Name: Site-Specific Lysine Lactylation via Genetic Code Expansion in E. coli and Mammalian Cells (Video) | JoVE Journal
Uploaded: 2026-02-24T13:25:00.000+00:00
Duration: 5 min 58 s
Description: Learn site-specific lysine lactylation using genetic code expansion in E. coli and mammalian cells. This method enables precise incorporation of Klac to study protein function and tumor metabolism.

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5 minutes

This research was supported by the National Natural Science Foundation of China (22377058), the Interdisciplinary Funding for Synthetic Biology in Traditional Chinese Medicine by Nanjing University of Chinese Medicine, and the National High-Level Talent Special Support Programs (10,000 Talents Program) - Young Talents.

1. Amber stop codon mutagenesis for genetic encoding of Lactyllysine into target gene
<ol>
	<li>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&#39;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.</li>
	<li>Thaw DH5&#945; competent cells on ice, mix 5 &#181;L of the recombination product with 50 &#181;L of cells, and incubate on ice for 30 min. Subject the mixture to a 45 s heat shock at 42 &#176;C, then place it on ice for 2-3 min. Add 350 &#181;L of LB medium and incubate at 37 &#176;C with shaking (220 rpm) for 1 h.</li>
	<li>Spread the cells onto a pre-warmed LB plate containing ampicillin and incubate at 37 &#176;C for 12-16 h. Pick two single colonies for Sanger sequencing to confirm the mutation.</li>
</ol>
2. Expression of Lactylated protein in E. coli cells
<ol>
	<li>Co-transform 50 &#181;L 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.</li>
	<li>Incubate the mixture on ice for 25 min, heat-shock at 42 &#176;C for 45 s, and return to ice for 3 min. Add 350 &#181;L of room temperature LB medium, shake vigorously at 220 rpm for 1 h at 37 &#176;C.</li>
	<li>Spread the cell culture onto an LB/plate supplemented with 100 &#181;g/mL of Ampicillin (Amp100) and 50 &#181;g/mL of Chloramphenicol (Cm50). Incubate the plates at 37 &#176;C for 12-16 h.</li>
	<li>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 &#176;C vigorously until OD 600 reaches 0.6-0.8.</li>
	<li>Induce with 0.2% L-arabinose and 1 mM Klac; incubate at 30 &#176;C, 220 rpm for an additional 16 h.</li>
</ol>
3. Genetic encoding of Lactyllysine in HEK 293T cells
<ol>
	<li>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.</li>
	<li>For preparing a diluted DNA solution, add 0.75 &#181;g of pcDNA-sfGFP-N149TAG and pNeu-KlacRS (Addgene plasmid # 232155) in a 1:1 ratio into 38 &#181;L of serum-free DMEM with High Glucose. Gently pipette up and down or vortex briefly to mix.</li>
	<li>For preparing diluted transfection reagent, add 2.25 &#181;L of transfection reagent into 38 &#181;L 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&#39;s instructions, as its serum content may interfere with transfection efficiency.</li>
	<li>Add the diluted transfection reagent immediately to the diluted DNA solution all at once. 
	NOTE: Do not mix the solutions in the reverse order.</li>
	<li>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.</li>
	<li>Add the 50 &#181;L transfection/ DNA mixture drop-wise onto the medium in each well and homogenize the mixture by gently swirling the plate.</li>
	<li>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.</li>
</ol>
4. His-tag purification of Lactylated protein in E. coli cells
<ol>
	<li>Harvest the cells by centrifugation at 6,900 x g for 10 min at 4 &#176;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 &#176;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.</li>
	<li>Filter supernatant through a 0.45 &#181;m 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,&#160; 
	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.</li>
	<li>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 &#181;L in each tube, flash freeze, and store at -80 &#176;C for the following analysis.</li>
</ol>
5. Biochemical characterization of the lactylated protein
<ol>
	<li>SDS-PAGE and Western Blotting
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>Incubate with primary antibodies (Anti-His 1:10,000; Anti-L-Lactyl Lysine 1:1,000) at 4 &#176;C overnight. Wash 3x with TBST. 
		&#8203;NOTE: Optimal dilution ratios should be determined empirically for antibodies obtained from different commercial sources.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Mass spectrometry analyses
	<ol>
		<li>For intact mass analysis, dilute the proteins to a final concentration of 0.1 &#181;g/&#181;L 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&#160;in water) and solvent B (acetonitrile, ACN) at a flow rate of 0.3 mL/min.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Separate peptides on a reversed-phase C18 capillary column (75 &#181;m 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.</li>
	</ol>
	</li>
	<li>Flow cytometry analysis
	<ol>
		<li>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 &#181;L 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.</li>
	</ol>
	</li>
</ol>

