Open AccessMolecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase from Aspergillus fumigatus
Author(s) -
Reichard Utz,
Monod Michel,
Rüchel Reinhard
Publication year - 1995
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1995.tb07700.x
Subject(s) - biology , microbiology and biotechnology , genomic dna , aspergillus fumigatus , nucleic acid sequence , gene , complementary dna , genomic library , molecular cloning , sequence analysis , peptide sequence , aspergillus awamori , genetics , biochemistry , enzyme
Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus fumigatus . The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre‐proenzyme deduced has an N‐terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N‐terminal sequence of the mature enzyme matched the N‐terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre‐proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori . Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.