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Clinical & Experimental Allergy

High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris

Authors


Dr Alain Jacquet, Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Rue des Professeurs Jeener et Brachet, 12, B-6041 Gosselies, Belgium. E-mail: ajacquet@sga.ulb.ac.be

Summary

Background The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success.

Objective The aim of this study was to establish an efficient system for the production of recombinant Der p 1.

Methods The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the α mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity.

Results ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40–60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1.

Conclusion This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.

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