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 journalArticle

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 2005

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Aminoacylation
Anticodon
Protein Engineering
Codon
Transfer RNA
Amino Acids
Proteins
Transfer RNA Aminoacylation
Genetic Code
Esters
In Vitro Techniques

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids. / Sisido, Masahiko; Ninomiya, Keiko; Ohtsuki, Takashi; Hohsaka, Takahiro.

In: Methods, Vol. 36, No. 3, 07.2005, p. 270-278.

Research output: Contribution to journalArticle

Sisido, Masahiko ; Ninomiya, Keiko ; Ohtsuki, Takashi ; Hohsaka, Takahiro. / Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids. In: Methods. 2005 ; Vol. 36, No. 3. pp. 270-278.
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