Purification and immunochemical characterization of recombinant and native ragweed allergen Amb a II.
The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described [Rogers et al. (1991) J. Immun. 147, 2547-2552; Griffith et al. (1991a), Int. Archs Allergy appl. Immun. 96, 296-304]. In this study, we present results generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both. The specificity of two anti-Amb a II antipeptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb aI.1. These two sera, directed against separate regions of the Amb a II molecule, detected three individual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragweed pollen extract. These Amb a II isoforms have pI in the 5.5-5.85 range and can be easily distinguished from Amb a I isoforms with pI in the 4.5-5.2 range detected by an anti-Amb a I specific peptide antiserum. The Amb a II isoforms have also been individually purified from pollen, positively identified as Amb a II by amino acid sequencing, and visualized as separate bands on IEF gels. An analysis of Amb a II cDNA sequences generated by PCR led to the prediction of three Amb a II isoforms with pI of 5.74, 5.86 and 5.97 that are very similar to the pI deduced from 2D Western blot analysis. Recombinant Amb aI.1 and Amb a II have been expressed in E. coli, purified in their denatured form, and examined by ELISA for their capacity to bind pooled allergic human IgE. Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties. In contrast, Amb a II had a markedly reduced IgE-binding capacity as compared to Amb a I.1. These data suggest that recombinant Amb a I.1 and Amb a II, isolated in a denatured form, differ significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.
Kuo
M C
MC
ImmuLogic Pharmaceutical Corporation, Waltham, MA.
Zhu
X J
XJ
Koury
R
R
Griffith
I J
IJ
Klapper
D G
DG
Bond
J F
JF
Rogers
B L
BL
D000485:Allergens / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D052179:Antigens, Plant; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000276:immunology / Q000302:isolation & purification*; D011058:Pollen / Q000276:immunology*; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D010940:Plant Proteins; D011994:Recombinant Proteins; C074244:Amb a II protein, Ambrosia artemisiifolia; D007073:Immunoglobulin E