Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens
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Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens Milica Popovic • Radivoje Prodanovic • Raluca Ostafe • Stefan Schillberg • Rainer Fischer Marija Gavrovic-Jankulovic
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Published online: 24 December 2014 Ó Springer Science+Business Media New York 2014
Abstract High-throughput characterization of allergens relies often on phage display technique which is subject to the limitations of a prokaryotic expression system. Substituting the phage display platform with a yeast surface display could lead to fast immunological characterization of allergens with complex structures. Our objective was to evaluate the potential of yeast surface display for characterization of plant-derived food allergens. The coding sequence of mature actinidin (Act d 1) was cloned into pCTCON2 surface display vector. Flow cytometry was used to confirm localization of recombinant Act d 1 on the surface of yeast cells using rabbit polyclonal antisera IgG and IgE from sera of kiwifruit-allergic individuals. Immunological (dot blot, immunoblot ELISA and ELISA inhibition), biochemical (enzymatic activity in gel) and biological (basophil activation) characterization of Act d 1 after solubilization from the yeast cell confirmed that recombinant Act d 1 produced on the surface of yeast cell is similar to its natural counterpart isolated from green kiwifruit. Yeast surface display is a potent technique that enables fast immunochemical characterization of allergens in situ without the need for protein purification and offers
an alternative that could lead to improvement of standard immunodiagnostic and immunotherapeutic approaches. Keywords Actinidin Allergen characterization Cysteine protease Kiwifruit Yeast surface display Abbreviations Act d 1 Actinidin CRD Component-resolved diagnosis CRIT Component-resolved immunotherapy GPI Glycosylphosphatidylinositol PE Phycoerythrin YNB-CAA Yeast nitrogen base/casamino acids YPD Yeast extract/peptone/dextrose Glc Glucose Raf Raffinose Gal Galactose CBB Coomassie brilliant blue OD Optical density IOD Integrated optical density CCDs Cross-reactive carbohydrate determinants
Introduction M. Popovic (&) R. Prodanovic M. Gavrovic-Jankulovic Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia e-mail: [email protected] R. Ostafe R. Fischer Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany S. Schillberg R. Fischer Department of Plant Biotechnology, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany
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Recombinant DNA technology facilitated major developments in the field of molecular allergology [1], helping to improve component-resolved diagnosis (CRD) and component-resolved immunotherapy (CRIT) [2]. Using recombinant allergens for in vitro diagnostic tests allows establishment of a patients’ individual IgE reactivity profile and patient-tailored desensitization immunotherapy [3]. Cloning recombinant
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