Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 inEscherichia coli and its purification

A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutamin ase (EC 3.5.1.2) hom marine bacterium Micrococcus luteus K-3 was constructe d. pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing th e structural gene synthesized by polymerase chain reaction into the downstr eam region of the tac promoter of expression vector pKK223-3. The translati onal start codon was located 10 bases downstream of the Shine-Dalgarno sequ ence (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 t hrough three column chromatography steps with a final yield of 17.1%. The r ecombinant enzyme showed the same enzymatic properties, including salt tole rance, 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 crystall ization analysis. (C) 1999 Academic Press.