CHARACTERIZATION OF PURIFIED RECOMBINANT BET-V-1 WITH AUTHENTIC N-TERMINUS, CLONED IN FUSION WITH MALTOSE-BINDING PROTEIN

A gene encoding the pollen major allergen Bet v 1 from Betula verrucos a (White Birch) has been cloned and expressed in Escherichia coli as a fusion with maltose-binding protein and a Factor Xa proteolytic cleav age site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after c leavage. Fusion protein was isolated by amylose affinity chromatograph y and enzymatically cleaved by incubation with Factor Xa. Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS-polyacry lamide gel electrophoresis. N-terminal sequencing of the first 20 amin o acids showed complete agreement with the deduced Bet v 1 DNA sequenc e. Mass spectrometry showed that recombinant Bet v 1 has a molecular m ass of 17,440 +/- 2 Da; 86% of the recombinant Bet v 1 amino acid sequ ence could be verified by digestion with Lys-C and mass spectrometric peptide mapping, The yield of purified recombinant Bet v 1 was 10 mg p er liter E. coli cell culture. Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and o ne or two minor spots focusing at slightly different pHs. The immunoch emical properties of recombinant protein were indistinguishable from t hose of naturally occurring Bet v 1 when compared using a panel of mur ine monoclonal antibodies and serum IgE from birch pollen allergic pat ients. Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1. Thus, the methods used for bact erial expression and protein purification result in relatively high yi elds of folded recombinant Bet v 1 with correct N-terminal sequence an d molecular mass. Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein fro m pollen. (C) 1996 Academic Press, Inc.