Characterization of a Foldase, Protein Disulfide Isomerase A, in the Protein Secretory Pathway of Aspergillus niger
| Autor: | P.J. Punt, David B. Archer, C. Ngiam, C.A.M.J.J. van den Hondel, David J. Jeenes |
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| Jazyk: | angličtina |
| Rok vydání: | 2000 |
| Předmět: |
Protein Denaturation
Protein Folding Transcription Genetic Saccharomyces cerevisiae Protein Disulfide-Isomerases Down-Regulation Genetics and Molecular Biology Endoplasmic Reticulum Applied Microbiology and Biotechnology Fungal Proteins Transformation Genetic Gene Expression Regulation Fungal Microsomes Gene expression HSP70 Heat-Shock Proteins RNA Messenger Protein disulfide-isomerase Secretory pathway Calcimycin Heat-Shock Proteins Fungal protein Ecology biology Endoplasmic reticulum Ribonuclease Pancreatic biology.organism_classification Actins Recombinant Proteins Dithiothreitol Secretory protein Biochemistry Foldase Aspergillus niger Food Science Biotechnology |
| Popis: | Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA , encoding PDIA was previously isolated from Aspergillus niger , and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces cerevisiae Δpdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA , suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae , overexpression of PDIA did not increase secreted yields of HEWL in A. niger , suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%. |
| Databáze: | OpenAIRE |
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