title: Cloning and characterization of a novel aspartic protease gene from marine-derived Metschnikowia reukaufii and its expression in E. coli.
abstract: Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant acid protease had milk clotting activity.
authors.affiliation: Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Chi
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Liu
authors.forename: Lixi
authors.initials: L
authors.lastname: Yue
authors.forename: Ying
authors.initials: Y
authors.lastname: Peng
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D055163:Metschnikowia / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases