PROTEIN EXPRESSION AND PURIFICATION 8, 365–373 (1996) ARTICLE NO. 0112 Characterization of Purified Recombinant Bet v 1 with Authentic N-Terminus, Cloned in Fusion with Maltose-Binding Protein Michael D. Spangfort, 1 Henrik Ipsen, Susanne H. Sparholt, Stig Aasmul-Olsen, Martin R. Larsen,* Ejvind Mørtz,* Peter Roepstorff,* and Jørgen N. Larsen Research Department, ALK-ABELLO ´ Group, Bøge Alle ´ 10-12, DK-2970 Hørsholm, Denmark; and *Department of Molecular Biology, Odense University, DK-5230 Odense, Denmark Received March 15, 1996, and in revised form July 12, 1996 rification result in relatively high yields of folded re- combinant Bet v 1 with correct N-terminal sequence A gene encoding the pollen major allergen Bet v 1 and molecular mass. Furthermore, the B- and T-cell from Betula verrucosa (White Birch) has been cloned epitope structures of recombinant Bet v 1 closely re- and expressed in Escherichia coli as a fusion with semble those of the natural protein from pollen.  1996 maltose-binding protein and a Factor Xa proteolytic Academic Press, Inc. cleavage site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the au- thentic amino terminus of Bet v 1 was generated after Tree pollens are a major source of airborne allergens cleavage. Fusion protein was isolated by amylose af- causing IgE-mediated (Type I) allergic symptoms dur- finity chromatography and enzymatically cleaved by ing early spring (1). The major allergen of Birch (Betula incubation with Factor Xa. Recombinant Bet v 1 was verrucosa) pollen has been identified as a 17-kDa pro- isolated by gel filtration and gave rise to a single band tein denoted Bet v 1 (2–4). More than 95% of birch with apparent molecular weight of 17 kDa when ana- pollen allergic patient’s serum IgE reacts with Bet v 1 lyzed by SDS – polyacrylamide gel electrophoresis. N- when analyzed by crossed radioimmunoelectrophoresis terminal sequencing of the first 20 amino acids showed (5). Birch pollen allergic patient’s IgE show cross-reac- complete agreement with the deduced Bet v 1 DNA tivity between Bet v 1 and the major pollen allergens sequence. Mass spectrometry showed that recombi- nant Bet v 1 has a molecular mass of 17,440 { 2 Da; Aln g 1, Car b 1, and Cor a 1 from other tree species 86% of the recombinant Bet v 1 amino acid sequence of Fagales (6,7). This indicates similarities in B-cell could be verified by digestion with Lys-C and mass epitope structures in agreement with the approxi- spectrometric peptide mapping. The yield of purified mately 75% amino acid identity between the major al- recombinant Bet v 1 was 10 mg per liter E. coli cell lergens (8 – 11). A number of T-cell epitopes, evenly dis- culture. Two-dimensional gel electrophoresis of puri- tributed along the primary amino acid sequence of Bet fied recombinant protein gave rise to one major pro- v 1, have been mapped using overlapping synthetic tein spot and one or two minor spots focusing at peptides (12). The biological function of Bet v 1 is still slightly different pHs. The immunochemical proper- not fully clarified but it shows a high sequence homol- ties of recombinant protein were indistinguishable ogy to a family of pathogenesis-related proteins (8,13). from those of naturally occurring Bet v 1 when com- Furthermore, based on in vitro biological activity, it pared using a panel of murine monoclonal antibodies has recently been suggested that Bet v 1 is a pollen- and serum IgE from birch pollen allergic patients. Fur- specific RNase (14). When analyzed by 2-D gel electro- thermore, recombinant Bet v 1 elicited T-cell prolifera- phoresis and silver staining, birch pollen extract shows tion comparable to that of natural Bet v 1. Thus, the a large number of protein spots representing different methods used for bacterial expression and protein pu- proteins in various isoallergens. About 20 of these pro- tein spots can be identified as Bet v 1 isoallergens by use of monospecific antibodies raised against purified 1 To whom correspondence should be addressed. Fax: (45) 45 76 16 82. E-mail: Michaels@inet.uni-c.dk. naturally occurring Bet v 1 (15,16). Analysis of IgE 365 1046-5928/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.