Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1

FEBS J. 2005 Jan;272(1):217-27. doi: 10.1111/j.1432-1033.2004.04403.x.


Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1. Moreover, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allergens / genetics
  • Allergens / immunology*
  • Animals
  • Cell Line
  • Epitopes / chemistry*
  • Humans
  • Immunoglobulin E / immunology*
  • Mass Spectrometry
  • Phylogeny
  • Plant Proteins / genetics
  • Plant Proteins / immunology*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Spodoptera


  • Allergens
  • Epitopes
  • Plant Proteins
  • Recombinant Proteins
  • PHLPI protein, Phleum pratense
  • Immunoglobulin E