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^2,3,4,5, metastasis⁶, metabolic reprogramming⁷, and therapeutic resistance^8,9.

At the phenotypic level, elevated lactylation is closely associated with enhanced tumor aggressiveness. For example, in clear cell renal cell carcinoma (ccRCC), H3K18 lactylation promotes proliferation and metastasis by upregulating platelet-derived growth factor receptor β (PDGFRβ), establishing a positive feedback loop between lactate accumulation and oncogenic signaling¹⁰. Similarly, in colorectal cancer (CRC), elevated H3K18 lactylation drives tumor progression via enhanced transcription of Rubicon-like autophagy enhancer (RUBCNL), thereby promoting autophagosome maturation and supporting cancer cell survival¹¹.

Beyond direct effects on tumor aggressiveness, lactylation also mediates tumor metabolic reprogramming by modulating the expression and activity of metabolism-related genes⁷. In non-small cell lung cancer (NSCLC), histone lactylation downregulates glycolysis-related enzymes while upregulating tricarboxylic acid (TCA) cycle enzymes, enhancing tumor cell glucose uptake and exacerbating metabolic dysregulation¹². Consistently, global lactylome profiling reveals that lactylation preferentially targets enzymes involved in central metabolic pathways, including glucose metabolism, TCA cycle, amino acid/fatty acid metabolism, and these modifications correlate strongly with aggressive tumor phenotypes⁷.

Importantly, the biological consequences of lactylation are highly site-dependent, and this modification has emerged as a key determinant of tumor therapeutic resistance. In CRC, K673 lactylation of meiotic recombination 11 (MRE11) enhances homologous recombination (HR) repair, thereby conferring resistance to chemotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors¹³. In glioblastoma (GBM), interaction between aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and pyruvate kinase M2 (PKM2) elevates lactate production, subsequently inducing K247 lactylation of X-ray repair cross-complementing protein 1 (XRCC1) to enhance DNA damage repair, thereby driving radioresistance and temozolomide (TMZ) chemoresistance¹⁴. These studies collectively highlight that precise control of lactylation at defined lysine residues is essential for mechanistic dissection and therapeutic targeting.

Advances in high-resolution mass spectrometry have revealed widespread lactylation across metabolic enzymes, transcription factors, and signaling transducers, underlining its broad role in controlling metabolism, signal transduction, and cellular physiology^15,16,17,18. To date, several writer enzymes catalyzing lactylation have been reported - for example, AARS1 mediates p53 lactylation to promote tumourigenesis¹⁹, and AARS1/2 regulate cGAS lactylation to suppress innate immunity - yet these enzymes are substrate-selective and currently cannot achieve fully site-specific lactylation on arbitrary proteins²⁰. Thus, the field remains challenged by how to manipulate Klac in a directed manner for research or therapeutic purposes.

Functional studies still predominantly rely on site-directed mutagenesis-typically substituting a lysine (K) with glutamine (Q) to mimic the uncharged state of acyl-lysine modifications such as acetylation or lactylation^21,22,23,24. However, this approach has significant limitations in faithfully recapitulating native lactylation: the chemical structure and side-chain geometry of glutamine differ from those of a lactylated lysine, and importantly, a K to Q substitution cannot distinguish between acetylated and lactylated states when both modifications might occur at the same site.

Genetic code expansion (GCE) provides a powerful solution to these limitations by enabling the direct incorporation of noncanonical amino acids into proteins at defined sites. Through positive screening of a Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS) mutant library, we previously identified a variant exhibiting Klac-dependent growth. This mutant, harboring mutations L301M, Y306L, L309A, and C348F, was designated KlacRS1^25,26. KlacRS1 aminoacylates its cognate orthogonal tRNA with Klac, suppresses the amber stop codon, and enables efficient, site-specific incorporation of Klac into target proteins. In addition to KlacRS1, several other aaRS variants capable of Klac incorporation have been reported, including MbPylRS-derived LacKRS (L270I/Y271L/L274A/C313F) and LacKRS3 (Y271M/C313S)^19,27, as well as an MmPylRS-derived LacKRS (C348T/Y384F)²⁸. Together, these tools establish a versatile genetic toolkit for producing site-specifically lactylated protein variants for understanding the biological consequences of individual lactylation events.

