High-frequency binding of IgE to the Der p allergen expressed in yeast.
The production of allergens from cDNA clones will provide a clonally pure source of material for experimental and perhaps clinical studies. Attempts to produce the major mite allergen, Der p I, in a highly antigenic form in bacteria have, to date, had limited success. In this study, a high level of production of Der p I from a Cup1 gene cassette from pYELC5-13T in Saccharomyces cerevisiae is described. Although the protein was insoluble, it could be readily solubilized in a urea solution and remained in solution when it was returned to more physiologic buffers. An amount equivalent to about 1 mg/L of yeast culture could then be isolated by affinity chromatography with an immobilized monoclonal antibody. This product reacted strongly with IgE in 9/11 sera from mite-allergic patients compared to the 50% reactivity achieved for Der p I previously produced as a fusion by bacteria. Similarly, the intensity of binding and ability to absorb out Der p I specificities were much greater for the yeast, pYELC5-13T, product. Studies with monoclonal antibodies also demonstrated the yeast, Der p I, had a high degree of antigenicity, although clear differences with the native allergen were demonstrated. The high frequency of reactivity with IgE of the pYELC5-13T formally demonstrates that a single gene product of Der p I is a major allergen and demonstrates that even for Der p I, which is synthesized from a proenzyme, considerable antigenicity can be obtained by expressing the mature protein.
Chua
K Y
KY
Western Australian Research Institute for Child Health, Perth.
Kehal
P K
PK
Thomas
W R
WR
Vaughan
P R
PR
Macreadie
I G
IG
D000485:Allergens / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology*; D008925:Mites / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000276:immunology*
D000485:Allergens; D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D007073:Immunoglobulin E