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Cell-Free Protein Production for Structural Biology

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Advanced Methods in Structural Biology

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Abstract

Cell-free protein synthesis using E. coli cell extracts has successfully been applied to protein sample preparation for structure determination by X-ray crystallization and NMR spectroscopy. The standard reaction solution for E. coli cell-free protein synthesis by coupled transcription-translation contains the S30 extract of E. coli cells, T7 RNA polymerase, and the DNA template (either plasmid or PCR-amplified linear DNA). Milligram quantities of proteins can be synthesized by the dialysis mode of the cell-free reaction in several hours. The E. coli cell-free protein synthesis method is suitable for the production of mammalian proteins, heteromultimeric protein complexes, and integral membrane proteins and features numerous advantages over the recombinant protein expression methods with bacterial and eukaryotic host cells. We present examples of structure determinations of mammalian and bacterial heteromultimeric protein complexes prepared by the cell-free production method.

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Acknowledgments

We thank Mr. K. Ake, Ms. T. Imada, and Ms. T. Nakayama for their assistance in the manuscript preparation. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses, the Targeted Proteins Research Program, and the Platform for Drug Discovery, Informatics, and Structural Life Science, of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to S.Y.).

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Authors and Affiliations

  1. RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan

    Takaho Terada, Seisuke Kusano, Takayoshi Matsuda, Mikako Shirouzu & Shigeyuki Yokoyama

  2. RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan

    Takaho Terada, Seisuke Kusano & Shigeyuki Yokoyama

  3. Center Director’s Strategic Program, RIKEN Center for Life Science Technologies, Yokohama, 230-0045, Japan

    Takayoshi Matsuda

  4. Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama, 230-0045, Japan

    Mikako Shirouzu

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  1. Takaho Terada
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  2. Seisuke Kusano
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  3. Takayoshi Matsuda
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  4. Mikako Shirouzu
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  5. Shigeyuki Yokoyama
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Correspondence to Shigeyuki Yokoyama .

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Editors and Affiliations

  1. Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK) , Tsukuba, Ibaraki, Japan

    Toshiya Senda

  2. Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan

    Katsumi Maenaka

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Terada, T., Kusano, S., Matsuda, T., Shirouzu, M., Yokoyama, S. (2016). Cell-Free Protein Production for Structural Biology. In: Senda, T., Maenaka, K. (eds) Advanced Methods in Structural Biology. Springer Protocols Handbooks. Springer, Tokyo. https://doi.org/10.1007/978-4-431-56030-2_5

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  • DOI: https://doi.org/10.1007/978-4-431-56030-2_5

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