ISOLATION AND CHARACTERIZATION OF A NOVEL STRESS-INDUCIBLE PDI-FAMILYGENE FROM ASPERGILLUS-NIGER

Current strategies to improve the secretion of heterologous proteins i n Aspergillus niger include the manipulation of chaperones and foldase s specific to the endoplasmic reticulum (ER). A family of ER-specific protein s which share active-site homology wit protein disulfide isome rase (PDI) has been identified from other systems, many of which are i nducible by agents which cause malfolding of proteins in the ER. Here we report identification of tigA from Aspergillus niger and erp38 from Neurospora crassa, two novel members of the PDI superfamily of protei ns. TIGA and ERp38 show 66% identity at the amino acid level and are p utative ER proteins. Both proteins show tandemly linked thiol-oxidored uctase domains followed by a functionally uncharacterised C-terminal d omain. The most distal active site in TIGA is created by excision of a 66-bp intron. Although no Unfolded Protein Response elements can be s een in the tigA promoter, sequence homology has identified associated with protein trafficking (ERPTRE) in a gene encoding the related mamma lian protein, ERp72, as well as a second motif conserved amongst the g lucose-related protein family. Southern and dot blot analysis indicate that the tigA gene is present in single copy. Both the A. niger and N . crassa proteins show homology with a stress-inducible alfalfa, G1. T ranscription of tigA is induced 2-3-fold after treatment with tunicamy cin, an inhibitor of N-linked glycosylation. Strains overexpressing a heterologous protein show no increased tigA mRNA levels. (C) 1997 Else vier Science B.V.