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.     

Immobilization of ovomucoid on chitosan

The inhibitory activity of ovomucoid from duck egg white, immobilized on chitosan with the use of glutaraldehyde or carbodiimide as cross-linking agents, was studied. Glutaraldehyde proved to be a more preferable cross-linking agent than carbodiimide. When chitosan is used as a protein carrier, the possibility of shifting the pH optimum of these compounds should be taken into account.     

Blocking effect of trypsin associated with ovomucoid on the antibody binding to ovomucoid

Ovomucoid-trypsin association complex was prepared by incubating chicken egg white ovomucoid with bovine trypsin. The reactivity of ovomucoid-trypsin complex was investigated by immunodiffusion, quantitative precipitation and enzyme-linked immunosorbent assay (ELISA). It was demonstrated that the association of trypsin with ovomucoid hindered the binding of the specific antibody at some antigenic sites of ovomucoid by lowering the antibody-binding affinity of these sites. The anti-ovomucoid antiserum was absorbed with ovomucoid-trypsin complex, and non-absorbed antibody was collected by immunoaffinity chromatography of ovomucoid-coupled Sepharose 4B. The antibody blocked the trypsin-inhibitory activity of ovomucoid in a molar ratio (antibody/ovomucoid) of about 1.2:1. The findings suggested that at least one antigenic site is located near the reactive site of trypsin inhibition (Arg89 decreases Ala90) of ovomucoid.     

Immobilization of ovomucoid on chitosan

The inhibitory activity of ovomucoid from duck egg white, immobilized on chitosan with the use of glutaraldehyde or carbodiimide as cross-linking agents, was studied. Glutaraldehyde proved to be a more preferable cross-linking agent than carbodiimide. When chitosan is used as a protein carrier, the possibility of shifting the pH optimum of these compounds should be taken into account.     

Transglutaminase-mediated modification of ovomucoid: effects on its trypsin inhibitory activity and antigenic properties

Hen egg can cause food hypersensitivity in infants and young children, and ovomucoid is the most allergenic factor among proteins contained in egg white. Since proteinase treatment, a well-recognized strategy in reducing food allergenicity, is ineffective when applied to ovomucoid because of its ability to act as trypsin inhibitor, we investigated the possibility of reducing the ovomucoid antiprotease activity and antigenic properties by covalently modifying its structure. The present paper reports data showing the ability of the Gln115 residue of ovomucoid to act as an acyl donor substrate for the enzyme transglutaminase and, as a consequence, to give rise to a covalent monodansylcadaverine conjugate of the protein in the presence of both enzyme and the diamine dansylated derivative. Moreover, we demonstrated that the obtained structural modification of ovomucoid significantly reduced the capability of the protein to inhibit trypsin activity, also having impact on its anti-ovomucoid serum-binding properties.