An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli.
The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is 'orthogonal', meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA(Glu) sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.
Santoro
Stephen W
SW
The Scripps Research Institute, Department of Chemistry and the Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, SR202, La Jolla, CA 92037, USA.
Anderson
J Christopher
JC
Lakshman
Vishva
V
Schultz
Peter G
PG
D000215:Acylation; D019943:Amino Acid Substitution; D001105:Archaea / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D018389:Codon, Nonsense / Q000235:genetics; D016384:Consensus Sequence; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D005975:Glutamate-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D043902:Pyrococcus horikoshii / Q000201:enzymology; D012352:RNA, Transfer, Glu / Q000235:genetics / Q000378:metabolism*
D018389:Codon, Nonsense; D029968:Escherichia coli Proteins; D012352:RNA, Transfer, Glu; D005975:Glutamate-tRNA Ligase