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Use ofl-buthionine sulfoximine for the efficient expression of disulfide-containing proteins in cell-free extracts ofEscherichia coli

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Abstract

We have developed a technique to improve the formation of correct disulfide bonds within cell-free synthesized proteins. Via the use of a metabolic inhibitor of glutamate-cysteine ligase, the accumulation of glutathione was effectively prevented in cell-free extracts, thereby enabling the stable maintenance of redox potential for extended reaction periods. As a result, in a reaction in which a model protein contatining 9 disulfide bonds was synthesized under cell-free conditions, the final amount of active protein products was increased by 50%. The method presented in this study will provide a rapid and robust route to the high-throughput expression and screening of proteins which require multiple disulfide bonds for their activity.

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Authors and Affiliations

  1. School of Chemical and Biological Engineering, College of Engineering, Seoul National University, 151-742, Seoul, Korea

    In-Seok Oh, Tae-Wan Kim, Jung-Won Keum & Cha-Yong Choi

  2. Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, 151-742, Seoul, Korea

    **-Ho Ahn & Cha-Yong Choi

  3. Department of Fine Chemical Engineering and Chemistry, Chungnam National University, 305-764, Daejeon, Korea

    Dong-Myung Kim

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  1. In-Seok Oh
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  2. Tae-Wan Kim
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  3. **-Ho Ahn
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  4. Jung-Won Keum
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  5. Cha-Yong Choi
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  6. Dong-Myung Kim
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Correspondence to Dong-Myung Kim.

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Oh, IS., Kim, TW., Ahn, JH. et al. Use ofl-buthionine sulfoximine for the efficient expression of disulfide-containing proteins in cell-free extracts ofEscherichia coli . Biotechnol. Bioprocess Eng. 12, 574–578 (2007). https://doi.org/10.1007/BF02931357

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  • DOI: https://doi.org/10.1007/BF02931357

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