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Cell-free synthesis of zinc-binding proteins

  • Original Paper
  • Published:
Journal of Structural and Functional Genomics

Abstract

Cell-free protein synthesis has become one of the standard methods for protein expression. The cell-free method is suitable for the synthesis of a protein that requires a ligand for its enzymatic activity and/or structure formation and stabilization, since it is an open system, which allows us to add the proper ligand to the reaction mixture. A large number of proteins that require zinc for their function are involved in diverse cellular processes, including transcription, DNA replication, metabolism, and cell signaling. In this study, we analyzed the effects of zinc on the cell-free synthesis of plant-specific zinc-binding transcription factors. The solubility and/or stability of the proteins were significantly increased in the presence of the proper concentration of zinc during the cell-free reaction. NMR analyses confirmed that correctly folded proteins were synthesized by the cell-free method. These results indicate that the cell-free method can be used to synthesize correctly folded and functional zinc-binding proteins.

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Abbreviations

SPL:

SQUAMOSA promoter binding protein-like

DBD:

DNA-binding domain

HSQC:

Heteronuclear single quantum coherence

CAT:

chloramphenicol acetyltransferase

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Acknowledgements

The authors thank Yukako Miyata, Emi Nunokawa, Yukiko Fujikura, Natsuko Matsuda, Natsumi Suzuki, Yasuko Tomo, Eiko Seki, Masaomi Ikari, and Fumiko Hiroyasu at RIKEN for technical assistance. The authors also thank Takashi Yabuki for valuable suggestions and comments. The authors are grateful to Azusa Ishii, Kiyomi Yajima, and Tomoko Nakayama for expert secretarial assistance. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Authors and Affiliations

  1. Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan

    Takayoshi Matsuda, Takanori Kigawa, Seizo Koshiba, Makoto Inoue, Masaaki Aoki, Kazuhiko Yamasaki & Shigeyuki Yokoyama

  2. Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8502, Japan

    Takayoshi Matsuda & Takanori Kigawa

  3. Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566, Japan

    Kazuhiko Yamasaki

  4. Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba , 305-0074, Japan

    Motoaki Seki & Kazuo Shinozaki

  5. Plant Functional Genomics Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan

    Motoaki Seki & Kazuo Shinozaki

  6. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Shigeyuki Yokoyama

Authors
  1. Takayoshi Matsuda
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  2. Takanori Kigawa
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  3. Seizo Koshiba
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  4. Makoto Inoue
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  5. Masaaki Aoki
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  6. Kazuhiko Yamasaki
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  7. Motoaki Seki
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  8. Kazuo Shinozaki
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  9. Shigeyuki Yokoyama
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Corresponding author

Correspondence to Shigeyuki Yokoyama.

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Matsuda, T., Kigawa, T., Koshiba, S. et al. Cell-free synthesis of zinc-binding proteins. J Struct Funct Genomics 7, 93–100 (2006). https://doi.org/10.1007/s10969-006-9012-1

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  • DOI: https://doi.org/10.1007/s10969-006-9012-1

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