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

研究成果: ジャーナルへの寄稿学術誌査読

20 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)155-161
ページ数7
ジャーナルProtein Expression and Purification
15
2
DOI
出版ステータス出版済み - 3月 1999
外部発表はい

!!!All Science Journal Classification (ASJC) codes

  • バイオテクノロジー

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