A practical method for cell-free protein synthesis to avoid stable isotope scrambling and dilution

Jun Yokoyama, Takayoshi Matsuda, Seizo Koshiba, Naoya Tochio, Takanori Kigawa

Research output: Contribution to journalArticlepeer-review

38 Citations (Scopus)

Abstract

During recent years, the targets of protein structure analysis using nuclear magnetic resonance spectroscopy have become larger and more complicated. As a result, a complete and precise stable isotope labeling technique has been desired. A cell-free protein synthesis system is appropriate for this purpose. In the current study, we achieved precise and complete 15N and 2H labeling using an Escherichia coli cell extract-based cell-free protein synthesis system by controlling the metabolic reactions in the system with their chemical inhibitors. The addition of aminooxyacetate, d-malate, l-methionine sulfoximine, S-methyl-l-cysteine sulfoximine, 6-diazo-5-oxo-l- norleucine, and 5-diazo-4-oxo-l-norvaline was quite effective for precise amino acid-selective 15N labeling even for aspartic acid, asparagine, glutamic acid, and glutamine, which generally suffer from severe isotope scrambling and dilution when using the conventional cell-free system. For 2H labeling, the back-protonation of the Hα and Hβ positions, which commonly occurred in the conventional system, was dramatically suppressed by simply adding aminooxyacetate and d-malate to the cell-free system except for the Hα positions in methionine and cysteine.

Original languageEnglish
Pages (from-to)223-229
Number of pages7
JournalAnalytical Biochemistry
Volume411
Issue number2
DOIs
Publication statusPublished - 2011 Apr 15
Externally publishedYes

Keywords

  • Amino acid-selective labeling
  • Cell-free protein synthesis
  • Deuterium labeling
  • NMR
  • Stable isotope labeling

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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