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Immunologic Research

, Volume 61, Issue 3, pp 230–239 | Cite as

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
Article

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

Notes

Acknowledgments

The authors thank Dr. Dane Wittrup (MIT, USA) for the surface display vector and yeast surface display strain and Dr. Milica Grozdanovic (Faculty of Chemistry, University of Belgrade) for the rabbit polyclonal antisera against natural Act d 1. This work was supported by Grant 172049 from the Ministry of Education, Science and Technological Development of the Republic of Serbia and a FEBS Collaborative and Experimental Scholarship for Central and Eastern Europe.

Conflicts of interest

The authors do not have any conflict of interest to declare.

Ethical standard

The study did not require the approval of the ethics committee.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Milica Popovic
    • 1
  • Radivoje Prodanovic
    • 1
  • Raluca Ostafe
    • 2
  • Stefan Schillberg
    • 3
  • Rainer Fischer
    • 2
    • 3
  • Marija Gavrovic-Jankulovic
    • 1
  1. 1.Department of Biochemistry, Faculty of ChemistryUniversity of BelgradeBelgradeSerbia
  2. 2.Institute for Molecular BiotechnologyRWTH Aachen UniversityAachenGermany
  3. 3.Department of Plant BiotechnologyFraunhofer Institute for Molecular Biology and Applied Ecology (IME)AachenGermany

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