Site-Specific Lysine Lactylation via Genetic Code Expansion in <em>E. coli </em>and Mammalian Cells

Lactylation is a recently discovered post-translational modification (PTM) with diverse biological functions. Since its initial identification in 2019 as a regulator of macrophage polarization, histone lactylation has emerged as a pivotal link connecting cellular metabolism to functional regulation. Biochemically, it involves the covalent transfer of a lactyl group to lysine residues, mediated either by lactyltransferases using lactyl-CoA as the acyl donor or by aminoacyl-tRNA synthetases (AARSs) via a lactyl-AMP intermediate. Through these mechanisms, intracellular lactate levels are directly coupled with protein modification and function. Lactylation mediates a range of critical cellular processes, from physiological functions such as gene regulation, DNA repair, metabolic reprogramming, and immune modulation to pathological processes such as tumor progression and metastasis. Despite the identification of numerous lactylation sites by proteomics, achieving site-specific lysine lactylation through mutagenesis remains challenging. To address this, we employ genetic code expansion technology, using a lactyl-lysine-specific aminoacyl-tRNA synthetase (KlacRS) and its cognate tRNA to precisely incorporate lactyl-lysine (Klac) at designated residues. This strategy enables the generation of homogeneously lactylated protein variants for direct functional studies. Here, we describe a streamlined protocol for site-specific protein lactylation, validated in both Escherichia coli (E. coli) and mammalian cells, using human enolase-1 (hENO1) and superfolder GFP (sfGFP) as model systems.