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

Summary

This study established a method that utilizes genetic code expansion to successfully incorporate lactyl-lysine (Klac) at specific sites of the human enolase-1 (hENO1) and superfolder GFP (sfGFP) in Escherichia coli and mammalian cells.

Abstract

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.

Introduction

Lysine lactylation (Klac), originally identified on histones, has emerged as a key node linking aberrant tumor metabolism and epigenetic regulation¹. The accumulation of lactate resulting from enhanced aerobic glycolysis in tumor cells provides a metabolic basis for lactylation, enabling lactate to act not merely as a metabolic byproduct but as a signaling molecule that directly modulates protein function. Increasing evidence indicates that lactylation broadly participates in tumor initiation and progression by coordinating cancer cell proliferation^2,3,4

Protocol

1. Amber stop codon mutagenesis for genetic encoding of Lactyllysine into target gene

1. Introduce a TAG amber stop codon at position 89 of the hENO1 gene and position 149 of the sfGFP gene via site-directed mutagenesis following the manufacturer's protocol, purify the PCR product, and perform homologous recombination. Use the following primers:
   ENO1-K89TAG-F CACAGAACAAGAGtagATTGACAAACTGATGATCGAG
   ENO1-K89TAG-R TctaCTCTTGTTCTGTGACGTTCAGTTTCTTG
   sfGFP-N149TAG-F CAACAGCCATtagGTGTATATCACCGCCGACAAGC
   sfGFP-N149TAG-R CACctaATGGCTGTTGAAGTTATACTCCAGC
   NOTE: For site-specific incorporation of lactylated lysine, the....

Results

We first evaluated the incorporation specificity of Klac into proteins in E. coli. The gene for human enolase 1 (hENO1) was engineered to contain an amber stop codon at residue K89 (hENO1-89TAG), and this construct was co-expressed with the orthogonal KlacRS and its cognate tRNA pair in E. coli. In the absence of 1 mM Klac supplementation, no full-length hENO1 was detected; by contrast, when 1 mM Klac was added to the growth media, full-length hENO1 containing Klac at position 89 (hENO1-89Klac).......

Discussion

Here, we describe a strategy for site-specific incorporation of Klac into proteins in both prokaryotic and eukaryotic cells. By introducing an amber stop codon at the desired position of the target gene and co-expressing it with the orthogonal KlacRS/tRNA pair in host cells, authentic lactyl lysine can be precisely incorporated into proteins in living cells, providing a powerful tool to probe lactylation-specific biology.

We demonstrated the feasibility of this workflow using the representativ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6*his,His tag Recombinant monoclonal antibody	Proteintech	84814-1-RR	A class of specific immunoglobulins targeting the 6×His tag (a short peptide composed of six consecutive histidine residues, HHHHHH) in recombinant proteins.
ACQUITY UPLC system	Waters		UltraPerformance Liquid Chromatography
Anti-L-Lactyllysine Rabbit mAb	PTM Biolabs	PTM-1401RM	A class of specific immunoglobulins targeting L-Lactyl Lysine (Klac), a post-translational modification (PTM) of proteins.
ClonExpress II One Step Cloning Kit	Vazyme	C112	Based on simple, rapid, and efficient DNA seamless cloning technology, insert fragments can be directionally cloned into any site of any vector.
CytoFlex flow cytometer	Beckmann Coulter		Flow cytometer with high-sensitivity detection and multi-color analysis capability
DH10B	Biorabbit	EC005H-S	A type of competent cells for plasmid transformation, gene cloning, and blue-white screening.
DH5α	Vazyme	C502	A type of competent cells for plasmid transformation, gene cloning, and blue-white screening.
EASY-nano LC 1200 system	Thermo Fisher Scientific		nano-liquid chromatography for separating high-sensitivity, high-resolution complex samples
Orbitrap Eclipse Tribrid mass spectrometer	Thermo Fisher Scientific		a flagship high-end tribrid liquid chromatography-mass spectrometry (LC-MS) system
Phanta Max Super-Fidelity DNA Polymerase	Vazyme	P505	A high-fidelity single enzyme combining rapid amplification and fidelity
PolyJet	SignaGen	SL100688	PolyJet DNA In Vitro Tranfection Reagent is formulated to be a powerful transfection Reagent that ensures effective and reproducible transfection with less cytotoxicity.