IgE binding properties of the recombinant ovomucoid third domain expressed in Escherichia coli.
Ovomucoid, a major allergen in hen's egg white, consists of three tandem domains. The third domain (DIII) cDNA was sublconed into pGEMT-vector and the resultant plasmid (pGEMDIII) was inserted into a pGEM-4T-2 glutathione-S-transferase (GST) fusion vector. The GST-DIII fusion protein was expressed in Escherichia coli. The 56-residue fragment corresponding to DIII (Leu131-Cys186) was liberated using cyanogen bromide to cleave off the GST that had been hydrolized with thrombin, which left an additional peptide at the terminus of the recombinant protein. Measurement of circular dichroism spectra indicated that the recombinant third domain (DIII*) had a structure that was slightly less compact than that of the native form. Immunoblot analysis showed that the human IgE binding activity of DIII* was identical to that of native DIII, while its activity was significantly increased to IgE antibodies from egg-allergic patients when tested with an enzyme-linked immunosorbent assay. These results indicate that recombinant DIII* has similar sequential epitopes, but may have more predominant conformational epitopes than native analogues. This might have important implications in egg-allergic reactions.
Sasaki
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E
Department of Food Science, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Mine
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Y
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002942:Circular Dichroism; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005512:Food Hypersensitivity / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D010059:Ovomucin / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection
D000485:Allergens; D000939:Epitopes; D011994:Recombinant Proteins; D010059:Ovomucin; D007073:Immunoglobulin E