Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Effective Platform for the Production of Recombinant Outer Membrane Vesicles in Gram-Negative Bacteria

  • Received : 2020.03.13
  • Accepted : 2020.05.28
  • Published : 2022.05.28
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Abstract

Bacterial outer membrane vesicles (OMVs) typically contain multiple immunogenic molecules that include antigenic proteins, making them good candidates for vaccine development. In animal models, vaccination with OMVs has been shown to confer protective immune responses against many bacterial diseases. It is possible to genetically introduce heterologous protein antigens to the bacterial host that can then be produced and relocated to reside within the OMVs by means of the host secretion mechanisms. Accordingly, in this study we sought to develop a novel platform for recombinant OMV (rOMV) production in the widely used bacterial expression host species, Escherichia coli. Three different lipoprotein signal peptides including their Lol signals and tether sequences-from Neisseria meningitidis fHbp, Leptospira interrogans LipL32, and Campylobactor jejuni JlpA-were combined upstream to the GFPmut2 model protein, resulting in three recombinant plasmids. Pilot expression studies showed that the fusion between fHbp and GFPmut2 was the only promising construct; therefore, we used this construct for large-scale expression. After inducing recombinant protein expression, the nanovesicles were harvested from cell-free culture media by ultrafiltration and ultracentrifugation. Transmission electron microscopy demonstrated that the obtained rOMVs were closed, circular single-membrane particles, 20-200 nm in size. Western blotting confirmed the presence of GFPmut2 in the isolated vesicles. Collectively, although this is a non-optimized, proof-of-concept study, it demonstrates the feasibility of this platform in directing target proteins into the vesicles for OMV-based vaccine development.

Keywords

Acknowledgement

The authors gratefully acknowledge the financial support provided by Thammasat University Research Fund under the TU Research Scholar, Contract no. 2/45/2560. We also greatly thank Asst. Prof. Veerachai Thitapakorn for providing administrative assistance.

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