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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 11, No. 4, 2008
Bioline Code: ej08051
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 11, No. 4, 2008

 en Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa check for this species in other resources : solving the structure - function anomaly of a reported aminopeptidase
Islam, M.M.

Abstract

The N-terminal amino acid sequence of an aminopeptidase from Japanese edible mushroom, Grifola frondosa check for this species in other resources , was reported to have high similarity with that of a serine proteinase from basidiomycete, Agaricus bisporous check for this species in other resources (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.

Keywords
5'-RACE, cDNA, genomic DNA, maitake, refolding.

 
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