Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE binding and type I skin test activity.
Aspergillus fumigatus secretes an 18-kDa nonglycosylated IgE-binding protein. This protein was previously shown to be a ribotoxin, like alpha-sarcin and mitogillin. A 686-bp long A. fumigatus cDNA encoding an 18-kDa ribotoxin was cloned and expressed in Escherichia coli as a fusion protein with six adjacent histidines (rAsp f I/a). rAsp f I/a was purified to homogeneity by Ni(2+)-chelate affinity chromatography and refolded. The recombinant protein was enzymatically active resulting in the cleavage of 28S rRNA within a universally conserved region. rAsp f I/a was cytotoxic for EBV immortalized or PHA stimulated human PBMC. Furthermore, rAsp f I/a was recognized by murine mAb made against an 18-kDa ribotoxin. IgE of individuals allergic to A. fumigatus bound to rAsp f I/a as shown by ELISA, dot blots, and Western blots. rAsp f I/a elicited positive immediate type I skin reactions in individuals allergic to A. fumigatus but not in healthy control individuals. The results show that rAsp f I/a has similar functional characteristics when compared to the native 18-kDa ribotoxin. rAsp f I/a expressed in E. coli can therefore be used as a standardized Ag/allergen for serologic and clinical diagnosis of A. fumigatus-associated diseases.
Moser
M
M
Swiss Institute of Allergy and Asthma Research (SIAF), Davos.
Crameri
R
R
Menz
G
G
Schneider
T
T
Dudler
T
T
Virchow
C
C
Gmachl
M
M
Blaser
K
K
Suter
M
M
D000485:Allergens / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000943:Antigens, Differentiation / Q000235:genetics*; D001232:Aspergillus fumigatus / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D037502:Galectin 3; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis; D012882:Skin Tests
D000485:Allergens; D000943:Antigens, Differentiation; D005656:Fungal Proteins; D037502:Galectin 3; D011994:Recombinant Proteins