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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification
Published on: November 16, 2016
[Yersinia pseudotuberculosis mutant OmpF porins with deletions of the external loops: genetic constructions design,
Abstract:
Yersinia pseudotuberculosis outer membrane (OM) recombinant mutant OmpF porins with deletions of the external loops L1, L6 and L8 were obtained using site-directed mutagenesis of the recombinant plasmid including ompF gene. Heterologeous expression of the mutant proteins was carried out in strain Rosetta of Escherichia coli (Novagen, USA), porins with the deletions were isolated from the inclusion bodies. Mutant proteins in oligomeric form were obtained as result of dialysis and ion-exchange chromatography. Spatial structure of the mutant proteins was demonstrated to have special features in comparison with that of the full-structured OmpF porin on the level of both secondary and tertiary structure. Lacking of the loops L1, L6 and L8 didn't affect the conductivity level of Y pseudotuberculosis porin channel as shown using bilayer lipid membrane (BLM) technique. Lacking of the loops mentioned above has a significant influence on the antigenic structure of the mutant porins as demonstrated with use of immunoblotting technique and ELISA.
Insights
Mutant Yersinia pseudotuberculosis OmpF porins lacking external loops L1, L6, and L8 maintained channel conductivity but altered antigenic structure. These findings impact understanding of porin function and immune response.
Area of Science:
- Microbiology
- Structural Biology
- Immunology
Background:
- Yersinia pseudotuberculosis outer membrane (OM) proteins, specifically porins like OmpF, play crucial roles in nutrient transport and bacterial virulence.
- Understanding the structure-function relationship of OmpF porins is essential for developing targeted antimicrobial strategies.
Purpose of the Study:
- To investigate the structural and functional consequences of deleting external loops (L1, L6, L8) in Yersinia pseudotuberculosis OmpF porin.
- To assess the impact of these deletions on porin channel conductivity and antigenic properties.
Main Methods:
- Site-directed mutagenesis was used to create recombinant OmpF porins with specific loop deletions.
- Heterologous expression in Escherichia coli, followed by isolation from inclusion bodies.
- Purification using dialysis and ion-exchange chromatography.
- Structural analysis of secondary and tertiary structures.
- Channel conductivity was measured using the bilayer lipid membrane (BLM) technique.
- Antigenic structure was evaluated using immunoblotting and ELISA.
Main Results:
- Mutant OmpF porins with deletions in loops L1, L6, and L8 were successfully produced and purified.
- Structural analysis revealed distinct secondary and tertiary structures in mutant porins compared to the wild-type.
- Deletion of loops L1, L6, and L8 did not significantly alter the conductivity of the Yersinia pseudotuberculosis porin channel.
- Significant changes in the antigenic structure of the mutant porins were observed.
Conclusions:
- External loops L1, L6, and L8 are not critical for maintaining the ion conductivity of Yersinia pseudotuberculosis OmpF porin.
- These loops play a significant role in determining the antigenic profile of the OmpF porin.
- Modifications to these loops could potentially be exploited for diagnostic or therapeutic purposes targeting Yersinia pseudotuberculosis.