<ol><li>Zhang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+regulation+of+gene+expression+by+histone+lactylation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Metabolic regulation of gene expression by histone lactylation.</a> Nature. 574 (7779), 575-580 (2019).</li>
<li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+lactylation+drives+oncogenesis+by+facilitating+m6A+reader+protein+YTHDF2+expression+in+ocular+melanoma%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Histone lactylation drives oncogenesis by facilitating m6A reader protein YTHDF2 expression in ocular melanoma.</a> Genome Biol. 22 (1), 85(2021).</li>
<li>Pan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Demethylzeylasteral+targets+lactate+by+inhibiting+histone+lactylation+to+suppress+the+tumorigenicity+of+liver+cancer+stem+cells%5BTitle%5D)+AND+%22Pharmacol+Res%22%5BJournal%5D)">Demethylzeylasteral targets lactate by inhibiting histone lactylation to suppress the tumorigenicity of liver cancer stem cells.</a> Pharmacol Res. 181, 106270(2022).</li>
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<li>Chai, P., Zhao, F., Jia, R., Zhou, X., Fan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lactate%2Flactylation+in+ocular+development+and+diseases%5BTitle%5D)+AND+%22Trends+Mol+Med%22%5BJournal%5D)">Lactate/lactylation in ocular development and diseases.</a> Trends Mol Med. 31 (12), 1073-1076 (2025).</li>
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<li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor-derived+lactate+promotes+resistance+to+bevacizumab+treatment+by+facilitating+autophagy+enhancer+protein+Rubcnl+expression+through+histone+H3+lysine+18+lactylation+%28H3K18la%29+in+colorectal+cancer%5BTitle%5D)+AND+%22Autophagy%22%5BJournal%5D)">Tumor-derived lactate promotes resistance to bevacizumab treatment by facilitating autophagy enhancer protein Rubcnl expression through histone H3 lysine 18 lactylation (H3K18la) in colorectal cancer.</a> Autophagy. 20 (1), 114-130 (2024).</li>
<li>Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lactate+modulates+cellular+metabolism+through+histone+lactylation-mediated+gene+expression+in+non-small+cell+lung+cancer%5BTitle%5D)+AND+%22Front+Oncol%22%5BJournal%5D)">Lactate modulates cellular metabolism through histone lactylation-mediated gene expression in non-small cell lung cancer.</a> Front Oncol. 11, 647559(2021).</li>
<li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+regulation+of+homologous+recombination+repair+by+MRE11+lactylation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Metabolic regulation of homologous recombination repair by MRE11 lactylation.</a> Cell. 187 (2), 294-311 (2024).</li>
<li>Li, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycometabolic+reprogramming-induced+XRCC1+lactylation+confers+therapeutic+resistance+in+ALDH1A3-overexpressing+glioblastoma%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">Glycometabolic reprogramming-induced XRCC1 lactylation confers therapeutic resistance in ALDH1A3-overexpressing glioblastoma.</a> Cell Metab. 36 (8), 1696-1710.e10 (2024).</li>
<li>Liberti, M. V., Locasale, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+warburg+effect%3A+How+does+it+benefit+cancer+cells%5BTitle%5D)+AND+%22Trends+Biochem+Sci%22%5BJournal%5D)">The warburg effect: How does it benefit cancer cells.</a> Trends Biochem Sci. 41 (3), 211-218 (2016).</li>
<li>Varner, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+lactoyl-CoA+%28lactyl-CoA%29+by+liquid+chromatography+mass+spectrometry+in+mammalian+cells+and+tissues%5BTitle%5D)+AND+%22Open+Biol%22%5BJournal%5D)">Quantification of lactoyl-CoA (lactyl-CoA) by liquid chromatography mass spectrometry in mammalian cells and tissues.</a> Open Biol. 10 (9), 200187(2020).</li>
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<li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NBS1+lactylation+is+required+for+efficient+DNA+repair+and+chemotherapy+resistance%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">NBS1 lactylation is required for efficient DNA repair and chemotherapy resistance.</a> Nature. 631 (8021), 663-669 (2024).</li>
<li>Zong, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alanyl-trna+synthetase%2C+AARS1%2C+is+a+lactate+sensor+and+lactyltransferase+that+lactylates+p53+and+contributes+to+tumorigenesis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Alanyl-trna synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis.</a> Cell. 187 (10), 2375-2392.e33 (2024).</li>
<li>Ju, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+alanyl-tRNA+synthetase+AARS1+moonlights+as+a+lactyltransferase+to+promote+YAP+signaling+in+gastric+cancer%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">The alanyl-tRNA synthetase AARS1 moonlights as a lactyltransferase to promote YAP signaling in gastric cancer.</a> J Clin Invest. 134 (10), e174587(2024).</li>
<li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lactylation+of+METTl16+promotes+cuproptosis+via+m6A-modification+on+FDX1+mRNA+in+gastric+cancer%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Lactylation of METTl16 promotes cuproptosis via m6A-modification on FDX1 mRNA in gastric cancer.</a> Nat Commun. 14 (1), 6523(2023).</li>
<li>Mao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+induces+mitochondrial+protein+lactylation+to+limit+oxidative+phosphorylation%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">Hypoxia induces mitochondrial protein lactylation to limit oxidative phosphorylation.</a> Cell Res. 34 (1), 13-30 (2024).</li>
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<li>Shao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+code+expansion+reveals+site-specific+lactylation+in+living+cells+reshapes+protein+functions%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Genetic code expansion reveals site-specific lactylation in living cells reshapes protein functions.</a> Nat Commun. 16 (1), 227(2025).</li>
<li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+encoding+of+%CE%B5-N-L-lactyllysine+for+detecting+delactylase+activity+in+living+cells%5BTitle%5D)+AND+%22Chem+Commun+%28Camb%29%22%5BJournal%5D)">Genetic encoding of ε-N-L-lactyllysine for detecting delactylase activity in living cells.</a> Chem Commun (Camb). 58 (61), 8544-8547 (2022).</li>
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<li>Mills, E. M., Barlow, V. L., Jones, A. T., Tsai, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+mammalian+cell+logic+gates+controlled+by+unnatural+amino+acids%5BTitle%5D)+AND+%22Cell+Rep+Methods%22%5BJournal%5D)">Development of mammalian cell logic gates controlled by unnatural amino acids.</a> Cell Rep Methods. 1 (6), 100073(2021).</li>
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</ol>

