Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli.
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.
Unit of Clinical Immunology and Allergy, Karolinska Institutet, Stockholm, Sweden. hans.gronlund@ks.se
Hans
H
Grönlund
Tomas
T
Bergman
Kristofer
K
Sandström
Gunvor
G
Alvelius
Renate
R
Reininger
Petra
P
Verdino
Alexander
A
Hauswirth
Karin
K
Liderot
Peter
P
Valent
Susanne
S
Spitzauer
Walter
W
Keller
Rudolf
R
Valenta
Marianne
M
van Hage-Hamsten
D000485:Allergens / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002415:Cats; D002942:Circular Dichroism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007120:Immunochemistry; D007073:Immunoglobulin E / Q000097:blood; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization
D000485:Allergens; D004220:Disulfides; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E; C059296:Fel d 1 protein, Felis domesticus