Expanding the Genetic Code of Escherichia coli

Abstract

A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-l-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as determined by analysis of dihydrofolate reductase containing the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

“…24-26 or 3) nonsense expression using an evolved orthogonal tRNA/aminoacyl-tRNA synthetase (AARS) pair recognizing a nonsense (commonly UAG or "amber") codon. 27,28 The final 6 method has resulted in the generation of several evolved tRNA/aaRS pairs, and incorporation of a variety of unnatural amino acids into proteins in E. coli, yeast, and mammalian cells.…”

Genetic incorporation of d-lysine into diketoreductase in Escherichia coli cells

“…Plasmids of pAC-ph△-AK3 and pETDuet- terminator genes were synthesized according to the patent [1] and ligated to pMD-18T vector. pMD-18T and pETDuet-1 vectors were obtained from TaKaRa (Japan) and Novagen (USA), respectively.…”

“…According to previous report and patent, [1,2] tRNA from Pyrococcus horikoshii ATCC 700860 was synthesized by GenScript Co. (China), and Lysyl-tRNA synthetase was amplified from the same strain by PCR as described in 1.4.…”

Antibody–Drug Conjugates for Tumor Targeting—Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

“…To make these reactions applicable to proteins, they must first be charged with aromatic azides, BCN or quinones in a stoichiometrically defined, site-specific manner. This no longer possible with a simple metstrain of E. coli, but requires amber-suppression technology for site-specific incorporation of BCN-lysine or other non-natural amino acids carrying functional groups like quinones or norbonenes [65][66][67][68][69][70][71][72] . So far, this technology was only used for imaging agents, as it shows unfavorably low expression yields 66 .…”