β-lactoglobulin mutation Ala86Gln improves its ligand binding and reduces its immunoreactivity.
β-Lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules. β-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 β-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 β-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 Ala86Gln 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 Ala86Gln mutant protein. Subsequently, the binding of various ligands such as retinol, palmitic acid, resveratrol and serotonin, with native, recombinant wild type and Ala86Gln mutant β-LGs were investigated by fluorescence spectroscopy and an improvement on the binding affinity of the mutated protein to various ligands was observed.
Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441, Iran.
Najmeh
N
Kazem-Farzandi
Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441, Iran. Electronic address: a.taheri@ast.ui.ac.ir.
Asghar
A
Taheri-Kafrani
UR 1268, INRA BIA-FIPL, B.P. 71627, 44316 Nantes Cedex 03, France.
Thomas
T
Haertlé
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003062:Codon; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology / Q000378:metabolism; D007782:Lactoglobulins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008024:Ligands; D008892:Milk / Q000009:adverse effects; D016269:Milk Hypersensitivity; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization
D003062:Codon; D007782:Lactoglobulins; D008024:Ligands; D011994:Recombinant Proteins; D007073:Immunoglobulin E
Ala; Alanine; Allergy; CD; CMA; Circular dichroism; Competitive ELISA; ELISA; ESI-MS; Gln; Glutamine; HPLC; Ligand binding; Mutation; SDS-PAGE; WT; circular dichroism; cow's milk allergy; electro-spray ionization mass spectrometry; enzyme-linked immunosorbent assay; high performance liquid chromatography; sodium dodecyl sulfate polyacrylamide gel electrophoresis; wild type; β-LG; β-Lactoglobulin; β-lactoglobulin