Expression in Escherichia coli of Sin a 1, the major allergen from mustard.
Sin a 1, the major yellow mustard allergen, is a seed storage protein that belongs to the 2S albumin family. It is composed of two disulfide-bonded polypeptide chains. The cloning of this allergen has been carried out by means of the polymerase chain reaction using non-degenerate oligonucleotides encoding the N-terminal and C-terminal regions of the mature protein as primers. Five genomic nucleotide sequences have been analyzed, encoding both mature polypeptide chains linked by the internal processed fragment. The sequence data show the existence of microheterogeneities at ten positions, demonstrating the polymorphism exhibited by the natural protein. One of the genomic clones was expressed in Escherichia coli by fusion to glutathione S-transferase from Schistosoma japonicum. The resulting chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B matrix, and digested with thrombin to release the recombinant allergen. The recombinant Sin a 1 is recognized by rabbit polyclonal and mouse monoclonal antisera raised against natural Sin a 1, as well as by the IgE of mustard-sensitive human sera. In addition, recombinant Sin a 1 possesses a high resistance to trypsin digestion, like the native mustard allergen.
González De La Peña
M A
MA
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Monsalve
R I
RI
Batanero
E
E
Villalba
M
M
Rodríguez
R
R
D000485:Allergens / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009149:Mustard Plant / Q000235:genetics* / Q000276:immunology*; D009500:Neutralization Tests; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology*; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D017931:DNA Primers; D018744:DNA, Plant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C079479:Sin a I protein, Sinapis alba