Characterization of Folded Recombinant Der p 5, a Potential Diagnostic Marker Allergen for House Dust Mite Allergy

Abstract

Background: Der p 5 was reported as an important allergen in Dermatophagoides pteronyssinus, which is particularly recognized by patients suffering from asthma. The aim of this study was to produce, by recombinant DNA technology, a folded Der p 5 allergen for diagnostic, therapeutic and preventive purposes. Methods: Der p 5-encoding cDNA was isolated from a λgt11 D. pteronyssinus expression cDNA library and expressed in Escherichia coli. rDer p 5 was purified to homogeneity and characterized by mass spectroscopy and circular dichroism. IgE reactivity was tested with sera from 117 mite-allergic patients and in a basophil histamine release experiment. Der p 5-specific rabbit IgG antibodies were produced for the ultrastructural localization of the allergen in mites by immunogold electron microscopy as well as for cross-reactivity studies. Results: rDer p 5 is a heat-stable protein with predominantly α-helical secondary structure which reacted with IgE from 31% of mite-allergic patients’ sera and showed no relevant cross-reactivity to group 5 allergens from storage mites and tropical mites. rDer p 5-specific rabbit IgG antibodies inhibited mite-allergic patients’ IgE binding to Der p 5 and allowed to localize the allergen in secretory granules of midgut epithelial cells of house dust mites. Conclusions: The described rDer p 5 molecule may be useful for diagnosis and immunotherapy of house dust mite-allergic patients.

Der p 5 Crystal Structure Provides Insight into the Group 5 Dust Mite Allergens

“…6) indicated that the Der p 21 allergen was dimeric (21). Studies of immuno-stained mites noted that Der p 5 appeared on fibrous structures in the food ball (10). In combination with the present results, these observations suggest that Der p 5 may be an integral structural component of these fibers given the apparent proclivity to polymerize.…”

“…The structure of Blo t 5 was determined in two studies using NMR (19,20). Both studies confirmed the results of CD spectroscopy and bioinformatics predictions of other group 5 and the highly similar group 21 mite allergens, which suggested that the proteins were largely helical in nature (10,(21)(22)(23). A superficial comparison of the two protein structures of Blo t 5 (2JMH and 2JRK in the Protein Data Bank) shows two elongated proteins, both with three ␣-helices all in a parallel orientation, and a disordered N terminus.…”

“…Furthermore, recent studies using polyclonal rabbit IgG antibodies raised against Der p 5 detected no binding with group 5 allergens from storage mites (i.e. Glycyphagus domesticus or Lepidoglyphus destructor) or B. tropicalis (10).…”

Identification of Der p 23, a Peritrophin-Like Protein, As a New Major Dermatophagoides Pteronyssinus Allergen Associated with the Peritrophic Matrix of Mite Fecal Pellets

“…The bacterial cells were grown overnight in LuriaBertani medium containing 100 mg/l ampicillin at 28°C, and expression of the recombinant protein was induced by adding isopropyl-β-thiogalactopyranoside to a final concentration of 0.5 mM. After cultivation for additional 3 h at 37°C, E. coli were harvested by centrifugation (15 min, 3000 rpm, 4°C; Sorvall RC5C) and lysed as described previously (26). The lysed bacterial cells were centrifuged at 18,000 rpm, 20 min, 4°C, and proteins of the soluble fraction containing Der p 23 were treated with 60% ammonium sulfate for 1.5 h at 4°C.…”

Advances in allergen-microarray technology for diagnosis and monitoring of allergy: The MeDALL allergen-chip

“…rDer p 5, rDer p 21 and rDer p 23 were purified as previously described [32][33][34]. Recombinant Der p 7 was purified by hydrophobic interaction chromatography and hydroxyapatite chromatography while the clone 16-derived protein was purified by anion and cation exchange chromatography.…”