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


OBJECTIVE
Cloning and sequencing a cDNA encoding a Sin a 2 subunit and producing the allergen as a recombinant protein.


METHODS
Sin 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.


RESULTS
Sin 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.


CONCLUSIONS
The recombinant 11S globulin from yellow mustard seeds produced in E coli retained the IgE-binding capability of the natural allergen.


CONCLUSIONS
The 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.
Palomares
Oscar
O
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense de Madrid, Spain.
Vereda
Andrea
A
Cuesta-Herranz
Javier
J
Villalba
Mayte
M
Rodríguez
Rosalía
R
D000328:Adult; D000368:Aged; D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005916:Globulins / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012639:Seeds / Q000737:chemistry / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D031226:Sinapis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
D000485:Allergens; D052179:Antigens, Plant; D005916:Globulins; D010940:Plant Proteins; D011994:Recombinant Proteins; C512826:Sin a 2 protein, Sinapis alba