Expression and secondary structure determination by NMR methods of the major house dust mite allergen Der p 2

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 beta-sheets and random coil, Based on long range distance constraints, a number of beta strands were aligned into two three-stranded, antiparallel beta-sheets.