Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE binding and type I skin test activity

• Published in: The Journal of immunology (1950) Vol. 149; no. 2; pp. 454 - 460
• Main Authors: Moser, M, Crameri, R, Menz, G, Schneider, T, Dudler, T, Virchow, C, Gmachl, M, Blaser, K, Suter, M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Am Assoc Immnol 15.07.1992; American Association of Immunologists
• Subjects: allergens; Allergens - analysis; Allergens - genetics; Allergens - immunology; Amino Acid Sequence; Antigens, allergens; Antigens, Differentiation - genetics; Asp f I antigen; Aspergillus fumigatus; Aspergillus fumigatus - immunology; Base Sequence; Biological and medical sciences; cDNA; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Fungal Proteins - analysis; Fungal Proteins - genetics; Fungal Proteins - immunology; Galectin 3; genes; Immediate hypersensitivity. Allergy. Anaphylaxis, etc; Immunobiology; immunoglobulin E-binding protein; Molecular Sequence Data; nucleotide sequence; predictions; Recombinant Proteins - analysis; Skin Tests; Human; IgE; Cytotoxicity; Fungi; Binding protein; Enzymatic activity; Fungi Imperfecti; Recombinant protein; Aspergillus fumigatus; Molecular cloning; Thallophyta; Allergen; Skin test
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.149.2.454

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.