PROTEIN EXPRESSION AND PURIFICATION 9, 33–39 (1997) ARTICLE NO. PT960671 High-Level Expression and Purification of the Major Birch Pollen Allergen, Bet v 1 Karin Hoffmann-Sommergruber,* ,1 Markus Susani,† Fatima Ferreira,‡ Peter Jertschin,† Horst Ahorn,§ Renate Steiner,* Dietrich Kraft,* Otto Scheiner,* and Heimo Breiteneder* *Institute of General and Experimental Pathology, University of Vienna, AKH Wa ¨ hringer Gu ¨ rtel 18-20, 1090 Vienna, Austria; †Advanced Biological Systems, Inc., Salzburg, Austria; ‡Institute of Genetics and General Biology, University of Salzburg, Salzburg, Austria; and §Ernst Bo ¨hringer Institute, Vienna, Austria Received June 24, 1996, and in revised form September 19, 1996 or more of the population of the Northern hemisphere Bet v 1, the single major allergen from birch pollen, from industrialized countries suffer from allergic dis- shares IgE epitopes with all major tree pollen aller- eases. Symptoms of pollinosis include rhinitis and rhi- gens from closely related species such as alder, hazel, noconjunctivitis, urticaria, atopic eczema, and asthma hornbeam, beech, and European chestnut. Because of and in some cases anaphylactic shock. high sequence homologies among these allergens and Pollens from trees of the order Fagales, in particular the well-studied cross-reactivities on B cell epitopes, birch, alder, hazel, hornbeam, and oak, are a major Bet v 1 is a representative model protein which can be source of allergenic proteins causing IgE-mediated dis- used for in vitro studies. eases in early springtime (2). Therefore, there is a ris- The cDNA coding for Bet v 1, the single major aller- ing need for materials for high-quality diagnostics con- gen from birch pollen, was cloned into the T7-based sisting of a range of several well-defined, standardized, Escherichia coli expression system pMW 175/ and purified allergenic molecules (3 – 5). Mixtures of BL21(DE3) and synthesized as a nonfusion protein. In allergenic proteins would help to improve differential contrast to other E. coli systems (e.g., pKK233-2/ diagnosis as well as to determine individual allergenic JM105), this system produces high levels of readily ex- profiles recognized by each patient. Total crude ex- tractable proteins corresponding to 5 – 10% of E. coli tracts are used so far for diagnosis and immunother- total protein, the percentage varying with culture con- apy. These natural raw extracts often consist of a ditions. The overall yield was 8–10 mg of purified re- poorly defined mixture of proteins, carbohydrates, and combinant protein per liter of culture medium. The organic compounds with varying amounts of allergenic recombinant allergen was purified by several steps, molecules in different preparations and batches. The including ion-exchange and hydrophobic interaction composition of allergenic extracts varies according to chromatography. The purified recombinant allergen extraction conditions such as temperature, extraction showed identical immunological properties with the period, extraction buffer, and pH of extraction buffer respective natural counterpart. The use of recombi- nant allergens of high purity is expected to result in (6, 7). For these reasons, the use of recombinant and more accurate diagnostic procedures, but possibly well-defined allergens is a necessity for standardiza- also in a superior immunotherapy of Type I allergic tion of diagnostic methods (8 – 10). diseases when compared with methods using crude al- Therefore, there is a rising need for adequate heterol- lergen extracts containing various amounts of aller- ogous expression systems which are capable of produc- gen concentrations.  1997 Academic Press ing large quantities of recombinant proteins and the establishing of suitable purification protocols (11 – 13). Nevertheless, the immunological parameters for re- combinant allergens have to be tested and compared In the past decade, Type I allergies have significantly to the naturally occurring counterparts by investigat- increased in developed countries (1). An estimated 20% ing the physicochemical properties of the purified re- combinant product (13 – 16). Bet v 1, the major birch pollen allergen, comprises a 1 To whom correspondence should be addressed. Fax: 43-1-40400/ 5130. range of closely related isoforms. Fourteen isoforms 33 1046-5928/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.