Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase from Aspergillus fumigatus

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...

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Bibliographic Details
Published in:FEMS microbiology letters 1995-07, Vol.130 (1), p.69-74
Main Authors: Reichard, Utz, Monod, Michel, Rüchel, Reinhard
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Aspartic Acid Endopeptidases - chemistry
Aspartic Acid Endopeptidases - genetics
Aspartic protease (EC 3.4.23)
Aspergillopepsin (EC 3.4.23.18)
Aspergillus fumigatus
Aspergillus fumigatus - enzymology
Aspergillus fumigatus - genetics
Base Sequence
Biological and medical sciences
Cloning, Molecular
DNA sequence
DNA, Fungal - analysis
Fundamental and applied biological sciences. Psychology
Gene, pep
Genes, Fungal - genetics
Genes. Genome
Isoelectric Point
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Molecular Weight
Protein Sorting Signals - genetics
Proteinase
Sequence Analysis, DNA
Sequence Homology, Amino Acid
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Summary: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.
ISSN:0378-1097
1574-6968
DOI:10.1111/j.1574-6968.1995.tb07700.x