Use of EF-Tu mutants for determining and improving aminoacylation efficiency and for purifying aminoacyl tRNAs with non-natural amino acids

Takashi Ohtsuki, Hiromichi Yamamoto, Yoshio Doi, Masahiko Sisido

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

We present three methods relating to tRNA aminoacylation with non-natural amino acids using an Escherichia coli EF-Tu E215A/D216A mutant that can bind tightly to aa-tRNAs carrying either non-natural or natural amino acids: (i) a method for improving aminoacylation efficiency, (ii) a rapid method for analysing aminoacylation efficiency without the use of radioisotope labelling and (iii) a method for purifying aminoacyl-tRNAs. Although the EF-Tu mutant may be incompatible with some kinds of non-natural amino acids, we confirmed that the EF-Tu mutant could efficiently bind to aa-tRNAs carrying various amino acids (Arg, Ser, O-methyltyrosine, Bodipy FL-aminophenylalanine and 2-acrydonylalanine). These methods may be used for the efficient in vitro synthesis of proteins containing various non-natural amino acids.

Original languageEnglish
Pages (from-to)239-246
Number of pages8
JournalJournal of biochemistry
Volume148
Issue number2
DOIs
Publication statusPublished - Aug 1 2010

Keywords

  • Aminoacyl RNA Synthesis
  • Biosynthesis
  • Elongation Factors
  • Ribonuclear Protein RNP
  • Transfer RNA
  • Translation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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