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Molecular characterization of a PDI-related gene prpA in Aspergillus niger var. awamori
Abstract:
A gene (prpA) homologous to the protein disulfide isomerase gene was isolated from Aspergillus niger by Southern hybridization using the pdi1 gene isolated from Trichoderma reesei as a DNA probe. The corresponding cDNA of the prpA gene has also been isolated from an A. niger var. awamori cDNA library. The prpA gene does not belong to any currently recognized family of protein disulfide isomerases since it contains only a single conserved thioredoxin domain at the N-terminus of the protein. The C-terminal two-thirds of the protein has no homology to any known proteins in the database. The PRPA protein contains an ER retention signal (HDEL) at its C-terminal end suggesting that it is located in the ER. Southern hybridization at high stringency showed that it was present as a single copy in the genome. Northern hybridization indicated that the transcript level of the prpA gene was higher if the cells were secreting a heterologous protein, bovine prochymosin. However, over-expression of the prpA gene from a multicopy integrated vector had little effect on chymosin secretion. A strain containing a deletion of the prpA gene was viable. However, deletion of the prpA gene appeared to cause a reduction of bovine chymosin production.
Insights
Researchers identified a novel Aspergillus niger gene, prpA, encoding a unique protein disulfide isomerase-like protein. Deletion of prpA reduced bovine prochymosin secretion, suggesting its role in protein production.
Area of Science:
- Molecular Biology
- Biochemistry
- Fungal Genetics
Background:
- Protein disulfide isomerases (PDIs) are crucial for protein folding.
- A novel PDI-homologous gene, prpA, was identified in Aspergillus niger.
- The prpA gene product (PRPA) has a unique structure with a single thioredoxin domain and an ER retention signal.
Purpose of the Study:
- To characterize the novel prpA gene and its encoded protein in Aspergillus niger.
- To investigate the function of PRPA in protein folding and secretion, particularly for heterologous proteins like bovine prochymosin.
- To determine the impact of prpA gene manipulation on cellular viability and protein production.
Main Methods:
- Southern and Northern hybridization were used to analyze prpA gene expression and copy number.
- cDNA library screening was performed to isolate the prpA gene and its transcript.
- Gene deletion and overexpression studies were conducted to assess the functional role of prpA.
Main Results:
- The prpA gene is present as a single copy in the A. niger genome.
- prpA transcript levels increased when cells secreted bovine prochymosin.
- Deletion of prpA reduced bovine prochymosin production, while overexpression had minimal impact.
Conclusions:
- The prpA gene encodes a unique PDI-like protein localized to the endoplasmic reticulum.
- PRPA plays a role in efficient secretion of heterologous proteins, influencing bovine prochymosin production.
- The prpA gene is essential for optimal protein secretion but not for cellular viability in Aspergillus niger.