Biochemical and immunological characterization of a recombinant precursor form of the house dust mite allergen Der p 1 produced by Drosophila cells

Background The major house dust mite allergen Der p 1 elicits strong IgE an tibody responses in patients suffering from mite allergy. Objective This study reports the expression and characterization of a recom binant precursor form of Der p 1 secreted as ProDer p 1 from insect cells. Methods The cDNA coding for ProDer p 1 was cloned downstream to the gp67 si gnal peptide, starting from commercial cDNA encoding Der p 1 and PCR-amplif ied ProDer p 1 genomic fragment. ProDer p 1, expressed in Drosophila cells and purified from culture medium, was compared to Der p 1 isolated from mit e culture, in terms of glycosylation, enzymatic activity as well as IgG- an d IgE-binding capacity. Results Sequence analysis of the genomic clone of ProDer p 1 revealed that, besides two introns in the mature Der p 1 coding sequence, two introns wer e also present in the propeptide coding sequence. ProDer p 1 was purifed to homogeneity by a combination of ion-exchange, hydroxyapatite and gel filtr ation chromatographies. The precursor form of Der p 1 could be processed in vitro into mature Der p 1 under acidic and reducing conditions. Carbohydra te analysis clearly indicated that ProDer p 1 expressed from insect cells w as glycosylated and that glycan structures were located only in the prosequ ence. ProDer p 1 displayed a similar immunoreactivity towards IgE, monoclon al and polyclonal IgG antibodies compared to natural Der p 1. Specific acti vity measurements using synthetic substrates clearly indicated that, contra ry to natural Der p 1, ProDer p 1 was totally enzymatically inactive. Conclusions The expression of an enzymatically inactive and highly antigeni c ProDer p 1 zymogen molecule could be a suitable strategy for the developm ent of in vitro diagnosis test as well as for specific immunotherapy.