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Practical cell-free protein synthesis system using purified wheat embryos

Nature Protocols volume 5, pages 227–238 (2010)Cite this article

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Abstract

Biochemical characterization of each gene product encoded in the genome is essential to understand how cells are regulated. The bottleneck has been and still is in how the gene products can be obtained. The wheat cell-free protein synthesis system we have developed is a powerful method for preparation of many different proteins at a time and also for preparation of large amounts of specific proteins for biochemical and structural analyses. Here, we show a method for preparation of the wheat embryo extract useful for the cell-free reactions, by which 5 ml of a high-activity extract is obtained in 4–5 d. We also describe the methods for small- and large-scale protein synthesis by hands-down operations with the use of mRNAs prepared by transcription of PCR products and pEU plasmids harboring the target cDNAs, which need 2–4 d excepting the time required for plasmid preparation.

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Figure 1
Figure 2: Schematic representations of DNA molecules.
Figure 3: Selection of good embryo particles by eye.
Figure 4: An example of the sample after the first 30,000g centrifugation.
Figure 5: A schematic representation of the bilayer to be formed at the start of the translation reaction (a) and a typical result of translation (b).
Figure 6: A typical result of the small-scale bilayer synthesis of DHFR.

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References

  1. Spirin, A.S., Baranov, V.I., Ryabova, L.A., Ovodov, S.Y. & Alakhov, Y.B. A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162–1164 (1988).

    Article  CAS  Google Scholar 

  2. Madin, K., Sawasaki, T., Ogasawara, T. & Endo, Y. A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proc. Natl. Acad. Sci. USA 97, 559–564 (2000).

    Article  CAS  Google Scholar 

  3. Sawasaki, T., Ogasawara, T., Morishita, R. & Endo, Y. A cell-free protein synthesis system for high-throughput proteomics. Proc. Natl. Acad. Sci. USA 99, 14652–14657 (2002).

    Article  CAS  Google Scholar 

  4. Sawasaki, T. et al. A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS Lett. 514, 102–105 (2002).

    Article  CAS  Google Scholar 

  5. Endo, Y. & Sawasaki, T. Cell-free expression systems for eukaryotic protein production. Curr. Opin. Biotechnol. 17, 373–380 (2006).

    Article  CAS  Google Scholar 

  6. Takai, K., Sawasaki, T. & Endo, Y. Development of key technologies for high-throughput cell-free protein production with the extract from wheat embryos. Adv. Protein Chem. Struct. Biol. 75, 53–84 (2008).

    CAS  PubMed  Google Scholar 

  7. Goshima, N. et al. Human protein factory for converting the transcriptome into an in vitro-expressed proteome. Nat. Methods 5, 1011–1017 (2008).

    Article  CAS  Google Scholar 

  8. Tsuboi, T. et al. Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates. Infect. Immun. 76, 1702–1708 (2008).

    Article  CAS  Google Scholar 

  9. Sawasaki, T. et al. The wheat germ cell-free expression system: methods for high-throughput materialization of genetic information. Methods Mol. Biol. 310, 131–144 (2005).

    Article  CAS  Google Scholar 

  10. Sawasaki, T. & Endo, Y. Chapter 6. Protein expression in the wheat-germ cell-free system. In Expression Systems (eds. Dyson, M.R. & Durocher, Y.) 87–108 (Scion Publishing Ltd., Oxfordshire, Oxford, UK, 2007).

  11. Endo, Y., Dohi, N. & Nakagawa, M. Germ extract for cell-free protein synthesis and process for producing the same. International Patent WO/2003/064671 filed 31 January 2003.

  12. Kamura, N., Sawasaki, T., Kasahara, Y., Takai, K. & Endo, Y. Selection of 5′-untranslated sequences that enhance initiation of translation in a cell-free protein synthesis system from wheat embryos. Bioorg. Med. Chem. Lett. 15, 5402–5406 (2005).

    Article  CAS  Google Scholar 

  13. Masaoka, T., Nishi, M., Ryo, A., Endo, Y. & Sawasaki, T. The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta. FEBS Lett. 582, 1795–1801 (2008).

    Article  CAS  Google Scholar 

  14. Makino, S., Sawasaki, T., Tozawa, Y., Endo, Y. & Takai, K. Covalent circularization of exogenous RNA during incubation with a wheat embryo cell extract. Biochem. Biophys. Res. Commun. 347, 1080–1087 (2006).

    Article  CAS  Google Scholar 

  15. Hirano, N., Sawasaki, T., Tozawa, Y., Endo, Y. & Takai, K. Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: limited interdomain misfolding in a eukaryotic translation system. Proteins 64, 343–354 (2006).

    Article  CAS  Google Scholar 

  16. Nureki, O. et al. Deep knot structure for construction of active site and cofactor binding site of tRNA modification enzyme. Structure 12, 593–602 (2004).

    Article  CAS  Google Scholar 

  17. Ikeda, K. et al. Immobilization of diverse foreign proteins in viral polyhedra and potential application for protein microarrays. Proteomics 6, 54–66 (2006).

