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 PopovicEmail author
  • Radivoje Prodanovic
  • Raluca Ostafe
  • Stefan Schillberg
  • Rainer Fischer
  • Marija Gavrovic-Jankulovic


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.


Actinidin Allergen characterization Cysteine protease Kiwifruit Yeast surface display 


Act d 1



Component-resolved diagnosis


Component-resolved immunotherapy






Yeast nitrogen base/casamino acids


Yeast extract/peptone/dextrose








Coomassie brilliant blue


Optical density


Integrated optical density


Cross-reactive carbohydrate determinants



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.


  1. 1.
    Valenta R, Kraft D. Recombinant allergens: from production and characterization to diagnosis, treatment, and prevention of allergy. Methods. 2004;32(3):207–8.CrossRefPubMedGoogle Scholar
  2. 2.
    Valenta R, Niespodziana K, Focke-Tejkl M, Marth K, Huber H, Neubauer A, et al. Recombinant allergens: What does the future hold? J Allergy Clin Immunol. 2011;127(4):860–4.CrossRefPubMedGoogle Scholar
  3. 3.
    Valenta R, Lidholm J, Niederberger V, Hayek B, Kraft D, Gronlund H. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT). Clin Exp Allergy. 1999;29(7):896–904.CrossRefPubMedGoogle Scholar
  4. 4.
    Kleber-Janke T, Crameri R, Appenzeller U, Schlaak M, Becker WM. Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology. Int Arch Allergy Immunol. 1999;119(4):265–74.CrossRefPubMedGoogle Scholar
  5. 5.
    Rhyner C, Weichel M, Flückiger S, Hemmann S, Kleber-Janke T, Crameri R. Cloning allergens via phage display. Methods. 2004;32(3):212–8.CrossRefPubMedGoogle Scholar
  6. 6.
    Graumann K, Premstaller A. Manufacturing of recombinant therapeutic proteins in microbial systems. Biotechnol J. 2006;1(2):164–86.CrossRefPubMedGoogle Scholar
  7. 7.
    Bowley DR, Labrijn AF, Zwick MB, Burton DR. Antigen selection from an HIV-1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage. Protein Eng Des Sel. 2007;20(2):81–90.CrossRefPubMedGoogle Scholar
  8. 8.
    Pepper LR, Cho YK, Boder ET, Shusta EV. A decade of yeast surface display technology: Where are we now? Comb Chem High Throughput Screen. 2008;11(2):127–34.CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Bidlingmaier S, Liu B. Construction of yeast surface-displayed cDNA libraries. In: Lu C, Browse J, Wallis JG, editors. cDNA libraries: methods in molecular biology. New York: Humana Press; 2011. p. 199–210.CrossRefGoogle Scholar
  10. 10.
    Antier D, Vantyghem C, Triaud F, Le Verger M, Grassin J. Costs related to drugs produced by recombinant DNA technology. Am J Health Syst Pharm. 2000;57(1):78–9.PubMedGoogle Scholar
  11. 11.
    Kondo A, Ueda M. Yeast cell-surface display—applications of molecular display. Appl Microbiol Biotechnol. 2004;64(1):28–40.CrossRefPubMedGoogle Scholar
  12. 12.
    Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997;15(6):553–7.CrossRefPubMedGoogle Scholar
  13. 13.
    Boder ET, Wittrup KD. Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol. 2000;328:430–44.CrossRefPubMedGoogle Scholar
  14. 14.
    Grozdanovic M, Popovic M, Polovic N, Burazer L, Vuckovic O, Atanaskovic-Markovic M, et al. Evaluation of IgE reactivity of active and thermally inactivated actinidin, a biomarker of kiwifruit allergy. Food Chem Toxicol. 2012;50(3–4):1013–8.CrossRefPubMedGoogle Scholar
  15. 15.
    Pastorello EA, Ispano M, Pravettoni V, Farioli L, Incorvaia C, Ansaloni R, et al. Cross-reacting allergens in clinical syndromes. Monogr Allergy. 1996;32:57–62.PubMedGoogle Scholar
