Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids

Masahiko Sisido, Keiko Ninomiya, Takashi Ohtsuki, Takahiro Hohsaka

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.

Original languageEnglish
Pages (from-to)270-278
Number of pages9
JournalMethods
Volume36
Issue number3
DOIs
Publication statusPublished - Jul 1 2005

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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