Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification.
A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutaminase (EC 3.5.1.2) from marine bacterium Micrococcus luteus K-3 was constructed. pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing the structural gene synthesized by polymerase chain reaction into the downstream region of the tac promoter of expression vector pKK223-3. The translational start codon was located 10 bases downstream of the Shine-Dalgarno sequence (AGGA) of pKK223-3. Escherichia coli JM109 transformed with pKSGHE3-1 exhibited more than 190-fold higher glutaminase activity than M. luteus K-3 under optimal culture conditions. The enzyme was purified to homogeneity through three column chromatography steps with a final yield of 17.1%. The recombinant enzyme showed the same enzymatic properties, including salt tolerance, as those of M. luteus K-3. This glutaminase expression system allows the production of sufficient quantities of glutaminase for basic structure-function studies including chemical modification and future X-ray crystallization analysis.
Department of Applied Chemistry, Faculty of Engineering, Oita, 870-1192, Japan.
R
R
Nandakumar
M
M
Wakayama
Y
Y
Nagano
T
T
Kawamura
K
K
Sakai
M
M
Moriguchi
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D005972:Glutaminase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016982:Micrococcus luteus / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid; D012965:Sodium Chloride / Q000494:pharmacology
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D005972:Glutaminase