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

Jun Yokoyama, Takayoshi Matsuda, Seizo Koshiba, Takanori Kigawa

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)193-201
Number of pages9
JournalJournal of biomolecular NMR
Volume48
Issue number4
DOIs
Publication statusPublished - 2010 Dec
Externally publishedYes

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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