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

• DOI: 10.1128/aem.02567-17
• Publication Date: 2018-02-09T14:59:41Z
• Authors: H. Bart van den B...; Diane Houben; Marien I. de Jonge; Wouter S. P. Jong; Joen Luirink
• Keywords: Radboudumc 4: lnfectious Diseases and Global Health RIMLS: Radboud Institute for Molecular Life Sciences; Salmonella typhimurium; 0301 basic medicine; Outer membrane vesicle; Autodisplay; Membrane Proteins; Recombinant Proteins; 3. Good health; 03 medical and health sciences; Laboratory Medicine - Radboud University Medical Center; Bacterial Proteins; Protein ligation; Nanobody; Escherichia coli; Antigen display; Paediatrics - Radboud University Medical Center; SpyTag; Vaccine; Biotechnology

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. and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences genetically fused the Hbp core structure for optimal exposure immune system. Although platform is relatively tolerant, increasing number, size, complexity of integrated generally lowers expression constructs limits display. This due intricate mechanism secretion across efficient quality control translocation-incompetent chimeric molecules in periplasm. To address this shortcoming, we explored coupling purified proteins carrier after its translocation using recently developed SpyTag/SpyCatcher protein ligation As expected, fusion small SpyTag did not hamper OMVs. Subsequent addition SpyCatcher domain resulted covalent Hbp-SpyTag. Using orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could coupled sequential strategy. Not only antigens proved suitable Spy-mediated but also nanobodies. Addition functionality might allow targeting bacterial OMV vaccines certain tissues tailor responses.IMPORTANCE Outer (OMVs) from Gram-negative bacteria attract interest development therapeutic agents. We aim construct semisynthetic recombinant presentation OMVs attenuated displaying an adapted autotransporter, Hbp, at surface. autotransporter accepts substantial modifications, capacity with respect structural restricted. Here describe application SpyCatcher/SpyTag technology enzymatically link present Protein was apparently unobstructed by environment allowed antigens, property have shown important vaccine efficacy. procedure appears versatile robust, allowing fast production experimental agents through modular plug-and-display procedure.