- Title
- Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa : Solving the structure - Function anomaly of a reported aminopeptidase
- Creator
- Islam, Mohammed
- Date
- 2008
- Type
- Text; Journal article
- Identifier
- http://researchonline.federation.edu.au/vital/access/HandleResolver/1959.17/31530
- Identifier
- vital:426
- Identifier
-
https://doi.org/10.2225/vol11-issue4-fulltext-5
- Identifier
- ISSN:0717-3458
- Abstract
- The N-terminal amino acid sequence of an aminopeptidase from Japanese edible mushroom, Grifola frondosa, was reported to have high similarity with that of a serine proteinase from basidiomycete, Agaricus bisporous (Nishiwaki and Hayashi, 2001). The full-length cDNA and the corresponding genomic DNA of the enzyme were cloned, based on the reported N-terminal amino acid sequence. The predicted open reading frame (ORF) of the cloned cDNA, encoding a product of 379 amino acids, was expressed in E. coli using pET expression vector. The expressed pro-enzyme (40 kDa) underwent autolysis to produce the mature protein (30 kDa) and a pro-peptide (10 kDa). The mature protein and the pro-peptide remained tightly bound to each other and could not be separated by Ni-NTA metal affinity chromatography or Q-Sepharose ion-exchange chromatography. The enzyme was inactive in the bound form. Upon treatment with subtilisin, the bound pro-peptide was further hydrolyzed and a high serine proteinase activity was recovered. No aminopeptidase activity was detected at any stage of the protein processing. These results clearly indicated that the N-terminal amino acid sequence and the function of the reported aminopeptidase were not derived from the same protein entity and hence caused the structure-function anomaly. © 2008 by Pontificia Universidad Cato?lica de Valparai?so.
- Publisher
- Universidad Católica de Valparaíso and CONICYT
- Relation
- Electronic Journal of Biotechnology Vol. 11, no. 4 (2008), p.
- Rights
- Copyright Universidad Católica de Valparaíso and CONICYT
- Rights
- This metadata is freely available under a CCO license
- Subject
- 10 Technology; DNA; Genomic DNA; Maitake; Refolding
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