Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis

Authors

  • Dong-Myung Kim,

    1. Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA; telephone: 650-723-5398; fax: 650-725-0555
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  • James R. Swartz

    Corresponding author
    1. Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA; telephone: 650-723-5398; fax: 650-725-0555
    • Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA; telephone: 650-723-5398; fax: 650-725-0555
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Abstract

A new approach for adenosine triphosphate (ATP) regeneration in a cell-free protein synthesis system is described. We first show that pyruvate can be used as a secondary energy source to replace or supplement the conventional secondary energy source, phosphoenol pyruvate (PEP). We also report that glucose-6-phosphate, an earlier intermediate of the glycolytic pathway, can be used for ATP regeneration. These new methods provide more stable maintenance of ATP concentration during protein synthesis. Because pyruvate and glucose-6-phosphate are the first and last intermediates of the glycolytic pathway, respectively, the results also suggest the possibility of using any glycolytic intermediate, or even glucose, for ATP regeneration in a cell-free protein synthesis system. As a result, the methods described provide cell-free protein synthesis with greater flexibility and cost efficiency. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 309–316, 2001.

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