Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids.
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.
Sisido
Masahiko
M
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan. sisido@cc.okayama-u.ac.jp
Ninomiya
Keiko
K
Ohtsuki
Takashi
T
Hohsaka
Takahiro
T
D000596:Amino Acids / Q000737:chemistry; D000926:Anticodon / Q000737:chemistry*; D003062:Codon / Q000737:chemistry*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D012343:RNA, Transfer; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis; D046249:Transfer RNA Aminoacylation
D000596:Amino Acids; D000926:Anticodon; D003062:Codon; D020135:Peptide Nucleic Acids; D012346:RNA, Transfer, Amino Acyl; D012343:RNA, Transfer