Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles Using Protein Ligation.

5 years ago

Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles Using Protein Ligation.

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

TheEscherichia colivirulence factor Hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on liveE. coliandSalmonellaTyphimurium bacteria 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. Adding 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.IMPORTANCEOuter membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semi-synthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuatedSalmonellaTyphimurium bacteria displaying an adaptedEscherichia coliautotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

Publisher URL: http://doi.org/10.1128/AEM.02567-17

DOI: 10.1128/AEM.02567-17