The authors have no conflicts of interests to disclose.

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...

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 proliferation2,3,4,5, metastasis6, metabolic reprogramming7, and therapeutic resistance8,9.
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 &#946; (PDGFR&#946;), 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 physiology15,16,17,18. 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 lactylation21,22,23,24. 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 KlacRS125,26. 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 platform25,26. 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.

<table><tbody><tr><td>6*his,His tag Recombinant monoclonal antibody</td><td>Proteintech</td><td>84814-1-RR</td><td>A class of specific immunoglobulins targeting the 6&amp;times;His tag (a short peptide composed of six consecutive histidine residues, HHHHHH) in recombinant proteins.</td></tr><tr><td>ACQUITY UPLC system</td><td>Waters</td><td></td><td>UltraPerformance Liquid Chromatography</td></tr><tr><td>Anti-L-Lactyllysine Rabbit mAb</td><td>PTM Biolabs</td><td>PTM-1401RM</td><td>A class of specific immunoglobulins targeting L-Lactyl Lysine (Klac), a post-translational modification (PTM) of proteins.</td></tr><tr><td>ClonExpress II One Step Cloning Kit</td><td>Vazyme&amp;nbsp;</td><td>C112</td><td>Based on simple, rapid, and efficient DNA seamless cloning technology, insert fragments can be directionally cloned into any site of any vector.</td></tr><tr><td>CytoFlex flow cytometer</td><td>Beckmann Coulter</td><td></td><td>Flow cytometer with high-sensitivity detection and multi-color analysis capability</td></tr><tr><td>DH10B</td><td>Biorabbit</td><td>EC005H-S</td><td>A type of competent cells for plasmid transformation, gene cloning, and blue-white screening.</td></tr><tr><td>DH5&amp;alpha;</td><td>Vazyme&amp;nbsp;</td><td>C502</td><td>A type of competent cells for plasmid transformation, gene cloning, and blue-white screening.</td></tr><tr><td>EASY-nano LC 1200 system</td><td>Thermo Fisher Scientific</td><td></td><td>nano-liquid chromatography for separating high-sensitivity, high-resolution complex samples</td></tr><tr><td>Orbitrap Eclipse Tribrid mass spectrometer</td><td>Thermo Fisher Scientific</td><td></td><td>a flagship high-end tribrid liquid chromatography-mass spectrometry (LC-MS) system</td></tr><tr><td>Phanta Max Super-Fidelity DNA Polymerase</td><td>Vazyme&amp;nbsp;</td><td>P505</td><td>A high-fidelity single enzyme combining rapid amplification and fidelity</td></tr><tr><td>PolyJet</td><td>SignaGen</td><td>SL100688</td><td>PolyJet DNA In Vitro Tranfection Reagent is formulated to be a powerful transfection Reagent that ensures effective and reproducible transfection with less cytotoxicity.</td></tr></tbody></table>

site-specific lysine lactylation via genetic code expansion in e. coli and mammalian cells

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

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)...

