Extraction and Purification of Art v 1, the Mugwort Pollen Major Allergen

RATIONALE: Mugwort (Artemisia vulgaris) is widely spread in the temperate and humid climate zones of the northern hemisphere and considered one of the main causes of late summer pollinosis. More than 95% of mugwort allergic patients are sensitized to the glycoallergen Art v 1. Here we investigated extraction conditions from pollen and developed a rapid and efficient method for purification of this major allergen. METHODS: After optimization of pollen extraction conditions nArt v 1 was pre-purified by ultra-filtration (VivaCell 250). Final purification was performed by anion- and cation exchange chromatographies using volatile triethylamine acetate (pH 5) and ammoniumbicarbonate (pH 7.9) buffers. Protein quality and quantity were analyzed by Coomassie-, Silver-, and periodic acid Schiff's reagent staining, immunoblot analysis, and Bradford assay, respectively. RESULTS: The newly established purification method yields about 5 mg of nArt v 1 from 10 g of mugwort pollen. Pollen extraction revealed to be a crucial step within the purification procedure regarding protein quality. Extraction at temperatures above 4 centigrade lead to partial carbohydrate degradation as monitored by an alpha-arabinose specific antibody. Furthermore, ion exchange chromatography revealed different charge populations of nArt v 1 displaying similar IgE binding activities. CONCLUSIONS: Pollen extraction conditions might play a critical role for the purification of naturally occurring glycoproteins. In addition, the described rapid and efficient purification method might be adapted for Art v 1 homologous allergens. ACKNOWLEDGEMENTS: The work of the authors was supported by the Joint Research Project S8802-B01 of the Austrian Science Funds “FWF”.