Expression and analysis of recombinant Amb a V and Amb t V allergens. Comparison with native proteins by immunological assays and NMR spectroscopy.
The Amb V allergens are small, highly disulfide-bonded ragweed pollen allergens that serve as useful models for understanding the molecular basis of the human immune response. We have produced recombinant Amb a V and Amb t V (from short and giant ragweed pollens, respectively) in Escherichia coli and have compared their structural and functional characteristics to those of the native proteins. Recombinant Amb t V was indistinguishable from native Amb t V as determined by NMR spectroscopy and antibody-binding studies. Whereas inhibition analysis showed that recombinant Amb a V possessed only approximately 50% of the antibody-binding activity of native Amb a V, the two proteins were similarly effective in stimulating Amb a V-specific T-cells. Our results demonstrate that even highly homologous proteins exhibit different abilities to fold into their native three-dimensional conformations and establish the potential and limits of expressing the recombinant Amb V allergens intracellularly in E. coli.
Rafnar
T
T
Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224-6801.
Ghosh
B
B
Metzler
W J
WJ
Huang
S K
SK
Perry
M P
MP
Mueller
L
L
Marsh
D G
DG
D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000906:Antibodies; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011058:Pollen; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000485:Allergens; D000906:Antibodies; D052179:Antigens, Plant; D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C069543:group 5 allergens, Ambrosia