In our previous studies, we applied this GCE-based system to express ALDOA proteins bearing site-specific lactylation at Lys147 in both E. coli and mammalian cells. Functional analyses demonstrated that lactylation at Lys147 exerts a negative feedback effect on glycolysis, enhances the thermal and chemical stability of ALDOA, and promotes its translocation from the cytoplasm to the nucleus. These results not only revealed a previously unrecognized regulatory mechanism of ALDOA but also validated the robustness, versatility, and practical utility of this site-specific lactylation platform^25,26. Building on this foundation, here we use human enolase-1 (hENO1) and superfolder GFP (sfGFP) as representative examples to describe a streamlined strategy for precise Klac incorporation using our orthogonal KlacRS/tRNA system.

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 target site should be mutated into an amber stop codon (TAG), or other nonsense codons, and TAA or TGA should be used as the genuine stop codon.
2. Thaw DH5α competent cells on ice, mix 5 µL of the recombination product with 50 µL of cells, and incubate on ice for 30 min. Subject the mixture to a 45 s heat shock at 42 °C, then place it on ice for 2-3 min. Add 350 µL of LB medium and incubate at 37 °C with shaking (220 rpm) for 1 h.
3. Spread the cells onto a pre-warmed LB plate containing ampicillin and incubate at 37 °C for 12-16 h. Pick two single colonies for Sanger sequencing to confirm the mutation.

2. Expression of Lactylated protein in E. coli cells

1. Co-transform 50 µL of E. coli DH10B chemically competent cells with approximately 100 ng each of pBad-ENO1-K89TAG, which encodes the target gene, and pEvol-MmKlacRS1 (Addgene plasmid # 232157), which encodes the KlacRS and tRNA pair.
2. Incubate the mixture on ice for 25 min, heat-shock at 42 °C for 45 s, and return to ice for 3 min. Add 350 µL of room temperature LB medium, shake vigorously at 220 rpm for 1 h at 37 °C.
3. Spread the cell culture onto an LB/plate supplemented with 100 µg/mL of Ampicillin (Amp₁₀₀) and 50 µg/mL of Chloramphenicol (Cm₅₀). Incubate the plates at 37 °C for 12-16 h.
4. Pick a single colony to inoculate a 4 mL starter culture and incubate overnight. On the next day, expand the overnight culture into 400 mL of LB/Amp₁₀₀Cm₅₀ and then shake at 220 rpm, 37 °C vigorously until OD 600 reaches 0.6-0.8.
5. Induce with 0.2% L-arabinose and 1 mM Klac; incubate at 30 °C, 220 rpm for an additional 16 h.

3. Genetic encoding of Lactyllysine in HEK 293T cells

1. Seed cells in 12-well plates at a density of 1 x 10⁵ cells per well with 1 mL of DMEM medium. After 24 h of incubation, when the cell confluency reaches 70%-80%, perform transfection.
   NOTE: A confluency of 70%-80% is recommended for transfection, while colony formation assays require lower seeding densities, which should be determined empirically.
2. For preparing a diluted DNA solution, add 0.75 µg of pcDNA-sfGFP-N149TAG and pNeu-KlacRS (Addgene plasmid # 232155) in a 1:1 ratio into 38 µL of serum-free DMEM with High Glucose. Gently pipette up and down or vortex briefly to mix.
3. For preparing diluted transfection reagent, add 2.25 µL of transfection reagent into 38 µL of serum-free DMEM with High Glucose. Gently pipette up and down 3x-4x to mix.
   NOTE: Whether Opti-MEM can be used depends on the manufacturer's instructions, as its serum content may interfere with transfection efficiency.
4. Add the diluted transfection reagent immediately to the diluted DNA solution all at once.
   NOTE: Do not mix the solutions in the reverse order.
5. Immediately pipette up and down 3x-4x or vortex briefly to mix. Incubate for 10-15 min at room temperature to allow transfection/DNA complexes to form.
   NOTE: The transfection/ DNA complex should be used within 20 min of preparation.
6. Add the 50 µL transfection/ DNA mixture drop-wise onto the medium in each well and homogenize the mixture by gently swirling the plate.
7. At 6 h post-transfection, replace medium with fresh DMEM containing 1 mM Klac. Culture for 24-48 h.
   NOTE: Based on our previous data, Klac at concentrations of 1-5 mM showed no cytotoxic effects on cells²⁶. The working concentration of Klac should be optimized based on the protein expression level. If the target protein is prone to loss of the lactylation modification, administration of HDAC inhibitors (e.g., Trichostatin A) or the sirtuin inhibitor NAM (nicotinamide) is recommended.

