Production and characterization of recombinant Aspergillus fumigatus Cu,Zn superoxide dismutase and its recognition by immune human sera.
The Cu,Zn superoxide dismutase (SOD) of Aspergillus fumigatus has previously been purified and shown to be immunoreactive to the sera of patients with aspergillosis; however, the purification of large quantities of the enzyme for expanded immunological analysis is both difficult and time-consuming. Accordingly, a lambdaEMBL3 A. fumigatus genomic library was screened with degenerate oligonucleotides based on N-terminal amino acid sequence data; from this initial screen a 1,400-bp fragment was identified, labelled, and used to screen an A. fumigatus lambdagt11 cDNA library. A full-length cDNA encoding Cu,Zn SOD was subsequently identified and cloned. The cDNA encodes a protein of 154 amino acids, which does not have a signal peptide. The A. fumigatus Cu,Zn SOD possesses the typical metal binding ligands of fungal Cu,Zn SODs (six histidines and one aspartic acid) and has significant overall homology with Cu, Zn SODs in general. A recombinant A. fumigatus Cu,Zn SOD has been expressed in Pichia pastoris, is enzymatically active, and has biochemical and biophysical properties that are similar to those of the native enzyme. A sheep polyclonal antibody raised against purified native A. fumigatus Cu,Zn SOD was reactive to the recombinant enzyme by immunoenzyme development of Western blots. Sixty percent of serum samples from patients with A. fumigatus infections were reactive against the recombinant Cu,Zn SOD via immunoenzyme development of Western blots, indicating that the recombinant protein may be useful in the serodiagnostic identification of A. fumigatus infections.
Holdom
M D
MD
Service de Dermatologie (DHURDV), Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. m.holdmom@umds.ac.uk
Lechenne
B
B
Hay
R J
RJ
Hamilton
A J
AJ
Monod
M
M
D000595:Amino Acid Sequence; D001228:Aspergillosis / Q000276:immunology / Q000382:microbiology*; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D018076:DNA, Complementary; D015698:Genomic Library; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D018076:DNA, Complementary; D007106:Immune Sera; D011994:Recombinant Proteins; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc