Jerónimo Pachón
1Unit of Infectious Disease, Microbiology, and Preventive Medicine, Institute of Biomedicine of Sevilla, University Hospital Virgen del Rocío/CSIC/University of Sevilla, 41013 Sevilla, Spain. mcconnell.mike75@gmail.com
Abstract:
Infections caused by Acinetobacter baumannii have emerged as a significant clinical problem due to the increase in infections caused by antibiotic resistant strains. A. baumannii OmpA is a highly conserved membrane protein that has multiple roles in interacting with the host during infection, and thus represents an attractive target for the development of novel antibacterial therapies. In the present study, the coding sequence of the mature form of A. baumannii OmpA was cloned into the vector pET-15b and purified under denaturing conditions from Escherichia coli inclusion bodies using nickel affinity chromatography. A Triton X-114 wash step was incorporated into the purification method in order to remove endotoxin, resulting in endotoxin levels of <1.3 EU/mg of protein. A protocol was developed for refolding the purified protein by dilution into the non-ionic detergent n-octyl-β-D-glucopyranoside followed by dialysis to remove excess denaturant and detergent. Cytotoxicity assays demonstrated that refolded A. baumannii OmpA was able to induce cell death in A549 cells. In addition, a polyclonal antibody was raised against the refolded protein and used to assess extracellular secretion of OmpA by Western blot. This protein expression and purification system may be useful for further characterization of A. baumannii OmpA.
Insights
Researchers developed a method to purify and refold Acinetobacter baumannii outer membrane protein A (OmpA). This purified OmpA induced cell death in cancer cells, suggesting potential for new antibacterial therapies against resistant infections.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Acinetobacter baumannii infections pose a significant clinical challenge due to rising antibiotic resistance.
- The A. baumannii outer membrane protein A (OmpA) is crucial for host interaction during infection and a potential therapeutic target.
Purpose of the Study:
- To develop a robust system for expressing, purifying, and refolding A. baumannii OmpA.
- To assess the biological activity and potential therapeutic utility of the refolded OmpA.
Main Methods:
- Cloning and expression of mature A. baumannii OmpA in E. coli.
- Purification using nickel affinity chromatography with a Triton X-114 wash to remove endotoxins.
- Protein refolding via dilution into n-octyl-β-D-glucopyranoside followed by dialysis.
- Cytotoxicity assays on A549 cells and Western blot analysis for OmpA secretion.
Main Results:
- Successfully purified endotoxin-free A. baumannii OmpA.
- Refolded OmpA demonstrated cytotoxicity, inducing cell death in A549 cells.
- A polyclonal antibody was generated for OmpA detection, confirming its extracellular secretion.
Conclusions:
- Established a viable system for producing functional A. baumannii OmpA.
- The refolded OmpA exhibits cytotoxic properties, highlighting its potential in developing novel antibacterial strategies.
- This system facilitates further investigation into OmpA's role in A. baumannii pathogenesis and therapeutic applications.
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