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

Protein Expr Purif. 2004 Oct;37(2):336-43. doi: 10.1016/j.pep.2004.06.012.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography
  • Chromatography, High Pressure Liquid
  • Circular Dichroism
  • Electrophoresis, Polyacrylamide Gel
  • Genetic Vectors
  • Immunoglobulin G / chemistry
  • Magnetic Resonance Spectroscopy / methods*
  • Methionine / chemistry*
  • Oxygen / chemistry*
  • Pichia / metabolism*
  • Plant Proteins / biosynthesis*
  • Plant Proteins / chemistry*
  • Plant Proteins / isolation & purification
  • Pollen / chemistry
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry*
  • Time Factors
  • Ultraviolet Rays


  • Immunoglobulin G
  • OLE6 protein, Olea europaea
  • Plant Proteins
  • Recombinant Proteins
  • Methionine
  • Oxygen