    Article  CAS  Google Scholar 

  18. Matsumoto, K. et al. Production of yeast tRNA (m7G46) methyltransferase (Trm8-Trm82 complex) in a wheat germ cell-free translation system. J. Biotechnol. 133, 453–460 (2008).

    Article  CAS  Google Scholar 

  19. Kanno, T., Kasai, K., Ikejiri-Kanno, Y., Wakasa, K. & Tozawa, Y. In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2. Plant Mol. Biol. 54, 11–22 (2004).

    Article  CAS  Google Scholar 

  20. Takahashi, H., Nozawa, A., Seki, M., Shinozaki, K., Endo, Y. & Sawasaki, T. A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis. BMC Plant Biol. 9, 39 (2009).

    Article  Google Scholar 

  21. Kanno, T. et al. Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system. Protein Expr. Purif. 52, 59–65 (2007).

    Article  CAS  Google Scholar 

  22. Kobayashi, T., Kodani, Y., Nozawa, A., Endo, Y. & Sawasaki, T. DNA-binding profiling of human hormone nuclear receptors via fluorescence correlation spectroscopy in a cell-free system. FEBS Lett. 582, 2737–2744 (2008).

    Article  CAS  Google Scholar 

  23. Sawasaki, T. et al. Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate. FEBS Lett. 582, 221–228 (2008).

    Article  CAS  Google Scholar 

  24. Kanno, T. et al. Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway. Plant Physiol. 138, 2260–2268 (2005).

    Article  CAS  Google Scholar 

  25. Morita, E.H., Sawasaki, T., Tanaka, R., Endo, Y. & Kohno, T. A wheat germ cell-free system is a novel way to screen protein folding and function. Protein Sci. 12, 1216–1221 (2003).

    Article  CAS  Google Scholar 

  26. Vinarov, D.A. et al. Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat. Methods 1, 149–153 (2004).

    Article  CAS  Google Scholar 

  27. Vinarov, D.A., Loushin Newman, C.L. & Markley, J.L. Wheat germ cell-free platform for eukaryotic protein production. FEBS J. 273, 4160–4169 (2006).

    Article  CAS  Google Scholar 

  28. Morita, E.H. et al. A novel way of amino acid-specific assignment in 1H-15N HSQC spectra with a wheat germ cell-free protein synthesis system. J. Biomol. NMR 30, 37–45 (2004).

    Article  CAS  Google Scholar 

  29. Kohno, T. Production of proteins for NMR studies using the wheat germ cell-free system. Methods Mol. Biol. 310, 169–185 (2005).

    Article  CAS  Google Scholar 

  30. Kohno, T. & Endo, Y. Production of protein for nuclear magnetic resonance study using the wheat germ cell-free system. Methods Mol. Biol. 375, 257–272 (2007).

    CAS  PubMed  Google Scholar 

  31. Kainosho, M. et al. Optimal isotope labelling for NMR protein structure determinations. Nature 440, 52–57 (2006).

    Article  CAS  Google Scholar 

  32. Miyazono, K. et al. Novel protein fold discovered in the PabI family of restriction enzymes. Nucleic Acids Res. 35, 1908–1918 (2007).

    Article  CAS  Google Scholar 

  33. Abe, M. et al. Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing. Proteins 67, 643–652 (2007).

    Article  CAS  Google Scholar 

  34. Kawasaki, T., Gouda, M.D., Sawasaki, T., Takai, K. & Endo, Y. Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ. Eur. J. Biochem. 270, 4780–4786 (2003).

    Article  CAS  Google Scholar 

  35. Nozawa, A. et al. A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters. Plant Cell Physiol. 48, 1815–1820 (2007).

    Article  CAS  Google Scholar 

  36. Goren, M.A. & Fox, B.G. Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. Protein Expr. Purif. 62, 171–178 (2008).

    Article  CAS  Google Scholar 

  37. Kaiser, L., Graveland-Bikker, J., Steuerwald, D., Vanberghem, M., Herlihy, K. & Zhang, S. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses. Proc. Natl. Acad. Sci. USA 105, 15726–15731 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by the Special Coordination Funds for Promoting Science and Technology (Y.E.) and in part by a Grant-in-Aid for Scientific Research on the Priority Areas (No. 20034040 to K.T.) by the Ministry of Education, Culture, Sports, Science and Technology, Japan. We are grateful to Dr. R. Morishita and Mr. Y. Tanaka of CFS for providing pictures and for checking the paper.

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Authors and Affiliations

  1. Cell-Free Science and Technology Research Center and Venture Business Laboratory, Ehime University, Matsuyama, Japan

    Kazuyuki Takai, Tatsuya Sawasaki & Yaeta Endo

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  1. Kazuyuki Takai
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  2. Tatsuya Sawasaki
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  3. Yaeta Endo
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Contributions

K.T. collected information and wrote the paper; T.S. prepared the data and pictures; and Y.E. supervised the study.

Corresponding author

Correspondence to Yaeta Endo.

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Takai, K., Sawasaki, T. & Endo, Y. Practical cell-free protein synthesis system using purified wheat embryos. Nat Protoc 5, 227–238 (2010). https://doi.org/10.1038/nprot.2009.207

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