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.

Lactylation is a recently discovered post-translational modification (PTM) with diverse biological functions. Since its initial identification in 2019 as ...

We developed a clock/RS tRNA system to overcome mimicry and enzyme limitations, enabling for precise lysine lactylation incorporation to study its biological This protocol can be applied to study tumor metabolism, enzyme functions, and clock sites. To begin, co-transform 50 microliters of Escherichia coli DH10B chemically competent cells with approximately 100 nanograms each of the desired plasmids. Incubate the transformation mixture on ice for 25 minutes, then heat shock it at 42 degrees Celsius for 45 seconds.Return the tube to ice for three minutes. Add 350 microliters of Luria bertani medium and shake the tube vigorously at 220 revolutions per minute for one hour at 37 degrees Celsius. Now spread the transformed cell culture onto a Luria bertani agar plate supplemented with ampicillin and chloramphenicol.Incubate the plate at 37 degrees Celsius for 12 to 16 hours. Then pick a single colony from the plate to inoculate a four-milliliter starter culture and incubate it overnight. The next day, expand the overnight culture into 400 milliliters of Luria bertani medium containing ampicillin and chloramphenicol.Shake the culture vigorously at 220 revolutions per minute and 37 degrees Celsius until the optical density at 600 nanometers reaches between 0.6 and 0.8. Next induce the culture with 0.2%L-Arabinose and one millimolar lactyl lysine and incubate at 30 degrees Celsius and 220 revolutions per minute for 16 hours. Seed cells om a 12-well plate at a density of one times 10 to the power of five cells per well, using one milliliter of DMEM.Incubate for 24 hours until cell confluency reaches between 70 and 80%then proceed to transfection. To prepare the diluted DNA solution, add 0.75 micrograms of plasmid containing sfGFP reporter and pNeu-KlacRS in a one-to-one ratio into 38 microliters of serum-free DMEM with high glucose. Gently pipette up and down or vortex briefly to mix.To prepare the diluted transfection reagent, add 2.25 microliters of transfection reagent into 38 microliters of serum-free DMEM with high glucose. Gently pipette up and down three to four times to mix thoroughly. Immediately add the diluted transfection reagent to the diluted DNA solution all at once.Now pipette up and down three to four times to mix the contents and incubate the solution at room temperature for 10 to 15 minutes to allow the transfection and DNA complexes to form. Add 50 microliters of the transfection and DNA complex dropwise onto the medium in each well and gently swirl the plate to homogenize the mixture. Six hours after transfection, replace the medium with fresh DMEM containing one millimolar lactyl lysine and culture the cells for 24 to 48 hours.In Escherichia coli, full-length hENO1-89Klac was produced when one millimolar lactyl lysine was added to the growth medium. Immunoblotting confirmed the presence of full-length hENO1-89Klac using anti-His tag and anti-L-lactyl lysine antibodies. Electrospray ionization time of flight mass spectrometry revealed a dominant peak at 48, 178 Daltons, matching the expected mass of hENO1-89Klac lacking the initiating methionine.Tandem mass spectrometry of the triptych digest confirmed site-specific incorporation of Klac at Residue 89 through the presence of a complete B&Y ion series mapping to the modified peptide. In mammalian HEK293T cells, fluorescence microscopy showed strong sfGFP expression throughout the cells when Klac was added for 24 hours and 48 hours, while no fluorescence was observed without Klac supplementation. Flow cytometry revealed that approximately 31%of cells expressed sfGFP after 24 hours, increasing to about 37%after 48 hours of Klac supplementation.This protocol enables investigation of site-specific lysine lactylation effects on protein function and facilitates measurement of resulting biological and functional outcomes. Following this procedure, researchers can perform mechanistic studies, conduct high-throughput screening assays, and generate stable cell lines for extended functional analysis. Our future studies aim to improve efficiency, enable stable integration, and develop clock biosynthesis.

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JoVE Journal

Biochemistry

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A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

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Learn site-specific lysine lactylation using genetic code expansion in E. coli and mammalian cells. This method enables precise incorporation of Klac to study protein function and tumor metabolism.

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

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