Steffen Ernst

Recominant Expression and Immunological Characterization of House Dust Mite Allergen Der P 1

The cysteine protease Der p1 from dust mite of the genus Dermatophagoides pteronyssinus is a major type I allergen. About 80% of house dust mite (HDM) allergic individuals are reactive to this protease in standard assays for detection of IgE. A curative treatment for atopic allergy is immunotherapy (IT) with HDM extracts which are complex mixtures occasionally resulting in anaphylactic reactions. Novozymes focuses on developing a recombinant variant of Der p1 which exhibit lowered risk of IgE‐mediated allergic reactions, while maintaining its ability to trigger proper Th‐cell responses. This may provide a safer alternative for specific IT of HDM allergy. A secreted recombinant form of pro‐Der p 1 expressed by Saccharamyces cerevisiae was obtained by fusion of the pro‐enzyme to a fungal signal peptide. The N‐glycosylation site of Der p1 was mutated resulting in a deglycosylated pro‐enzyme with a molecular mass of 35 kDa. Protein purification procedure was developed to obtain nearly pure Der p1 protein followed by determination of concentration by active‐site‐titration with the cysteine protease inhibitor E64. The deglycosylated recombinant pro‐Der p 1 revealed immunologic similarity to the native Der p 1 molecule when compared in basophile histamine release, IgE‐binding assays and T‐cell proliferation assays. By in silico epitope mapping of a modelled 3‐dimensional structure of Der p1, five putative IgG and IgE epitopes were predicted. By protein engineering, the predicted epitopes were removed one by one in Der p1 and screening for hypoallergenic variants was performed.