Expression of the major olive pollen allergen Ole e 10 in the yeast Pichia pastoris: evidence of post-translational modifications.
Olive pollen allergy is a clinical disorder that affects around 20% of the population in Mediterranean areas. The major olive pollen allergen, Ole e 10, is involved in cross-reactivity phenomena and asthma induction in allergic patients, and, besides its clinical interest, Ole e 10 is the first member of a new family of plant proteins. Ole e 10-specific cDNA has been cloned in the plasmid pPICZalphaA and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has been purified in a two chromatographic-step procedure. N-Terminal sequencing, mass spectrometry, IgG, and IgE binding assays were employed to characterize the recombinant allergen. These analyses revealed that the product undergoes a proteolytic cleavage in the N-terminal end with the loss of the first six residues. Different strategies were used to solve this problem, such as changes in the fermentation conditions and the employment of protease-deficient yeast strains. Proteolytic cleavage was minimized and about 51% of rOle e 10 was obtained as a full-length protein. Moreover, a covalent modification was found in the N-terminal end of the full-length rOle e 10. Peptide mapping and mass spectrometry analyses pointed to the existence of a phosphorylation located in a serine residue of the N-terminal segment of rOle e 10 and it was confirmed after treatment of the sample with alkaline phosphatase. Finally, both full-length and truncated rOle e 10 retained most of the IgG- and IgE-binding capabilities of the natural protein isolated from the pollen.
Barral
Patricia
P
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain.
Batanero
Eva
E
Villalba
Mayte
M
Rodríguez
Rosalía
R
D000485:Allergens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D014162:Transfection
D000485:Allergens; D052179:Antigens, Plant; D007074:Immunoglobulin G; C487628:Ole e 10 allergen, Olea europaea; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D010447:Peptide Hydrolases