Jeenes D J, Pfaller R, Archer D B
Department of Genetics and Microbiology, Institute of Food Research, Colney, Norwich, UK.
Gene. 1997 Jul 9;193(2):151-6. doi: 10.1016/s0378-1119(97)00098-x.
Current strategies to improve the secretion of heterologous proteins in Aspergillus niger include the manipulation of chaperones and foldases specific to the endoplasmic reticulum (ER). A family of ER-specific proteins which share active-site homology wit protein disulfide isomerase (PDI) has been identified from other systems, many of which are inducible 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 proteins. TIGA and ERp38 show 66% identity at the amino acid level and are putative ER proteins. Both proteins show tandemly linked thiol-oxidoreductase domains followed by a functionally uncharacterised C-terminal domain. The most distal active site in TIGA is created by excision of a 66-bp intron. Although no Unfolded Protein Response elements can be seen in the tigA promoter, sequence homology has identified associated with protein trafficking (ERPTRE) in a gene encoding the related mammalian protein, ERp72, as well as a second motif conserved amongst the glucose-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. Transcription of tigA is induced 2-3-fold after treatment with tunicamycin, an inhibitor of N-linked glycosylation. Strains overexpressing a heterologous protein show no increased tigA mRNA levels.
目前，提高黑曲霉中异源蛋白分泌的策略包括对内质网（ER）特异性伴侣蛋白和折叠酶的调控。已从其他系统中鉴定出一类与蛋白二硫键异构酶（PDI）具有活性位点同源性的内质网特异性蛋白，其中许多可被导致内质网中蛋白质错误折叠的试剂诱导。在此，我们报告了从黑曲霉中鉴定出的tigA和从粗糙脉孢菌中鉴定出的erp38，它们是PDI蛋白超家族的两个新成员。TIGA和ERp38在氨基酸水平上具有66%的同一性，并且是假定的内质网蛋白。这两种蛋白均显示串联连接的硫醇氧化还原酶结构域，随后是功能未明确的C末端结构域。TIGA中最远端的活性位点是通过切除一个66bp的内含子产生的。尽管在tigA启动子中未发现未折叠蛋白反应元件，但序列同源性已鉴定出与相关哺乳动物蛋白ERp72编码基因中的蛋白转运（ERPTRE）相关，以及葡萄糖相关蛋白家族中保守的第二个基序。Southern杂交和斑点印迹分析表明，tigA基因以单拷贝形式存在。黑曲霉和粗糙脉孢菌的这两种蛋白均与应激诱导的苜蓿蛋白G1具有同源性。用衣霉素（一种N-连接糖基化抑制剂）处理后，tigA的转录诱导2-3倍。过表达异源蛋白的菌株未显示tigA mRNA水平增加。
