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Characterization and mutagenesis of the IgE-binding epitopes of recombinant Ber e 1

Anna Oommen, University of Nebraska - Lincoln

Abstract

The Brazil nut is the seed of the Bertholletia excelsa tree and grows in the Amazon basin. Over the years there has been an increase in the number of individuals describing allergic symptoms following ingestion of Brazil nuts. The reason for this increased prevalence is not known though some researchers attribute this to an increased exposure to these nuts. Brazil nuts have one of the highest naturally occurring concentrations of selenium and some people ingest them for health reasons associated with this mineral. ^ The Brazil nut 2S albumin has a high content of sulfur containing amino acids, which makes it attractive as a source to nutritionally enhance plants deficient in these amino acids. ^ Brazil nut 2S albumin, Ber e 1, is the major allergen of Brazil nuts. To characterize Ber e 1, overlapping recombinant proteins were expressed in E coli and five linear IgE epitopes were mapped to the large subunit of the Brazil nut 2 S Albumin using sera from 6 Brazil nut allergic subjects. The overall pattern of IgE recognition of the epitopes amongst the different subjects was polymorphic. Out of these five epitopes, Epitope 3 was recognized in 3 out of 6, of the subjects suggesting it to be immunodominant. While all five epitopes appear to be linear, we cannot eliminate the existence of conformational epitopes as 2 subjects recognized the full-length recombinant protein with IgE immunoblotting but failed to recognize smaller segments of the protein. ^ With the aim of better defining epitope 3 and generating hypoallergenic variants of Ber e 1, each amino acid in this epitope was mutated to encode an alanine by oligonucleotide-directed mutagenesis. Fourteen mutants were generated and the mutations were confirmed by DNA sequencing. The wild type and mutant plasmids were then directionally cloned into a prokaryotic expression vector, pRSET that encodes an N-terminal 6X HIS tag. Proteins from the wild type and mutants were expressed and purified by metal-affinity chromatography on a nickel resin column via the HIS tag. IgE binding assays were performed on the purified proteins with sera from 2 Brazil nut allergic subjects that had previously recognized epitope 3. Attenuated IgE binding was established with a number of the mutants. While the two subjects had 4 amino acid residues in common that were sensitive to the modifications, the overall pattern of recognition of the individual amino acids in this single epitope is polymorphic. This is interesting as it suggests that even within a single epitope, of Ber e 1, the pattern of IgE binding varies amongst the different patients. (Abstract shortened by UMI.)^

Subject Area

Biology, Molecular|Agriculture, Food Science and Technology|Health Sciences, Immunology

Recommended Citation

Oommen, Anna, "Characterization and mutagenesis of the IgE-binding epitopes of recombinant Ber e 1" (2003). ETD collection for University of Nebraska - Lincoln. AAI3117804.
http://digitalcommons.unl.edu/dissertations/AAI3117804

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