Rapid and efficient purification of Phleum pratense major allergens Phl p 1 and group Phl p 2/3 using a two-step procedure

The standardization of natural allergenic extracts and the characterization of recombinant allergens ensures a continuing requirement for highly purif ied natural allergens. The extraction and purification methods have to be r eproducible and also preserve the biological and immunological activity of the allergen. A simple two-step purification system has been established in order to provide milligram amounts of purified natural Phl p 1 and Phl p 2 /3. Both major allergens were separated from other proteins of timothy gras s pollen extract in one step by hydrophobic interaction chromatography (HIC ) under mild conditions. The allergens elute in the flow-through fraction w hile the rest of the proteins remain bound to the column. The very differen t molecular weights of Phl p 1 and Phl p 2/3 permitted separation of the al lergens by a second step using gel filtration. (C) 1999 Elsevier Science B. V. All rights reserved.