Display of recombinant proteins on bacterial outer membrane vesicles by using protein ligation

H. Bart van den Berg van Saparoea, Diane Houben, Marien I. de Jonge, Wouter S.P. Jong, Joen Luirink*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

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The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/ SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.

Original languageEnglish
Article numbere02567-17
Pages (from-to)1-17
Number of pages17
JournalApplied and Environmental Microbiology
Issue number8
Early online date2 Apr 2018
Publication statusPublished - Apr 2018


  • Antigen display
  • Autodisplay
  • Nanobody
  • Outer membrane vesicle
  • Protein ligation
  • SpyTag
  • Vaccine


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