Cell-free complements in vivo expression of the E. coli membrane proteome

Authors

  • David F. Savage,

    1. Graduate Group in Biophysics, University of California at San Francisco, San Francisco, California 94158-2517, USA
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    • These authors contributed equally to this work.

  • Corey L. Anderson,

    1. Centre for Structure of Membrane Proteins, University of California at San Francisco, San Francisco, California 94158-2517, USA
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    • These authors contributed equally to this work.

  • Yaneth Robles-Colmenares,

    1. Centre for Structure of Membrane Proteins, University of California at San Francisco, San Francisco, California 94158-2517, USA
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  • Zachary E. Newby,

    1. Chemistry and Chemical Biology Graduate Program, University of California at San Francisco, San Francisco, California 94158-2517, USA
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  • Robert M. Stroud

    Corresponding author
    1. Centre for Structure of Membrane Proteins, University of California at San Francisco, San Francisco, California 94158-2517, USA
    • Department of Biochemistry and Biophysics, University of California at San Francisco, 600 16th Street, San Francisco, CA 94158-2517, USA; fax: (415) 476-1902.
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Abstract

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.

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