Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation
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
DOI:
10.1128/aem.02567-17
Publication Date:
2018-02-09T14:59:41Z
AUTHORS (5)
ABSTRACT
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.
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