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

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)183-192
Number of pages10
JournalApplied Microbiology and Biotechnology
Volume70
Issue number2
DOIs
Publication statusPublished - Mar 1 2006

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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    Takakura, T., Ito, T., Yagi, S., Notsu, Y., Itakura, T., Nakamura, T., Inagaki, K., Esaki, N., Hoffman, R. M., & Takimoto, A. (2006). High-level expression and bulk crystallization of recombinant L-methionine γ-lyase, an anticancer agent. Applied Microbiology and Biotechnology, 70(2), 183-192. https://doi.org/10.1007/s00253-005-0038-2