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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

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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.

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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.

References

  1. Swartz, J. Developing cell-free biology for industrial applications. J. Ind. Microbiol. Biotechnol. 33, 476–485 (2006).

    Article  CAS  Google Scholar 

  2. Terada, T., Murata, T., Shirouzu, M. & Yokoyama, S. Cell-free expression of protein complexes for structural biology. Methods Mol. Biol. 1091, 151–159 (2014).

    Article  CAS  Google Scholar 

  3. Takai, K., Sawasaki, T. & Endo, Y. Practical cell-free protein synthesis system using purified wheat embryos. Nat. Protoc. 5, 227–238 (2010).

    Article  CAS  Google Scholar 

  4. Jackson, R.J. & Hunt, T. Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 96, 50–74 (1983).

    Article  CAS  Google Scholar 

  5. Ezure, T., Suzuki, T., Shikata, M., Ito, M. & Ando, E. A cell-free protein synthesis system from insect cells. Methods Mol. Biol. 607, 31–42 (2010).

    Article  Google Scholar 

  6. Mikami, S., Kobayashi, T., Masutani, M., Yokoyama, S. & Imataka, H. A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr. Purif. 62, 190–198 (2008).

    Article  CAS  Google Scholar 

  7. Carlson, E.D., Gan, R., Hodgman, C.E. & Jewett, M.C. Cell-free protein synthesis: applications come of age. Biotechnol. Adv. 30, 1185–1194 (2012).

    Article  CAS  Google Scholar 

  8. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).

    Article  CAS  Google Scholar 

  9. Shimizu, Y., Kanamori, T. & Ueda, T. Protein synthesis by pure translation systems. Methods 36, 299–304 (2005).

    Article  CAS  Google Scholar 

  10. Kuruma, Y., Stano, P., Ueda, T. & Luisi, P.L. A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. Biochim. Biophys. Acta 1788, 567–574 (2009).

    Article  CAS  Google Scholar 

  11. Kuruma, Y., Suzuki, T., Ono, S., Yoshida, M. & Ueda, T. Functional analysis of membranous Fo-a subunit of F1Fo-ATP synthase by in vitro protein synthesis. Biochem. J. 442, 631–638 (2012).

    Article  CAS  Google Scholar 

  12. Ozaki, Y., Suzuki, T., Kuruma, Y., Ueda, T. & Yoshida, M. UncI protein can mediate ring-assembly of c-subunits of FoF1-ATP synthase in vitro. Biochem. Biophys. Res. Commun. 367, 663–666 (2008).

    Article  CAS  Google Scholar 

  13. Noireaux, V. & Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl. Acad. Sci. USA 101, 17669–17674 (2004).

    Article  CAS  Google Scholar 

  14. Kalmbach, R. et al. Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. J. Mol. Biol. 371, 639–648 (2007).

    Article  CAS  Google Scholar 

  15. Soga, H. et al. In vitro membrane protein synthesis inside cell-sized vesicles reveals the dependence of membrane protein integration on vesicle volume. ACS Synth. Biol. 3, 372–379 (2014).

    Article  CAS  Google Scholar 

  16. Matsubayashi, H., Kuruma, Y. & Ueda, T. In vitro synthesis of E. coli Sec translocon from DNA. Angew. Chem. Int. Ed. 53, 7535–7538 (2014).

    Article  CAS  Google Scholar 

  17. Kuruma, Y., Suzuki, T. & Ueda, T. Production of multi-subunit complexes on liposome through an E. coli cell-free expression system. Methods Mol. Biol. 607, 161–171 (2010).

    Article  CAS  Google Scholar 

  18. Niwa, T. et al. Bimodal protein solubility distribution revealed by an aggregation analysis of the entire ensemble of Escherichia coli proteins. Proc. Natl. Acad. Sci. USA 106, 4201–4206 (2009).

    Article  CAS  Google Scholar 

  19. Soga, N., Kinosita, K. Jr., Yoshida, M. & Suzuki, T. Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase. FEBS J. 278, 2647–2654 (2011).

    Article  CAS  Google Scholar 

  20. Ohashi, H., Shimizu, Y., Ying, B.W. & Ueda, T. Efficient protein selection based on ribosome display system with purified components. Biochem. Biophys. Res. Commun. 352, 270–276 (2007).

