HIGH-LEVEL EXPRESSION AND PURIFICATION OF THE MAJOR BIRCH POLLEN ALLERGEN, BET-V-1

Bet v 1, the single major allergen from birch pollen, shares IgE epito pes with all major tree pollen allergens from closely related species such as alder, hazel, hornbeam, beech, and European chestnut. Because of high sequence homologies among these allergens and the well-studied cross-reactivities on B cell epitopes, Bet v 1 is a representative mo del protein which can be used for in vitro studies. The cDNA coding fo r Bet v 1, the single major allergen from birch pollen, was cloned int o the T7-based Escherichia coli expression system pMW 175/BL21(DE3) an d synthesized as a nonfusion protein. In contrast to other E. coil sys tems (e.g., pKK233-2/JM105), this system produces high levels of readi ly extractable proteins corresponding to 5-10% of E. coli total protei n, the percentage varying with culture conditions. The overall yield w as 8-10 mg of purified recombinant protein per liter of culture medium . The recombinant allergen was purified by several steps, including io n-exchange and hydrophobic interaction chromatography. The purified re combinant allergen showed identical immunological properties with the respective natural counterpart. The use of recombinant allergens of hi gh purity is expected to result in more accurate diagnostic procedures , but possibly also in a superior immunotherapy of Type I allergic dis eases when compared with methods using crude allergen extracts contain ing various amounts of allergen concentrations. (C) 1997 Academic Pres s.