Molecular Biology and Genetics

Microbial Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 11 No. 4, Issue of October 15, 2008
© 2008 by Pontificia Universidad Católica de Valparaíso -- Chile Received October 17, 2007 / Accepted May 22, 2008
DOI: 10.2225/vol11-issue4-fulltext-5

Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa: solving the structure - function anomaly of a reported aminopeptidase

Mohammed M. Islam#
Enzyme Laboratory
National Food Research Institute
Tsukuba, 2-1-2 Kannondai
Ibaraki 305-8642, Japan
Tel: 61-3-5320-2041
Fax: 61-3-5320-2408

Financial support: This work was supported by the Science and Technology Agency (STA) fellowship sponsored by Japan International Science & Technology Exchange Centre (JISTEC).

Keywords: 5’-RACE, cDNA, genomic DNA, maitake, refolding.

#Present address: Ballarat Cancer Research Centre, University of Ballarat, c/o-St. John of God Hospital, 101 Drummond Street, Ballarat, VIC 3350, Australia. Tel: 61-3-5320-2041; Fax: 61-3-5320-2408.


CAPS: N-cyclohexyl-3-aminopropanesulfonic acid
cDNA: omplementary DNA to mRNA
CHES: N-cyclohexyl-2-aminoethanesulfonic acid.
DTT: dithiothreitol
FPLC: fast protein liquid chromatography
IPTG: isopropylthio-β-D-galactoside
LB: Luria-Bertani
MES: 2-(N-morpholino)ethanesulfonic acid
Ni-NTA: Nickel-nitriloacetic acid
ORF: open reading frame
PCR: polymerase chain reaction
PMSF: phenylmethanesulphonylfluoride
PVDF: polyvinylidene fluoride
RACE: rapid amplification of cDNA ends
SDS-PAGE: sodium dodecyl sulfate - polyacrylamide gel electrophoresis
TAPS: N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid
UTR: untranslated region

Abstract   Full Text

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.

Supported by UNESCO / MIRCEN network