Formation of Disulfide Bonds and Homodimers of the Major Cat Allergen Fel d 1 Equivalent to the Natural Allergen by Expression in Escherichia coli

• Published in: The Journal of biological chemistry Vol. 278; no. 41; pp. 40144 - 40151
• Main Authors: Grönlund, Hans, Bergman, Tomas, Sandström, Kristofer, Alvelius, Gunvor, Reininger, Renate, Verdino, Petra, Hauswirth, Alexander, Liderot, Karin, Valent, Peter, Spitzauer, Susanne, Keller, Walter, Valenta, Rudolf, van Hage-Hamsten, Marianne
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.10.2003; American Society for Biochemistry and Molecular Biology
• Subjects: Allergens - chemistry; Allergens - genetics; Amino Acid Sequence; Animals; Cats; Circular Dichroism; Dimerization; Disulfides - chemistry; Escherichia coli - genetics; Gene Expression; Glycoproteins - chemistry; Glycoproteins - genetics; Glycoproteins - immunology; Humans; Hypersensitivity - immunology; Immunochemistry; Immunoglobulin E - blood; In Vitro Techniques; Lymphocyte Activation; Medicin och hälsovetenskap; Molecular Sequence Data; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Spectrometry, Mass, Electrospray Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M301416200

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.