Johnson, D. B. F. et al. Nat. Chem. Biol. 7, 779–786 (2011).
Probes can be site-specifically introduced into proteins by reassigning the stop codon UAG to encode an unnatural amino acid. But incorporation efficiency is low because the stop codon continues to serve as a stop signal. Johnson et al. fully reassign the UAG codon in Escherichia coli by knocking out the essential release factor 1 and fixing the expression of release factor 2. This greatly improves incorporation of unnatural amino acids, allowing incorporation at multiple sites.
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Incorporating multiple unnatural amino acids. Nat Methods 8, 897 (2011). https://doi.org/10.1038/nmeth.1755
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DOI: https://doi.org/10.1038/nmeth.1755