Production and characterization of recombinant Aspergillus fumigatus Cu,Znsuperoxide dismutase and its recognition by immune human sera

The Cu,Zn superoxide dismutase (SOD) of Aspergillus fumigatus has previousl y 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 lambda EMBL3 A. fumigatus genomic library was screened with degenerate oligonucleotides based on N-terminal amino acid sequence data; f rom this initial screen a 1,400-bp fragment was identified, labelled, and u sed to screen an A. fumigatus lambda gt11 cDNA library. A full-length cDNA. encoding Cu,Zn SOD was subsequently identified and cloned. The cDNA encode s 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 over all homology with Cu,Zn SODs in general. ii 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 na tive 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 SO D via immunoenzyme development of Western blots, indicating that the recomb inant protein may be useful in the serodiagnostic identification of A. fumi gatus infections.