title: Immunochemical characterization of two Pichia pastoris-derived recombinant group 5 Dactylis glomerata isoallergens.
abstract: BACKGROUND
  Grass pollen of the Poaceae grasses are known to be highly allergenic. Major allergens from the species Lolium, Phleum, Poa and Holcus have been cloned and expressed as recombinant proteins, but of the important species Dactylis glomerata no recombinants are available.
  
  
  METHODS
  Dac g 5 was cloned by PCR on the basis of homology with Lol p 5 and expressed in Pichia pastoris. Recombinant Dac g 5 (rDac g 5) was affinity purified and compared to natural Dac g 5 (nDac g 5) by immunoblot, radioallergosorbent test (RAST), RAST inhibition, basophil histamine release assay (HRA), competitive radioimmunoassay (RIA) and sandwich enzyme-linked immunosorbent assay (ELISA). In addition, N-terminal sequencing, concanavalin A (Con A) binding, circular dichroism spectrum measurements and matrix-assisted laser desorption ionization-time of flight mass-spectrometric analysis were performed.
  
  
  RESULTS
  Clones were obtained that coded for pro-Dac g 5 and two mature isoforms of Dac g 5; the deduced amino acid sequences of both isoforms differed by 4 amino acids. Both mature isoforms were expressed in Pichia at a concentration of approximately 15 mg/l. SDS-PAGE analysis showed that rDac g 5 had an apparent M(r) approximately 10 kD above nDac g 5. By mass spectrometry this difference was shown to be around 2.5 kD. Positive Con A staining suggested (O-linked) glycosylation as an explanation for this increase in M(r). Whereas both purified recombinants showed a tendency to dimerize, purified nDac g 5 contained a 12-kD peptide not observed for rDac g 5. RAST, RAST inhibition and HRA showed that the IgE reactivity of rDac g 5 was similar to that of nDac g 5. A small subgroup, however, clearly demonstrated decreased IgE reactivity to rDac g 5.02. Differences in immune reactivity of both isoforms were confirmed by monoclonal antibody (mAb)-based sandwich ELISA.
  
  
  CONCLUSIONS
  Dac g 5 was successfully cloned and expressed in P. pastoris. Minor differences in primary structure between isoforms influence their immune reactivity.
authors.affiliation: CLB Department of Immunopathology, Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: van Oort
authors.forename: P G
authors.initials: PG
authors.lastname: de Heer
authors.forename: P
authors.initials: P
authors.lastname: Lerouge
authors.forename: L
authors.initials: L
authors.lastname: Faye
authors.forename: R C
authors.initials: RC
authors.lastname: Aalberse
authors.forename: R
authors.initials: R
authors.lastname: van Ree
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology*; D052179:Antigens, Plant; D003001:Cloning, Molecular; D007120:Immunochemistry; D010843:Pichia / Q000235:genetics / Q000276:immunology; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology*; D006109:Poaceae / Q000235:genetics / Q000276:immunology*; D011058:Pollen / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C444200:Dac g 5 allergen; D010940:Plant Proteins; D011994:Recombinant Proteins