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Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2003 Oct 10; Vol. 278 (41), pp. 40144-51. Date of Electronic Publication: 2003 May 05. - Publication Year :
- 2003
-
Abstract
- Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.
- Subjects :
- Allergens genetics
Amino Acid Sequence
Animals
Cats
Circular Dichroism
Dimerization
Disulfides chemistry
Escherichia coli genetics
Gene Expression
Glycoproteins genetics
Glycoproteins immunology
Humans
Hypersensitivity immunology
Immunochemistry
Immunoglobulin E blood
In Vitro Techniques
Lymphocyte Activation
Molecular Sequence Data
Protein Folding
Protein Structure, Quaternary
Protein Structure, Secondary
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins genetics
Spectrometry, Mass, Electrospray Ionization
Allergens chemistry
Glycoproteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 278
- Issue :
- 41
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 12732623
- Full Text :
- https://doi.org/10.1074/jbc.M301416200