Efficient incorporation of unnatural amino acids into proteins in Escherichia coli.
We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins containing unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.
Ryu
Youngha
Y
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Schultz
Peter G
PG
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018389:Codon, Nonsense; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D005975:Glutamate-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D017017:Methanococcus / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012365:RNA, Transfer, Tyr / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D018389:Codon, Nonsense; D029968:Escherichia coli Proteins; D012365:RNA, Transfer, Tyr; D001616:beta-Galactosidase; D014448:Tyrosine-tRNA Ligase; D005975:Glutamate-tRNA Ligase