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

41 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 2006

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Lyases
Crystallization
Methionine
Antineoplastic Agents
Enzymes
Plasmids
Endotoxins
Crystallizers
Column chromatography
Pyridoxal Phosphate
Initiator Codon
Ammonium Sulfate
Bioreactors
Binding sites
Sodium chloride
Nucleotides
Polishing
Ribosomes
Genetic Promoter Regions
Sodium Chloride

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering
  • Microbiology (medical)

Cite this

High-level expression and bulk crystallization of recombinant L-methionine γ-lyase, an anticancer agent. / Takakura, Tomoaki; Ito, Takaomi; Yagi, Shigeo; Notsu, Yoshihide; Itakura, Takashi; Nakamura, Takumi; Inagaki, Kenji; Esaki, Nobuyoshi; Hoffman, Robert M.; Takimoto, Akio.

In: Applied Microbiology and Biotechnology, Vol. 70, No. 2, 03.2006, p. 183-192.

Research output: Contribution to journalArticle

Takakura, T, Ito, T, Yagi, S, Notsu, Y, Itakura, T, Nakamura, T, Inagaki, K, Esaki, N, Hoffman, RM & Takimoto, A 2006, 'High-level expression and bulk crystallization of recombinant L-methionine γ-lyase, an anticancer agent', Applied Microbiology and Biotechnology, vol. 70, no. 2, pp. 183-192. https://doi.org/10.1007/s00253-005-0038-2
Takakura, Tomoaki ; Ito, Takaomi ; Yagi, Shigeo ; Notsu, Yoshihide ; Itakura, Takashi ; Nakamura, Takumi ; Inagaki, Kenji ; Esaki, Nobuyoshi ; Hoffman, Robert M. ; Takimoto, Akio. / High-level expression and bulk crystallization of recombinant L-methionine γ-lyase, an anticancer agent. In: Applied Microbiology and Biotechnology. 2006 ; Vol. 70, No. 2. pp. 183-192.
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