Purification and characterization of recombinant Bet v I, the major birch pollen allergen. Immunological equivalence to natural Bet v I.

F.D Ferreira,K Hoffmann-Sommergruber,H Breiteneder,K Pettenburger,C Ebner,W Sommergruber,R Steiner,B Bohle,W.R Sperr,P Valent

doi:10.1016/s0021-9258(19)36554-8

Abstract

Pollen from trees of the order Fagales (e.g. birch, alder, hazel, oak, and hornbeam) are a major cause of Type I allergies observed in early spring. Previously, we reported the cloning and sequencing of Bet v I, the major birch pollen allergen, which showed high sequence similarities to a family of plant pathogen-activated genes (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935-1938). Here, we present the results on the expression, purification, and characterization of recombinant Bet v I produced in Escherichia coli as fusion and non-fusion protein, respectively. The purified recombinant proteins were analyzed to verify purity and structural integrity, and their immunological properties were compared to those of Bet v I isolated from birch pollen (natural Bet v I). Immunoblot analyses showed that the recombinant proteins are specifically recognized by monoclonal antibodies raised against natural Bet v I as well as by IgE from birch pollen-allergic patients. However, enzyme-linked immunosorbent assays revealed a decreased IgE-binding activity of the recombinant fusion Bet v I compared to the non-fusion and natural Bet v I proteins, which probably results from conformational changes due to the fusion tail. Recombinant non-fusion Bet v I was equivalent to natural Bet v I with respect to IgE-binding properties, the ability to induce in vitro proliferation of allergen-specific T-cell clones, and the ability to release histamine from basophils derived from birch pollen-allergic patients.

Highlights

From the SZnstitut fur Allgemeine und Experimentelk Pathologie, Uniuersitat Wien, Wahringer Gurtel18-20, A-I090 Vienna, Austria, the nErnstBohringer Znstitut fur Arzneimittelforschung, A-1120 Vienna,Austria, the IIAKH, Medizinische Abteilung, Hamatologie und Hamostaseologie, Uniuersitat Wien, A-1090 Vienna,Austria, the YlIZnstitutfur Genetik und Allgemeine Biologie, Universitat Salzburg, A-5020 Salzburg, Austria, and the #Max-Planck-Znstitut fur Biochemie am Klopferspitz, D W-8033Martinsried, Germany
The immunological properties of the purified recombinant proteins produced in Escherichia coli were compared to those of Bet u I isolated from birch pollen
Purification of Recombinant Bet u I-Fusion and non-fusion forms of the full-length Bet u I cDNA were expressed in E. coli as intracellularproteins

Summary

IMMUNOLOGICAL EQUIVALENCE TO NATURAL Bet u I*

The plates were incubated (4 h at room temperature and overnight at 4 "C) with alkaline phosphatase-conjugated horse anti-human IgE antibodies (Kallestad, Chaska) and developed with the substrate p-nitrophenyl phosphate.The optical density was measured at 405 nm (with 550 nm as reference wavelength), using an ELISA reader (Dynatech MR 600).Adequate controls were performed for each incubation step of the ELISA assay and a serum pool from 20 non-allergic individuals (NHS) assured the specificity of the IgEdetection system. T-cell Proliferation Assays-Isolation of Bet u I-specific T-cell clones from the peripheral blood of seven birch pollen-allergic patients (as indicated by typical case history, positive skin tests, and positive RAST) and proliferation assays were done as previously described (Parronchi et al, 1991; Ebner et al, 1993). Total histamine of cell suspensions was determined after cell lysis in distilled water

RESULTS

Characterization of Recombinant Bet u I

DISCUSSION

Blood basophils and mast cells play a major role in the