An unnatural base pair for incorporating amino acid analogs into proteins

Nat Biotechnol. 2002 Feb;20(2):177-82. doi: 10.1038/nbt0202-177.

Abstract

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry*
  • Base Sequence
  • Cell-Free System
  • Codon
  • DNA / chemistry
  • DNA-Directed RNA Polymerases / metabolism
  • Escherichia coli / metabolism
  • Gas Chromatography-Mass Spectrometry
  • Genetic Variation
  • Models, Chemical
  • Molecular Sequence Data
  • Plasmids / metabolism
  • Protein Biosynthesis
  • Protein Engineering / methods*
  • RNA, Messenger / metabolism
  • RNA, Transfer / metabolism
  • Time Factors
  • Transcription, Genetic
  • Tyrosine / analogs & derivatives*
  • Tyrosine / chemistry
  • Tyrosine / pharmacology
  • Viral Proteins

Substances

  • Amino Acids
  • Codon
  • RNA, Messenger
  • Viral Proteins
  • Tyrosine
  • DNA
  • RNA, Transfer
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • 3-chlorotyrosine