Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR

Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (p I 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast...

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Bibliographic Details
Published in:Protein expression and purification 2004-10, Vol.37 (2), p.336-343
Main Authors: Barral, Patricia, Tejera, Marı́a Luisa, Treviño, Miguel Ángel, Batanero, Eva, Villalba, Mayte, Bruix, Marta, Rodrı́guez, Rosalı́a
Format: Article
Language:English
Subjects:
Allergen
Chromatography
Chromatography, High Pressure Liquid
Circular Dichroism
Electrophoresis, Polyacrylamide Gel
Genetic Vectors
Immunoglobulin G - chemistry
Magnetic Resonance Spectroscopy - methods
Methionine - chemistry
NMR
Olive pollen
Oxygen - chemistry
Pichia - metabolism
Pichia pastoris
Plant Proteins - biosynthesis
Plant Proteins - chemistry
Plant Proteins - isolation & purification
Pollen - chemistry
Protein Binding
Protein Structure, Tertiary
Recombinant Proteins - chemistry
Time Factors
Ultraviolet Rays
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Summary:Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (p I 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2004.06.012