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.

Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules.

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.

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 (M_r) on SDS-PAGE of 33 kd (HM_r), 30 kd (IM_r) and 28 kd (LM_r). Extraction of IM_r Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HM_r and IM_r Dac g 1 differs at 2 positions, and LM_r 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 LM_r molecule. IM_r Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HM_r Dac g 1.

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.