title: Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40.
abstract: We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote.
authors.affiliation: Department of Applied Chemistry, Faculty of Engineering, Oita University, Dannoharu 700, Oita 870-1192, Japan.
authors.forename: Naohisa
authors.initials: N
authors.lastname: Masuo
authors.forename: Kotaro
authors.initials: K
authors.lastname: Ito
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Yoshimune
authors.forename: Mitsuyo
authors.initials: M
authors.lastname: Hoshino
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Matsushima
authors.forename: Yasuji
authors.initials: Y
authors.lastname: Koyama
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Moriguchi
mesh_terms: D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D005972:Glutaminase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016982:Micrococcus luteus / Q000201:enzymology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005972:Glutaminase