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Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis

Authors

  • C. Eric Hodgman,

    1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL
    2. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL
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  • Michael C. Jewett

    Corresponding author
    1. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL
    2. Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL
    • Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL
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Correspondence to: M.C. Jewett

ABSTRACT

Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7 ± 0.5 µg mL−1 with batch reactions lasting up to 2 h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (µg protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from ∼2 × 10−4 to ∼4 × 10−1 µg $−1, which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries. Biotechnol. Bioeng. 2013;110: 2643–2654. © 2013 Wiley Periodicals, Inc.

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