Four-base codon-mediated incorporation of non-natural amino acids into proteins in a eukaryotic cell-free translation system.
Various four-base codons have been shown to work for the introduction of non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Here, a four-base codon-mediated non-natural mutagenesis was applied to a eukaryotic rabbit reticulocyte cell-free translation system. Mutated streptavidin mRNAs containing four-base codons were prepared and added to a rabbit reticulocyte lysate in the presence of tRNAs that were aminoacylated with a non-natural amino acid and had the corresponding four-base anticodons. A Western blot analysis of translation products indicated that the four-base codons CGGU, CGCU, CCCU, CUCU, CUAU, and GGGU were efficiently decoded by the aminoacyl-tRNAs having the corresponding four-base anticodons. In contrast, the four-base codons AGGU, AGAU, CGAU, UUGU, UCGU, and ACGU were not decoded. The stop codon-derived four-base codons UAGU, UAAU, and UGAU were found to be inefficient, whereas the amber codon UAG and opal codon UGA were efficient for the incorporation of non-natural amino acids. The application of the expanded genetic code in a eukaryotic cell-free system opens the possibility of a four-base codon-mediated incorporation of non-natural amino acids into proteins in living eukaryotic cells.
Taira
Hikaru
H
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Fukushima
Masaharu
M
Hohsaka
Takahiro
T
Sisido
Masahiko
M
D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System / Q000378:metabolism; D002478:Cells, Cultured; D003062:Codon / Q000235:genetics*; D005057:Eukaryotic Cells; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000235:genetics; D046188:Protein Modification, Translational / Q000235:genetics*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000502:physiology*
D000596:Amino Acids; D003062:Codon; D011506:Proteins