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The PURE system for the cell-free synthesis of membrane proteins

Nature Protocols volume 10, pages 1328–1344 (2015)Cite this article

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A Corrigendum to this article was published on 25 February 2016

This article has been updated

Abstract

Cell-free gene expression systems are biotechnological tools for the in vitro production of proteins of interest. The addition of membrane vesicles (liposomes) enables the production of membrane proteins, including those in large-molecular-weight complexes, such as the SecYEG translocon or ATP synthase. Here we describe a protocol for the cell-free synthesis of membrane proteins using the protein synthesis using recombinant elements (PURE) system, and for subsequent quantification of products and analyses of membrane localization efficiency, product orientation in the membrane and complex formation in the membrane. In addition, measurements of ATP synthase activity are used as an example to demonstrate the functional nature of the cell-free synthesized proteins. This protocol allows the rapid production and the detailed analysis of membrane proteins, and the complete process from template DNA preparation to activity measurement can be accomplished within 1 d. In contrast to alternative methods using living cells, this protocol can also help to prevent the difficulties in membrane protein purification and the risks of protein aggregation during reconstitution into lipid membranes.

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Figure 1: Schematic overview of the workflow for the cell-free synthesis of membrane proteins in the PURE system.
Figure 2: Liposome preparation using Bio-Beads (Step 8B(iv)).
Figure 3: SecYEG synthesized using the PURE system.
Figure 4: FoF1-ATP synthase synthesized in the PURE system.

Change history

  • 06 January 2016

     In the version of this article initially published, the units for concentration of the FD component listed in Table 1 are incorrect. The correct unit should be µg/ml. The spelling of the 'PUREflex' kit was also incorrect and has been changed to PUREfrex throughout. These errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas (grant numbers 26119704 and 26103511 to Y.K.) and the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, to T.U. We thank T. Suzuki of Waseda University for valuable advice about FoF1-ATP synthase, H. Matsubayashi for the SecYEG translocon data and T. Kanamori of GeneFrontier for providing the PURE system.

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Authors and Affiliations

  1. Earth-Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan

    Yutetsu Kuruma

  2. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan

    Yutetsu Kuruma & Takuya Ueda

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  1. Yutetsu Kuruma
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  2. Takuya Ueda
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Contributions

Y.K. developed the methods presented in this study, and validated the protocol, wrote the manuscript and prepared the figures. T.U. supervised all of the work and prepared the final version of the manuscript.

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Correspondence to Yutetsu Kuruma.

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Kuruma, Y., Ueda, T. The PURE system for the cell-free synthesis of membrane proteins. Nat Protoc 10, 1328–1344 (2015). https://doi.org/10.1038/nprot.2015.082

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