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, JHN-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, anti-parallel beta-sheets.
Mueller
G A
GA
Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22908, USA.
Smith
A M
AM
Williams
D C
DC
Hakkaart
G A
GA
Aalberse
R C
RC
Chapman
M D
MD
Rule
G S
GS
Benjamin
D C
DC
D000485:Allergens / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D004391:Dust; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002479:Inclusion Bodies; D008925:Mites; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D000485:Allergens; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D004391:Dust; D006023:Glycoproteins; D011994:Recombinant Proteins