Isolation, characterization, and cloning of cDNA and the gene for an elastinolytic serine proteinase from Aspergillus flavus

M V Ramesh, T Sirakova, P E Kolattukudy
Infection and Immunity 1994, 62 (1): 79-85
An elastinolytic serine proteinase produced by Aspergillus flavus 28 that was isolated from a patient who died of aspergillosis has been purified and characterized. The enzyme was inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. The metal-chelating agents EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not severely inhibit the enzyme. A cDNA and a 2.95-kb segment of genomic DNA containing the proteinase gene were sequenced. The open reading frame that would code for a protein containing 403 amino acids was interrupted by three introns. The mature protein lacks 121 N-terminal amino acids including a putative 21-amino-acid signal peptide. The purified mature protein showed a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas that calculated from the deduced protein sequence was 30 kDa. This elastinolytic serine proteinase of A. flavus has 83 and 82% sequence homology to the similar proteinases from A. fumigatus and A. oryzae. The catalytic properties and the sequence homology around the putative catalytic amino acids suggest that this enzyme belongs to the serine proteinases of the subtilisin family.

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