Expression and Secondary Structure Determination by NMR Methods of the Major House Dust Mite Allergen Der p 2

Abstract

There exists a strong correlation between asthma and sensitization to indoor allergens. This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods. The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter. The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST). The secondary structure was determined by examining chemical shifts, short and long range NOESYs, J HN-HA coupling constants, and amide exchange rates. From these data, it is clear that Der p 2 is composed of ␤-sheets and random coil. Based on long range distance constraints, a number of ␤-strands were aligned into two three-stranded, antiparallel ␤-sheets.

Hydrogen Exchange Nuclear Magnetic Resonance Spectroscopy Mapping of Antibody Epitopes on the House Dust Mite Allergen Der p 2

“…Residue Cys-73 forms a disulfide bond with Cys-78, and mutants that lacked this disulfide, as well as chemically reduced Der p 2, showed reduced affinity for ␣DpX and IgE Ab (6, 7). The final mAb examined was 6D6, which belongs to a group of mAb that recognizes different naturally occurring Der p 2 isoforms at position 114, either asparagine or aspartic acid (9,10). The protected residues for mAb 6D6 were 111 and 116.…”

“…Amide peak intensities were obtained from two-dimensional HSQC spectra acquired using 256 t 1 points and 1024 t 2 points and with 64 transients (20). The three-dimensional HSQC-NOESY spectra (21) were obtained as previously described (9). All spectra were transformed and analyzed using Felix 95.0 (Biosym/Molecular Simulations, San Diego, CA) with standard protocols.…”

“…Competitive Inhibition ELISA-The ability of rDer p 2 and the various alanine mutants to inhibit mAb binding to solid phase rDer p 2 was measured using a goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) conjugated to horseradish peroxidase (9,24). The results were calculated as the percentage inhibition using the value of maximum inhibition by rDer p 2 as 100% inhibition.…”

“…Production of rDer p 2-Recombinant Der p 2 (rDer p 2) was expressed and purified from Escherichia coli cultures as previously described (9). Briefly, the protein was recovered from the insoluble fraction of the cell sonicate and resolubilized with 6 M guanidine (one-fifth the original cell culture volume), and after dialysis against buffer (10 mM Trizma (Tris base), 1 mM EDTA, pH 8.5), the protein was purified by mAb affinity chromatography.…”

Effect of Amino Acid Polymorphisms of House Dust Mite Der p 2 Variants on Allergic Sensitization

“…The V40L located inside the molecule interacts with several residues. 15 However, although valine and leucine are hydrophobic residues, differences in the size of sidechain may alter the structure of a particular epitope. As for the M111L, our results suggested that both residues M111L and D114N may be the components of the epitope that has been demonstrated by other studies.…”