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Cell-Free Translation of Biofuel Enzymes

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Cell-Free Protein Synthesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1118))

Abstract

In nature, bacteria and fungi are able to utilize recalcitrant plant materials by secreting a diverse set of enzymes. While genomic sequencing efforts offer exhaustive lists of genes annotated as potential polysaccharide-degrading enzymes, biochemical and functional characterizations of the encoded proteins are still needed to realize the full potential of this natural genomic diversity. This chapter outlines an application of wheat germ cell-free translation to the study of biofuel enzymes using genes from Clostridium thermocellum, a model cellulolytic organism. Since wheat germ extract lacks enzymatic activities that can hydrolyze insoluble polysaccharide substrates and is likewise devoid of enzymes that consume the soluble sugar products, the cell-free translation reactions provide a clean background for production and study of the reactions of biofuel enzymes. Examples of assays performed with individual enzymes or with small sets of enzymes obtained directly from cell-free translation are provided.

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Acknowledgments

This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494). The authors thank researchers in the University of Wisconsin Center for Eukaryotic Structural Genomics for advice and timely access to protocols and equipment (NIGMS U54 GM074901, U54 GM094584, U01 GM094622; J.L. Markley, G.N Phillips, B.G. Fox). The authors thank Dr. Paul Weimer (University of Wisconsin–Madison, Dept. of Bacteriology) for his scientific insights and the generous gifts of phosphoric acid-swollen cellulose and C. thermocellum genomic DNA, Dr. Bruce Dale for the generous gift of AFEX-pretreated switchgrass, Dr. Masood Hadi for the generous gift of ionic liquid-pretreated switchgrass, and Dr. George Phillips and Dr. Chris Bianchetti (Great Lakes Bioenergy Research Center) for the X-ray diffraction analysis of some cellulose substrates.

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Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin, Madison, WI, USA

    Taichi E. Takasuka, Johnnie A. Walker, Lai F. Bergeman, Kirk A. Vander Meulen, Shin-ichi Makino, Nathaniel L. Elsen & Brian G. Fox

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  1. Taichi E. Takasuka
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  2. Johnnie A. Walker
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  3. Lai F. Bergeman
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  4. Kirk A. Vander Meulen
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  5. Shin-ichi Makino
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  6. Nathaniel L. Elsen
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Editor information

Editors and Affiliations

  1. Institute for Molecular Bioscience (IMB), The University of Queensland, St. Lucia, Australia

    Kirill Alexandrov

  2. Institute for Molecular Bioscience (IMB), The University of Queensland, St. Lucia, Australia

    Wayne A. Johnston

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Takasuka, T.E. et al. (2014). Cell-Free Translation of Biofuel Enzymes. In: Alexandrov, K., Johnston, W. (eds) Cell-Free Protein Synthesis. Methods in Molecular Biology, vol 1118. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-782-2_5

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  • DOI: https://doi.org/10.1007/978-1-62703-782-2_5

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-781-5

  • Online ISBN: 978-1-62703-782-2

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