4. His-tag purification of Lactylated protein in E. coli cells

1. Harvest the cells by centrifugation at 6,900 x g for 10 min at 4 °C. Resuspend in 50 mL of lysis buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl). Lyse by ultrasonication on ice for 3 s pulse, 8 s interval at 200 W for 30 min. Centrifuge at 13,800 x g, 4 °C for 30 min and then collect supernatant.
   NOTE: If the target protein is susceptible to mechanical shearing, the ultrasonication step can be substituted with gentle cell lysis using a formulated protein extraction reagent suitable for E. coli.
2. Filter supernatant through a 0.45 µm membrane to collect the soluble fractions. Load the clarified lysates onto a 1 mL Ni-NTA gravity column. Wash with approximately 5 mL of Buffer A (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 
   5 mM imidazole) until no detectible protein is found in the eluate. Elute the protein with 1 mL of Buffer B (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 200 mM imidazole) for 5x. Save all fractions. Check for eluted protein with a quick Bradford assay.
3. Combine the purified protein fractions and perform buffer exchange into the storage buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl). Aliquot the concentrated proteins into 50 µL in each tube, flash freeze, and store at -80 °C for the following analysis.

5. Biochemical characterization of the lactylated protein

1. SDS-PAGE and Western Blotting
   1. Separate the protein by SDS-PAGE (90 V for 20 min, then 150 V for 50 min). Stain the gel with Coomassie Blue, heat for 15 s in a microwave, and destain with destaining solution (40% absolute ethanol, 10% glacial acetic acid, and 50% deionized water) on a shaker (50 rpm, 15 min). Repeat the destaining step until protein bands are clearly visible.
   2. Activate a PVDF membrane in methanol for 1 min. Assemble the transfer stack in the order: sponge, filter paper, gel, membrane, filter paper, sponge. Carry out the transfer at 250 mA for 1 h in a pre-chilled buffer. Block the membrane with 5% skim milk for 1.5 h, then wash 3x with TBST (10 min each).
   3. Incubate with primary antibodies (Anti-His 1:10,000; Anti-L-Lactyl Lysine 1:1,000) at 4 °C overnight. Wash 3x with TBST.
      ​NOTE: Optimal dilution ratios should be determined empirically for antibodies obtained from different commercial sources.
   4. Incubate with rabbit secondary antibody (1:10,000) for 1.5 h at room temperature. Wash 3x with TBST. Image using chemiluminescence and save data in Image Lab.
2. Mass spectrometry analyses
   1. For intact mass analysis, dilute the proteins to a final concentration of 0.1 µg/µL with water containing 0.1% formic acid (FA) and then subject them to an ultra-high performance liquid chromatography (UPLC) system. The mobile phase consisted of solvent A (0.1% FA in water) and solvent B (acetonitrile, ACN) at a flow rate of 0.3 mL/min.
   2. Measure the molecular weights of hENO1-89Klac using a high-resolution quadrupole-time-of-flight (Q-TOF) mass spectrometer with the m/z range set to 300-2000. Collect and process all data using the MassLynx software.
   3. For proteomic analysis, analyze the digested cell lysates as previously described, using a nano-flow liquid chromatography system coupled to a high-resolution tribrid mass spectrometer equipped with an analyzer. The mobile phase consisted of solvent A (0.1% FA in water) and solvent B (ACN/water, 8:2, v/v) at a flow rate of 300 nL/min.
   4. Separate peptides on a reversed-phase C18 capillary column (75 µm x 250 mm) with a 90 min gradient: 3%-8% B (0-5 min), 8%-28% B (5-60 min), 28%-38% B (60-75 min), 38%-100% B (75-80 min), and 100% B (80-90 min). Acquire MS1 spectra over m/z 350-1800 with a resolution of 120,000 and an AGC target of 4e5. Generate MS2 spectra by higher-energy collisional dissociation (HCD) fragmentation (collision energy = 32%), with a first mass of m/z 110, a resolution of 30,000, and an AGC target of 5e4.
3. Flow cytometry analysis
   1. Wash the cells 2x with pre-cooled PBS buffer, followed by centrifugation at 1,380 x g for 3 min after each wash. Resuspend the cell pellet obtained after centrifugation in 500 µL of PBS buffer and gently pipette to mix. Filter the cell suspension through a 300-mesh sterile gauze, collect the filtrate into a flow cytometry tube to minimize interference from cell aggregates during detection. Analyze the prepared sample using a flow cytometer with dual lasers, with excitation at 405 nm and detection at 488 nm for the corresponding fluorescence signals.

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.