Reversible lysine acetylation is one of the most widely distributed post‐translational modifications; it is involved in a variety of biological processes and can be found in all three domains of life. Acetyltransferases and deacetylases work coordinately to control levels of protein acetylation. In this work, we applied the genetic code expansion strategy to site‐specifically incorporate Nε‐thioacetyl‐l‐lysine (TAcK) as an analog of Nε‐acetyl‐l‐lysine (AcK) into green fluorescent protein and malate dehydrogenase in Escherichia coli. We showed that TAcK could serve as an ideal functional mimic for AcK. It could also resist the bacterial sirtuin‐type deacetylase CobB. Thus, genetic incorporation of TAcK as a non‐deacetylatable analog of AcK into proteins will facilitate in vivo studies of protein acetylation.

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