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Cell-Free Protein Synthesis for Structure Determination by X-ray Crystallography

  • Miki Watanabe
  • Ken-ichi Miyazono
  • Masaru Tanokura
  • Tatsuya Sawasaki
  • Yaeta Endo
  • Ichizo Kobayashi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 607)

Abstract

Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

Key words

Structural genomics Toxic protein Wheat germ Restriction enzyme Selenomethionine SAD 

Notes

Acknowledgments

The study of PabI was carried out in collaboration with Jan Kosinski, Ken Ishikawa, Masayuki Kamo, Koji Nagata, and Janusz M. Bujnicki. We are grateful to Kazuyuki Takai for encouragements and patience. IK was supported, during writing, by twenty-first century COE program “Genome and Language” from MEXT and “Grants-in-Aid for Scientific Research” (19657002, 21370001) from JSPS.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Miki Watanabe
    • 1
  • Ken-ichi Miyazono
    • 2
  • Masaru Tanokura
    • 2
  • Tatsuya Sawasaki
    • 3
  • Yaeta Endo
    • 3
  • Ichizo Kobayashi
    • 4
  1. 1.Department of Medical Genome Sciences, Graduate School of Frontier ScienceUniversity of TokyoTokyoJapan
  2. 2.Department of Applied Biological Chemistry, Graduate School of Agricultural and Life SciencesUniversity of TokyoTokyoJapan
  3. 3.Cell-Free Science and Technology Research Center and Venture Business LaboratoryEhime UniversityEhimeJapan
  4. 4.Department of Medical Genome Sciences, Graduate School of Frontier Science, Graduate Program in Biophysics and Biochemistry, Graduate School of Science, and Institute of Medical ScienceUniversity of TokyoTokyoJapan

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