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 (pI 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.
Barral
Patricia
P
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain.
Tejera
María Luisa
ML
Treviño
Miguel Angel
MA
Batanero
Eva
E
Villalba
Mayte
M
Bruix
Marta
M
Rodríguez
Rosalía
R
D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008715:Methionine / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011058:Pollen / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D013997:Time Factors; D014466:Ultraviolet Rays
D007074:Immunoglobulin G; C107504:OLE6 protein, Olea europaea; D010940:Plant Proteins; D011994:Recombinant Proteins; D008715:Methionine; D010100:Oxygen