Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens
- 388 Downloads
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.
KeywordsActinidin Allergen characterization Cysteine protease Kiwifruit Yeast surface display
- Act d 1
Yeast nitrogen base/casamino acids
Coomassie brilliant blue
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.
The study did not require the approval of the ethics committee.
- 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
- 24.Dreidi R. Cloning of actinidin from green kiwifruit into pQE9 vector for protein expression. Belgrade: Faculty of Chemistry; 2010.Google Scholar
- 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
- 29.Baker PR, Clauser KR, http://prospector.ucsf.edu.