  16. 16.
    Shewry P. Tuber storage proteins. Ann Bot. 2003;91(7):755–69.CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Praekelt UM, McKee RA, Smith H. Molecular analysis of actinidin, the cysteine proteinase of Actinidia chinensis. Plant Mol Biol. 1988;10(3):193–202.CrossRefPubMedGoogle Scholar
  18. 18.
    Pastorello EA, Conti A, Pravettoni V, Farioli L, Rivolta F, Ansaloni R, et al. Identification of actinidin as the major allergen of kiwi fruit. J Allergy Clin Immunol. 1998;101(4):531–7.CrossRefPubMedGoogle Scholar
  19. 19.
    Palacin A, Rodriguez J, Blanco C, Lopez-Torrejon G, Sanchez-Monge R, Varela J, et al. Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms. Clin Exp Allergy. 2008;38(7):1220–8.CrossRefPubMedGoogle Scholar
  20. 20.
    Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol. 2004;114(5):1169–75.CrossRefPubMedGoogle Scholar
  21. 21.
    Bublin M, Pfister M, Radauer C, Oberhuber C, Bulley S, Dewitt AM, et al. Component-resolved diagnosis of kiwifruit allergy with purified natural and recombinant kiwifruit allergens. J Allergy Clin Immunol. 2010;125(3):687–94 94 e1.CrossRefPubMedGoogle Scholar
  22. 22.
    Taylor RG, Walker DC, Mclnnes RR. E.coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res. 1993;21(7):1677–8.CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Harboe N, Ingild A. Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol Suppl. 1973;1:161–4.CrossRefPubMedGoogle Scholar
  24. 24.
    Dreidi R. Cloning of actinidin from green kiwifruit into pQE9 vector for protein expression. Belgrade: Faculty of Chemistry; 2010.Google Scholar
  25. 25.
    Gietz RD, Woods R. Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method. In: Xiao W, editor. Yeast protocol: methods in molecular biology. New York: Humana Press; 2006. p. 107–20.Google Scholar
  26. 26.
    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–5.CrossRefPubMedGoogle Scholar
  27. 27.
    Shevchenko A. Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry. Anal Biochem. 2001;296(2):279–83.CrossRefPubMedGoogle Scholar
  28. 28.
    Gattiker A, Bienvenut WV, Bairoch A, Gasteiger E. FindPept, a tool to identify unmatched masses in peptide mass fingerprinting protein identification. Proteomics. 2002;2(10):1435–44.CrossRefPubMedGoogle Scholar
  29. 29.
    Baker PR, Clauser KR,
  30. 30.
    Sorensen HP. Towards universal systems for recombinant gene expression. Microb Cell Fact. 2010;9:27.CrossRefPubMedCentralPubMedGoogle Scholar
  31. 31.
    Ceriotti A, Duranti M, Bollini R. Effects of N-glycosylation on the folding and structure of plant proteins. J Exp Bot. 1998;49(324):1091–103.CrossRefGoogle Scholar
  32. 32.
    van der Veen MJ, van Ree R, Aalberse RC, Akkerdaas J, Koppelman SJ, Jansen HM, et al. Poor biologic activity of cross-reactive IgE directed to carbohydrate determinants of glycoproteins. J Allergy Clin Immunol. 1997;100(3):327–34.CrossRefPubMedGoogle Scholar
  33. 33.
    van Ree R. Clinical importance of cross-reactivity in food allergy. Curr Opin Allergy Clin Immunol. 2004;4(3):235–40.CrossRefPubMedGoogle Scholar
  34. 34.
    Altmann F. The role of protein glycosylation in allergy. Int Arch Allergy Immunol. 2007;142(2):99–115.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Milica Popovic
    • 1
    Email author
  • 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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