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

17 Citations (Scopus)

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
Pages (from-to)155-161
Number of pages7
JournalProtein Expression and Purification
Volume15
Issue number2
DOIs
Publication statusPublished - Jan 1 1999

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Micrococcus luteus
Glutaminase
Escherichia coli
Salt-Tolerance
Initiator Codon
Enzymes
Crystallization
Genetic Promoter Regions
Chromatography
Plasmids
X-Rays
Bacteria
Polymerase Chain Reaction
DNA
Genes
salt-tolerant glutaminase

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification. / Nandakumar, Renu; Wakayama, Mamoru; Nagano, Yoshio; Kawamura, Tatsuro; Sakai, Kenji; Moriguchi, Mitsuaki.

In: Protein Expression and Purification, Vol. 15, No. 2, 01.01.1999, p. 155-161.

Research output: Contribution to journalArticle

Nandakumar, Renu ; Wakayama, Mamoru ; Nagano, Yoshio ; Kawamura, Tatsuro ; Sakai, Kenji ; Moriguchi, Mitsuaki. / Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification. In: Protein Expression and Purification. 1999 ; Vol. 15, No. 2. pp. 155-161.
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