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

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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-l 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-l 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.

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Acknowledgements

The authors thank Mr. Kensaku Akita for his helpful suggestions. We are also grateful to our colleagues at the Discovery Research Laboratories and the Manufacturing Technology R & D Laboratories of Shionogi & Co., Ltd. for their warm support.

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Authors and Affiliations

  1. Discovery Research Laboratories, Shionogi & Co., Ltd., 1-3, Kuise Terajima 2-chome, Amagasaki, Hyogo, 660-0813, Japan

    Tomoaki Takakura, Takaomi Ito, Yoshihide Notsu, Takashi Itakura, Takumi Nakamura & Akio Takimoto

  2. AntiCancer Inc., 7917 Ostrow Street, San Diego, CA, 92111, USA

    Shigeo Yagi & Robert M. Hoffman

  3. Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama, Okayama, 700-8530, Japan

    Kenji Inagaki

  4. Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan

    Nobuyoshi Esaki

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  1. Tomoaki Takakura
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  2. Takaomi Ito
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  3. Shigeo Yagi
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  4. Yoshihide Notsu
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  5. Takashi Itakura
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  6. Takumi Nakamura
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  7. Kenji Inagaki
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  8. Nobuyoshi Esaki
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  9. Robert M. Hoffman
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  10. Akio Takimoto
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Correspondence to Tomoaki Takakura.

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Takakura, T., Ito, T., Yagi, S. et al. High-level expression and bulk crystallization of recombinant l-methionine γ-lyase, an anticancer agent. Appl Microbiol Biotechnol 70, 183–192 (2006). https://doi.org/10.1007/s00253-005-0038-2

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  • DOI: https://doi.org/10.1007/s00253-005-0038-2

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