Cloning, sequencing, and recombinant production of Sin a 2, an allergenic 11S globulin from yellow mustard seeds

BackgroundAmong allergenic spices, mustard represents 1 of the most important in terms of allergenic potency and widespread use. An 11S globulin (Sin a 2) has been isolated from yellow mustard seeds and identified as a new major allergen.ObjectiveCloning and sequencing a cDNA encoding a Sin a 2 subunit and producing the allergen as a recombinant protein.MethodsSin a 2 subunit–encoding cDNA was amplified by polymerase chain reaction, cloned, and sequenced. The allergen was produced as a recombinant protein in Escherichia coli and used for enzyme-linked immunosorbent assay, immunoblotting, and inhibition experiments. Sera from patients with mustard allergy and an anti-polyhistidine monoclonal antibody were used.ResultsSin a 2–specific cDNA comprises an open reading frame that encodes a protein of 510 amino acids, in which the first 23 residues correspond to the signal peptide. Sequence alignment with other allergenic 11S globulins showed levels of sequence identity ranging between 27% and 38%. Three peptides described as epitopes in Ara h 3 were moderately conserved in Sin a 2. Approximately 87% of the IgE binding to natural Sin a 2 was inhibited by the recombinant allergen using sera from patients with mustard allergy.ConclusionThe recombinant 11S globulin from yellow mustard seeds produced in E coli retained the IgE-binding capability of the natural allergen.Clinical implicationsThe availability of Sin a 2 sequence and its recombinant production could help to develop future therapeutic approaches and might well open new investigation lines to resolve whether 11S globulins are proteins implicated in cross-reactivity processes involving mustard seeds.