cDNA cloning and heterologous expression of the major allergens from peach and apple belonging to the lipid-transfer protein family.
BACKGROUND
Lipid-transfer proteins (LTPs) have been identified as major allergens of Rosaceae fruits in populations living in areas virtually free of Fagales trees, such as several Mediterranean communities. Pru p 3 and Mal d 3, the allergens from peach and apple, respectively, have a main clinical relevance in these areas.


OBJECTIVE
To isolate and characterize cDNAs for Pru p 3 and Mal d 3, and to produce recombinant Pru p 3 in the yeast Pichiapastoris.


METHODS
cDNAs for both allergens were isolated by polymerase chain reaction using nondegenerated primers. Expression of Pru p 3 was performed in P. pastoris using the pPIC 9 vector. The recombinant product was purified by gel-filtration chromatography followed by RP-HPLC. Immunodetection and immunoblot inhibition assays were carried out with sera from peach-allergic patients.


RESULTS
The cDNAs for both Pru p 3 and Mal d 3 showed a 273 open reading frame coding for the 91 amino acid mature polypeptides. The deduced amino acid sequences exhibited N-terminal regions fully identical to those previously determined for the natural peach and apple allergens. Pru p 3 was expressed in P. pastoris at 20 mg/L of culture medium. The recombinant allergen showed the same N-terminal sequence (plus a glutamic acid added for proper extracellular expression) and apparent molecular size as natural Pru p 3. Both the recombinant and natural forms of Pru p 3 displayed similar immunoglobulin (Ig)E-binding capacity in immunodetection and immunoblot inhibition assays.


CONCLUSIONS
Comparison of the complete primary structures of mature Pru p 3 and Mal d 3 deduced from their corresponding cDNA clones supports the close relationship between both allergens. Recombinant Pru p 3 binds IgE in vitro like its natural counterpart. Therefore, it can be a useful tool for specific diagnosis and structural studies.
Diaz-Perales
A
A
Unidad de Bioquímica, Departamento de Biotecnología, ETS Ingenieros Agrónomos, Madrid, Spain.
Garcia-Casado
G
G
Sanchez-Monge
R
R
Garcia-Selles
F J
FJ
Barber
D
D
Salcedo
G
G
D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence / Q000235:genetics; D052179:Antigens, Plant; D001483:Base Sequence / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005512:Food Hypersensitivity / Q000097:blood; D006801:Humans; D027845:Malus / Q000276:immunology*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010940:Plant Proteins; D027861:Prunus / Q000276:immunology*
D000485:Allergens; D052179:Antigens, Plant; D002352:Carrier Proteins; D018076:DNA, Complementary; C463180:Mal d 3 allergen; D010940:Plant Proteins; C463181:Pru p 3 allergen; C511920:lipid transfer proteins, plant