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

Renu Nandakumar, Mamoru Wakayama, Yoshio Nagano, Tatsuro Kawamura, Kenji Sakai, Mitsuaki Moriguchi

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)155-161
Number of pages7
JournalProtein Expression and Purification
Volume15
Issue number2
DOIs
StatePublished - Mar 1999

ASJC Scopus subject areas

  • Biotechnology

Fingerprint Dive into the research topics of 'Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification'. Together they form a unique fingerprint.

  • Cite this