Combining Protein Ligation Systems to Expand the Functionality of Semi-Synthetic Outer Membrane Vesicle Nanoparticles

H. Bart van den Berg van Saparoea, Diane Houben, Coen Kuijl, Joen Luirink, Wouter S.P. Jong*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


Bacterial outer membrane vesicles (OMVs) attract increasing interest as immunostimulatory nanoparticles for the development of vaccines and therapeutic agents. We previously engineered the autotransporter protein Hemoglobin protease (Hbp) into a surface display carrier that can be expressed to high density on the surface of Salmonella OMVs. Moreover, we implemented Tag-Catcher protein ligation technology, to obtain dense display of single heterologous antigens and nanobodies on the OMVs through coupling to the distal end of the Hbp passenger domain. Here, we aimed to further expand the versatility of the Hbp platform by enabling the coupling of heterologous proteins to internal sites of the Hbp passenger. Inserted SpyTags were shown to be accessible at the Salmonella OMV surface and to efficiently couple SpyCatcher-equipped fusion proteins. Next, we combined distally placed SnoopCatcher or SnoopTag sequences with internal SpyTags in a single Hbp molecule. This allowed the coupling of two heterologous proteins to a single Hbp carrier molecule without obvious steric hindrance effects. Since coupling occurs to Hbp that is already exposed on the OMVs, there are no limitations to the size and complexity of the partner proteins. In conclusion, we constructed a versatile modular platform for the development of bivalent recombinant OMV-based vaccines and therapeutics.

Original languageEnglish
Article number32477305
Pages (from-to)890
JournalFrontiers in Microbiology
Publication statusPublished - 12 May 2020


Part of this research was carried out in the framework of the Bac-Vactory program that was funded by the domain of Applied and Engineering Sciences (TTW) of the Netherlands Organization for Scientific Research (NWO). Part of this work was funded by an EU/Eureka Eurostars grant to project OptiFemVac (ref: 11019).

FundersFunder number
EU/Eureka Eurostars
Netherlands Organization for Scientific Research
Nederlandse Organisatie voor Wetenschappelijk Onderzoek


    • autodisplay
    • nanoparticle
    • outer membrane vesicle
    • protein display
    • protein ligation
    • Snoop
    • Spy
    • vaccine


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