Isolation and characterisation of a gene encoding protein disulphide isomerase, pdiA, from Aspergillus niger.
Current strategies to improve the secretion of heterologous proteins from Aspergillus niger include the manipulation of chaperones and foldases specific to the endoplasmic reticulum (ER). Here we report the isolation of a gene, pdiA, encoding a putative protein disulphide isomerase (PDI) from A. niger using the Saccharomyces cerevisiae PDI gene as a probe. Sequencing of a genomic clone and RT-PCR products predict a 515-aa protein comprising a 20-aa ER-translocation signal sequence and a 495-aa mature protein (Mr = 54.3 kDa). The predicted protein also contains two thiol oxidoreductase active sites with a -CGHC- motif and a carboxy terminal -HDEL ER-retention signal. Three introns were identified within the pdiA gene and Southern- and dot-blot analysis indicates that the gene is present in a single copy. Northern-blot analysis shows a transcript of the predicted size. Sequence homology to a motif associated with protein trafficking and the induction of chaperones has been identified in the pdiA promoter. Transcription of pdiA is induced 3-4-fold after treatment with tunicamycin, an inhibitor of N-linked glycosylation. The kinetics of induction suggest that pdiA expression is not part of the primary stress response.
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Genetics and Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
Jeenes
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Archer
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D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001234:Aspergillus niger / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D007438:Introns; D007535:Isomerases / Q000235:genetics*; D007700:Kinetics; D018832:Molecular Chaperones / Q000235:genetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014415:Tunicamycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; D014415:Tunicamycin; D010088:Oxidoreductases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases