Expression and Purification of Site-Specifically Lysine-Acetylated and Natively-Folded Proteins for Biophysical Investigations.

N-(ε)-lysine-acetylation (short: lysine-acetylation) is a dynamic and powerful posttranslational modification to regulate protein function. Mutational approaches are often poor to access the real mechanistic impact of lysine-acetylation at the molecular level. Therefore, the ability to site-specifically incorporate N-(ε)-acetyl-L-lysine (short: AcK) into proteins dramatically increased our understanding how lysine-acetylation regulates protein function by using diverse molecular mechanisms going far beyond neutralizing a positive charge at the lysine-side chain. Genetically encoding AcK is a powerful way to introduce AcK into proteins, resulting in homogenously, quantitatively, and site-specifically lysine-acetylated proteins. Thereby, lysine-acetylated proteins can be produced in their natively-folded state in a high quality and in a yield sufficient to perform biophysical studies, including X-ray crystallography. This protocol describes the expression and purification of site-specifically lysine-acetylated proteins in Escherichia coli using the genetic-code expansion concept (GCEC) and subsequent steps to assess the successful incorporation of AcK by immunoblotting and mass-spectrometry.