    Article  CAS  Google Scholar 

  21. Shimizu, Y., Kuruma, Y., Kanamori, T. & Ueda, T. The PURE system for protein production. Methods Mol. Biol. 1118, 275–284 (2014).

    Article  CAS  Google Scholar 

  22. Fujii, S. et al. Liposome display for in vitro selection and evolution of membrane proteins. Nat. Protoc. 9, 1578–1591 (2014).

    Article  CAS  Google Scholar 

  23. Fujiwara, K., Katayama, T. & Nomura, S.M. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system. Nucleic Acids Res. 41, 7176–7183 (2013).

    Article  CAS  Google Scholar 

  24. Asahara, H. & Chong, S. In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme. Nucleic Acids Res. 38, e141 (2010).

    Article  Google Scholar 

  25. Chizzolini, F., Forlin, M., Cecchi, D. & Mansy, S.S. Gene position more strongly influences cell-free protein expression from operons than T7 transcriptional promoter strength. ACS Synth. Biol. 3, 363–371 (2014).

    Article  CAS  Google Scholar 

  26. Schuler, M.A., Denisov, I.G. & Sligar, S.G. Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol. Biol. 974, 415–433 (2013).

    Article  CAS  Google Scholar 

  27. du Plessis, D.J., Nouwen, N. & Driessen, A.J. The Sec translocase. Biochim. Biophys. Acta 1808, 851–865 (2011).

    Article  CAS  Google Scholar 

  28. Kedrov, A., Kusters, I., Krasnikov, V.V. & Driessen, A.J. A single copy of SecYEG is sufficient for preprotein translocation. EMBO J. 30, 4387–4397 (2011).

    Article  CAS  Google Scholar 

  29. Schagger, H. & von Jagow, G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223–231 (1991).

    Article  CAS  Google Scholar 

  30. Fillingame, R.H., Jiang, W. & Dmitriev, O.Y. Coupling H+ transport to rotary catalysis in F-type ATP synthases: structure and organization of the transmembrane rotary motor. J. Exp. Biol. 203, 9–17 (2000).

    CAS  PubMed  Google Scholar 

  31. Mitome, N., Suzuki, T., Hayashi, S. & Yoshida, M. Thermophilic ATP synthase has a decamer c-ring: indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling. Proc. Natl. Acad. Sci. USA 101, 12159–12164 (2004).

    Article  CAS  Google Scholar 

  32. Diez, M. et al. Proton-powered subunit rotation in single membrane-bound FoF1-ATP synthase. Nat. Struct. Mol. Biol. 11, 135–141 (2004).

    Article  CAS  Google Scholar 

  33. Aggeler, R., Ogilvie, I. & Capaldi, R.A. Rotation of a γ-ɛ subunit domain in the Escherichia coli F1F0-ATP synthase complex. The gamma-epsilon subunits are essentially randomly distributed relative to the α3β3δ domain in the intact complex. J. Biol. Chem. 272, 19621–19624 (1997).

    Article  CAS  Google Scholar 

  34. Shimizu, Y. & Ueda, T. PURE technology. Methods Mol. Biol. 607, 11–21 (2010).

    Article  CAS  Google Scholar 

  35. Green, M.R. & Sambrook, J. Molecular Cloning: A Laboratory Manual 4th edn. (Cold Spring Harbor Laboratory Press, 2012).

  36. Veenendaal, A.K., van der Does, C. & Driessen, A.J. The protein-conducting channel SecYEG. Biochim. Biophys. Acta 1694, 81–95 (2004).

    Article  CAS  Google Scholar 

  37. Weber, J. ATP synthase: subunit-subunit interactions in the stator stalk. Biochim. Biophys. Acta 1757, 1162–1170 (2006).

    Article  CAS  Google Scholar 

  38. Suzuki, T. et al. FoF1-ATPase/synthase is geared to the synthesis mode by conformational rearrangement of epsilon subunit in response to proton motive force and ADP/ATP balance. J. Biol. Chem. 278, 46840–46846 (2003).

    Article  CAS  Google Scholar 

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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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