Improving cell-free protein synthesis for stable-isotope labeling

Takayoshi Matsuda, Seizo Koshiba, Naoya Tochio, Eiko Seki, Noriyuki Iwasaki, Takashi Yabuki, Makoto Inoue, Shigeyuki Yokoyama, Takanori Kigawa

Research output: Contribution to journalArticlepeer-review

57 Citations (Scopus)

Abstract

Cell-free protein synthesis is suitable for stable-isotope labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium L-glutamate (L-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium D-Glu system). The productivity of the D-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the 1H-15N HSQC spectrum of the uniformly stable-isotope labeled Ras protein, prepared with the D-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the D-Glu system. These results indicate that the D-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of proteins.

Original languageEnglish
Pages (from-to)225-229
Number of pages5
JournalJournal of biomolecular NMR
Volume37
Issue number3
DOIs
Publication statusPublished - 2007 Mar 1
Externally publishedYes

Keywords

  • Cell-free protein synthesis
  • Invitro translation
  • Potassium D-glutamate
  • Stable-isotope labeling

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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