High-level expression and bulk crystallization of recombinant L-methionine γ-lyase, an anticancer agent

Tomoaki Takakura, Takaomi Ito, Shigeo Yagi, Yoshihide Notsu, Takashi Itakura, Takumi Nakamura, Kenji Inagaki, Nobuyoshi Esaki, Robert M. Hoffman, Akio Takimoto

研究成果査読

56 被引用数 (Scopus)

抄録

L-Methionine γ-lyase is a pyridoxal 5′-phosphate-dependent enzyme which has tumor selective anticancer activity. An efficient production process for the recombinant enzyme was constructed by using the overexpression plasmid in Escherichia coli, large-scale cultivation, and practical crystallization on an industrial scale. The plasmid was optimized with a promoter and the region of the ribosome-binding site. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The transformants produced the enzyme, which intracellularly accumulated at 2.1 mg/ml as an active form and accounted for 43% of the total proteins in the soluble fraction by simple batch fermentation using a 500-1 fermentor. The crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100-1 crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. We prepared 600 g of purified enzyme with a low endotoxin content of sufficient quality for therapeutical use, with a 41% overall yield in the purification process.

本文言語English
ページ(範囲)183-192
ページ数10
ジャーナルApplied Microbiology and Biotechnology
70
2
DOI
出版ステータスPublished - 3月 2006

ASJC Scopus subject areas

  • バイオテクノロジー
  • 応用微生物学とバイオテクノロジー

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