β-lactoglobulin mutation Ala86Gln improves its ligand binding and reduces its immunoreactivity

Abstract

beta-Lactoglobulin (beta-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules. beta-LG is also one of the major allergens in milk. Despite a lot of researches on decreasing of cow's milk allergenicity, the effects of the mutation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) patients have not been investigated. We described here the expression in the yeast Pichia pastoris of a mutant beta-LG, in which Alanine 86 was changed into Glutamine (Ala86Gln; a mutation on one of the major epitopes of the protein). The purity and native like folded structure of the recombinant Ala86GIn have been demonstrated using circular dichroism, HPLC, SDS-PAGE and mass spectrometry. The effect of the mutation on the binding of IgE from CMA patients to mutant protein was evaluated by ELISA methods and the results showed that the mutation of Ala-86 was associated with weaker binding of IgE from CMA patients to Ala86GIn mutant protein. Subsequently, the binding of various ligands such as retinol, palmitic acid, resveratrol and serotonin, with native, recombinant wild type and Ala86GIn mutant beta-LGs were investigated by fluorescence spectroscopy and an improvement on the binding affinity of the mutated protein to various ligands was observed. (C) 2015 Elsevier B.V. All rights reserved.