Escherichia coli expression and purification of recombinant dog albumin, a cross-reactive animal allergen.
BACKGROUND
Animal hair-dander represents an important source of indoor allergens. Diagnosis and therapy of animal allergy would benefit from the availability of defined recombinant allergens. They may be preferred to animal-derived proteins, particularly for in vivo application in patients.


OBJECTIVE
The purpose of this study was to express and purify recombinant dog albumin, an important cross-reactive animal allergen.


METHODS
Complementary (c)DNA sequences coding for dog albumin were obtained by reverse transcription and subsequent PCR amplification from dog liver RNA. Dog albumin-encoding cDNA sequences were inserted into phage lambdagt11, and IgE-reactive phage clones were isolated by immunoscreening with serum IgE from a patient with dog allergy. Dog albumin was expressed as IgE-reactive recombinant protein in Escherichia coli and purified by inclusion body preparation, resolubilization, and diethylaminoethyl cellulose chromatography. Cross-reactivity of dog albumin with cat and human albumin was examined by immunoblot, as well as ELISA inhibition experiments.


RESULTS
A cDNA sequence coding for complete dog albumin was obtained by reverse transcription and subsequent PCR amplification from dog liver. The cDNA and deduced amino acid sequence of dog albumin was highly homologous to the sequences of albumins from animals to human subjects, thus explaining the extensive cross-reactivities among albumins. Recombinant dog albumin was expressed in E coli and purified. It reacted with serum IgE from patients allergic to dog albumin and a monoclonal anti-human albumin antibody. Immunologic competition experiments performed with serum IgE from patients allergic to dog albumin and a mouse monoclonal antihuman albumin antibody indicated the presence of similar epitopes on dog, cat, and human albumin.


CONCLUSIONS
Recombinant dog albumin may be used for diagnostic purposes to identify patients who are cross-sensitized to many animal species and perhaps for specific immunotherapy of sensitized individuals.
Pandjaitan
B
B
Institute of Medical and Chemical Laboratory Diagnostics, AKH, University of Vienna, Vienna, Austria.
Swoboda
I
I
Brandejsky-Pichler
F
F
Rumpold
H
H
Valenta
R
R
Spitzauer
S
S
D000418:Albumins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D000485:Allergens / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002415:Cats; D003429:Cross Reactions; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid
D000418:Albumins; D000485:Allergens; D018076:DNA, Complementary; D000939:Epitopes; D011994:Recombinant Proteins; D007073:Immunoglobulin E