Journal of Biomolecular NMR

, Volume 48, Issue 4, pp 193–201

An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system

  • Jun Yokoyama
  • Takayoshi Matsuda
  • Seizo Koshiba
  • Takanori Kigawa
Article

Abstract

Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein using Escherichia coli cell extract-based CF. This method takes advantage of endogenous metabolic conversions to generate SI-labeled asparagine, glutamine, cysteine, and tryptophan, which are much more expensive than the other 16 kinds of SI-labeled amino acids, from inexpensive sources, such as SI-labeled algal amino acid mixture, SI-labeled indole, and sodium sulfide, during the CF reaction. As compared with the conventional method employing 20 kinds of SI-labeled amino acids, highly enriched uniform SI-labeling with similar labeling efficiency was achieved at a greatly reduced cost with the newly developed method. Therefore, our method solves the cost problem of the SI labeling of proteins using the CF.

Keywords

Cell-free protein synthesis In vitro translation Stable-isotope labeling Metabolic conversion 

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Jun Yokoyama
    • 1
    • 2
    • 3
  • Takayoshi Matsuda
    • 1
  • Seizo Koshiba
    • 1
    • 4
  • Takanori Kigawa
    • 1
    • 2
  1. 1.RIKEN Systems and Structural Biology CenterTsurumi, YokohamaJapan
  2. 2.Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and EngineeringTokyo Institute of TechnologyYokohamaJapan
  3. 3.Tsukuba LaboratoryTaiyo Nippon Sanso Corp.Tsukuba, IbarakiJapan
  4. 4.Department of Supramolecular Biology, Graduate School of NanobioscienceYokohama City UniversityTsurumi, YokohamaJapan

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