Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40.
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.
Masuo
Naohisa
N
Department of Applied Chemistry, Faculty of Engineering, Oita University, Dannoharu 700, Oita 870-1192, Japan.
Ito
Kotaro
K
Yoshimune
Kazuaki
K
Hoshino
Mitsuyo
M
Matsushima
Kenichiro
K
Koyama
Yasuji
Y
Moriguchi
Mitsuaki
M
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
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005972:Glutaminase