ISOLATION AND CHARACTERIZATION OF A GENE ENCODING PROTEIN DISULFIDE-ISOMERASE, PDIA, FROM ASPERGILLUS-NIGER

Current strategies to improve the secretion of heterologous proteins f rom Aspergillus niger include the manipulation of chaperones and folda ses specific to the endoplasmic reticulum (ER). Here we report the iso lation of a gene, pdiA, encoding a putative protein disulphide isomera se (PDI) from A. niger using the Saccharomyces cerevisiae PDI gene as a probe. Sequencing of a genomic clone and RT-PCR products predict a 5 15-aa protein comprising a 20-aa ER-translocation signal sequence and a 495-aa mature protein (M-r= 54.3 kDa). The predicted protein also co ntains two thiol oxidoreductase active sites with a -CGHC- motif and a carboxy terminal -HDEL ER-retention signal. Three introns were identi fied within the pdiA gene and Southern- and dot-blot analysis indicate s that the gene is present in a single copy. Northern-blot analysis sh ows a transcript of the predicted size. Sequence homology to a motif a ssociated with protein trafficking and the induction of chaperones has been identified in the pdiA promoter. Transcription of pdiA is induce d 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.