Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus.

摘要: A 32-kDa nonglycosylated alkaline protease (EC 3.4.1.14) with elastolytic activity, secreted by the opportunistic pathogen Aspergillus fumigatus ATCC 42202, is suggested to be a virulence factor of this fungus. The enzyme serine subtilisin family, and its cDNA nucleotide sequence has recently been reported. We have cloned encoding mature into high-level Escherichia coli expression plasmid produced recombinant as fusion protein six-adjacent-histidine affinity tag at carboxy terminus. Subsequently, was purified homogeneity, chromatography yielding 30 40 mg per liter E. culture. Refolded protease, in comparison native demonstrated weak enzymatic activity but similar immunochemical characteristics analyzed antigen-specific enzyme-linked immunosorbent assay (ELISA), competition ELISA, immunoblotting assays. To assess allergenic potential sera from patients allergic bronchopulmonary aspergillosis healthy control individuals were ELISA techniques. Sera did not protease-specific immunoglobulin E (IgE) antibodies and, remarkably, show significantly elevated IgG antibody levels compared those individuals. This suggests that A. does elicit IgE immunogenicity, property which may explain fungus persistence