Purification and characterization of recombinant Bet v I, the major birch pollen allergen. Immunological equivalence to natural Bet v I.
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.
Ferreira
F D
FD
Institut für Allgemeine und Experimentelle Pathologie, Universität Wien, Vienna, Austria.
Hoffmann-Sommergruber
K
K
Breiteneder
H
H
Pettenburger
K
K
Ebner
C
C
Sommergruber
W
W
Steiner
R
R
Bohle
B
B
Sperr
W R
WR
Valent
P
P
D000485:Allergens / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006967:Hypersensitivity; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000378:metabolism; D007525:Isoelectric Focusing; D008027:Light; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012542:Scattering, Radiation; D012882:Skin Tests; D013601:T-Lymphocytes / Q000276:immunology*; D014197:Trees / Q000276:immunology*
D000485:Allergens; D052179:Antigens, Plant; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C061312:Bet v 1 allergen, Betula; D007073:Immunoglobulin E