title: Characterization of natural Dac g 1 variants: an alternative to recombinant group 1 allergens.
abstract: BACKGROUND
  Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy.
  
  
  OBJECTIVE
  Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules.
  
  
  METHODS
  Dac g 1 was cloned and expressed in the yeast Pichia pastoris. Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests.
  
  
  RESULTS
  Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr). Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LMr molecule. IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr Dac g 1.
  
  
  CONCLUSIONS
  Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.
authors.affiliation: Department of Immunopathology, Sanquin Research, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
authors.forename: Erica
authors.initials: E
authors.lastname: van Oort
authors.forename: Pleuni G
authors.initials: PG
authors.lastname: de Heer
authors.forename: Miranda
authors.initials: M
authors.lastname: Dieker
authors.forename: Astrid W
authors.initials: AW
authors.lastname: van Leeuwen
authors.forename: Rob C
authors.initials: RC
authors.lastname: Aalberse
authors.forename: Ronald
authors.initials: R
authors.lastname: van Ree
mesh_terms: D000485:Allergens / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D003001:Cloning, Molecular; D006031:Glycosylation; D006801:Humans; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C057603:Dac g I protein, Dactylis glomerata; D010940:Plant Proteins; D011994:Recombinant Proteins