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Practical cell-free protein synthesis system using purified wheat embryos

Nature Protocols volume 5, pages 227238 (2010) | Download Citation

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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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.

Author information

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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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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DOI

https://doi.org/10.1038/nprot.2009.207

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