Expression, purification, cross-reactivity and homology modeling of peanut profilin

Protein Expr Purif. 2010 Sep;73(1):36-45. doi: 10.1016/j.pep.2010.03.005. Epub 2010 Mar 15.

Abstract

Plant profilins are known pan-allergens involved in the cross-reactions between pollen and plant foods. Peanut profilin, Ara h 5, is one of the important peanut allergens. Presently, most immunological, biochemical and structural studies on peanut allergens have focused on the three major allergens (Ara h 1, 2 and 3). Here Ara h 5 was cloned, expressed in Escherichia coli, Rosetta2(DE3) (Novagen), purified using a combination of ammonium sulfate fractionation and size-exclusion chromatography and yielded a total of 29 mg/l of culture. IgE reactivity was assayed using multiplexed microarray with other peanut allergens (Ara h 1, 2, 3, and 8) and birch (Bet v 2) and timothy (Phl p 2) profilin using sera from peanut allergic Swedish patients. Using homology modeling, Ara h 5 structure was also generated, compared against other profilins and utilized to predict surface-exposed residues potentially forming epitopes. The allergen was recognized by 3 out of 33 sera (9.1%). IgE reactivity to Ara h 5 also coincided with that of two other profilins, Phl p 12 and Bet v 2, confirming cross-reactivity. Interestingly, IgE reactivity to Ara h 5 was higher than above-mentioned profilins which may be indicating specificity of sera towards peanut profilin. Eight surface-exposed epitopes were predicted and verified against experimentally validated sequential epitopes. Three epitopes (#1, 5 and 7) mostly located at the accessible loops and neutral to relatively electropositive sites were found common among profilins, which should be involved in cross-reactivity. A specific putative epitope (#4) was also found which may explain the relative high IgE reactivity to Ara h 5 as compared to the other profilins. Due to its close relation to other allergenic profilins, Ara h 5 could be used as a model and allergen of choice for profilin allergy diagnosis.

MeSH terms

  • Allergens / chemistry
  • Allergens / genetics
  • Allergens / immunology
  • Allergens / metabolism*
  • Amino Acid Sequence
  • Arachis / immunology
  • Arachis / metabolism*
  • Chemical Precipitation
  • Chromatography, Gel
  • Cloning, Molecular
  • Cross Reactions
  • Epitopes, B-Lymphocyte / chemistry
  • Escherichia coli / genetics
  • Humans
  • Immunoglobulin E / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / immunology
  • Plant Proteins / metabolism*
  • Profilins / chemistry
  • Profilins / genetics
  • Profilins / immunology
  • Profilins / metabolism*
  • Protein Array Analysis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism*
  • Sequence Alignment

Substances

  • Allergens
  • Epitopes, B-Lymphocyte
  • Plant Proteins
  • Profilins
  • Recombinant Proteins
  • Immunoglobulin E