Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus

An aspartic proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fr action of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS- PAGE, was not detected in the culture supernatant. A specific gene probe wa s designed on the basis of the N-terminal sequence of the native protein, a nd the PEP2 genomic and cDNA were isolated from corresponding libraries. Th e deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include th e two DTG-motifs of the active site common to almost all pepsin-like enzyme s; PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Sacc haromyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger. Recombinant PEP2 was overexpressed in Pichia pastori s and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morph ology. Identification of proteinases, such as PEP2, which are apparently as sociated with the Aspergillus cell wall raises new interest in these molecu les with respect to their possible function in the pathogenesis of invasive aspergillosis.