The bioconjugation of proteins with small molecules has revolutionised the fields of chemical medicine, chemical biology, and cell biology, enabling scientists to probe and perturb dynamic cellular processes. The general use of chemical methods for the functionalisation of proteins is still limited however as they often require complicated reaction partners and/or non-physiological pH, which is incompatible with many protein scaffolds. Herein we describe efficient organocatalyst-mediated reaction on proteins using simple chemical probes which can afford a range of stable C-C linked bioconjugates at physiological pH. Herein we showcase the OPAL methodology in the &lsquo;biotinylation&rsquo; of green fluorescent protein (GFP).

Organocatalyst-mediated protein aldol ligations (OPAL) of proteins

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins like growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of the targeting proteins (antibodies or receptor ligands) connected to the highly cytotoxic drugs (too potent for conventional chemotherapy) by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. 
One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. The recent efforts of scientists brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monometyl auristatin E and methotrexate as model cytotoxic drugs. Both strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Preparation of site-specific cytotoxic protein conjugates via maleimide-thiol chemistry and sortase A-mediated ligation.

Lactylation is a recently identified post-translational modification with significant biological functions. Although numerous lactylation targets and sites have been discovered by proteomic studies, elucidating their biological functions by conventional biochemical methods remains challenging. To overcome this, we utilize genetic code expansion technology, which employs a lactylated-lysine-specific tRNA synthetase (KlacRS) and tRNA pair, enabling the precise incorporation of lactylated lysine (Klac) at a user-defined residue. By deploying this approach, one can produce precisely lactylated variants of proteins for direct functional characterization. Here, we present a streamlined protocol for site-specific incorporation of lactylation into proteins of interest. This workflow is validated in both Escherichia coli and mammalian cells, and illustrated using green fluorescent protein (GFP) as a model system.

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>

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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 representative functional protein hENO1 and sfGFP as model systems. hENO1 is a multifunctional glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate. It is involved in diverse biological processes, including cell growth, hypoxia adaptation, allergic responses, and immune regulation. Notably, multiple lactylation sites have been identified on ENO1 in tumor cells, including K89, characterized in this study, highlighting its potential roles as a transcriptional repressor and tumor suppressor. sfGFP is a robust fluorescent reporter protein widely used for labeling and tracking in live-cell studies. The residue at position 149 serves as a permissive site for site-specific incorporation of unnatural amino acids, making sfGFP an ideal model for visualizing and validating genetic code expansion systems across biological contexts. By using our workflow, Klac was site-specifically incorporated into hENO1 at position 89 in E. coli, as confirmed by SDS-PAGE, immunoblot, and mass spectrometry analysis. In mammalian cells, Klac was efficiently incorporated into sfGFP at position 149, verified by fluorescence microscopy and flow cytometry. This approach effectively overcomes the limitation of lysine-to-glutamine substitution, which cannot faithfully reproduce the chemical structure or biological function of authentic lactylation in living cells.
The GCE-based lactylation platform has been widely applied to investigate molecular mechanisms underlying protein function and to enable high-throughput functional screening. For instance, researchers have used this system to generate site-specifically lactylated p53 variants, such as p53K120Klac, p53K139Klac, and p53Dual-Klac, demonstrating that lactylation markedly reduces p53 binding affinity for the p53 response element (p53RE-DNA)19. Compromised DNA binding prevents lactylated p53 from effectively recruiting transcriptional machinery, inhibiting LLPS-mediated formation of transcriptional condensates and ultimately promoting tumorigenesis.
Beyond mechanistic studies, the GCE platform has been adapted for high throughput monitoring of enzyme activity. For example, a dual-color fluorescent probe (EGFP-K85Klac-mCherry) was developed by site-specifically incorporating Klac at Lys85 of enhanced green fluorescent protein (EGFP), enabling real-time reporting of de-lactylase activity in live cells27. This probe has also been used to monitor deacetylase activity in real time29, illustrating the versatility of the system for both mechanistic and functional screening applications.
Despite its utility, the current methodology has limitations. First, the incorporation efficiency of Klac remains suboptimal. Only about 37% of transfected mammalian cells expressed full-length lactylated protein, likely due to tRNA orthogonality, Klac membrane permeability, and intracellular stability. Future efforts could employ directed evolution to enhance the activity and specificity of the KlacRS/tRNA pair, improving UAG suppression efficiency and protein yield25,26. Second, the reliance on transient transfection limits application in hard-to-transfect cells and long-term studies30,31,32. Stable genomic integration of the KlacRS/tRNA system and the TAG-containing target gene-using PiggyBac or CRISPR/Cas9-would facilitate the generation of stable cell lines and support prolonged lactylation studies25,33. Third, unlike acetyllysine (AcK), which has been successfully biosynthesized in engineered cells and applied in screening deacetylase inhibitors and generating live epigenetic sensors in mouse models29, Klac biosynthesis remains underdeveloped. Site-specific lactylation still relies primarily on exogenous Klac supplementation, limiting large-scale production of homogeneous lactylated proteins and their application in complex biological systems. Addressing this bottleneck is critical for expanding the utility of GCE-based lactylation in mechanistic studies and high-throughput functional screening.
In summary, we have established a genetic code expansion platform for site-specific protein lactylation in prokaryotic and eukaryotic systems. This approach not only validates the KlacRS1 system but also provides a precise and scalable tool to overcome key technical barriers in lactylation research, supporting future mechanistic studies of this physiologically relevant modification.

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>

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) was produced at a yield of ~1.2 mg/L (Figure 1A). The presence of full-length hENO1 and selective incorporation of Klac was confirmed by immunoblotting using anti-His tag and anti-L-lactyl lysine antibodies (Figures 1B-C). The purified hENO1-89Klac was subjected to electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS; Figure 2A). A major peak observed at 48178.00 Da corresponds well to the intact hENO1 containing 89Klac and lacking the initiating methionine (hENO1-89Klac-Met: expected 48175.14 Da). Further analysis by tandem MS of the tryptic digest of hENO1-89Klac confirmed site-specific incorporation: the spectrum contained a complete series of b- and y-ions that map unambiguously to modification of residue 89 by Klac (Figure 2B).
We next tested Klac incorporation into proteins in mammalian cells. We co-transfected HEK293T cells with plasmid pNeu-KlacRS, which encoded both KlacRS and tRNA genes, along with an sfGFP reporter bearing an amber stop codon at position 149 (sfGFP-149 TAG). Fluorescence confocal microscopy (Figure 3A) revealed strong sfGFP fluorescence throughout the cells when Klac was added, while no fluorescence was observed in the absence of Klac. Cell morphology remained normal under Klac-supplemented conditions. No fluorescence signal was detected when Klac was not added. Using flow cytometry, we quantified fluorescence and found that ~31% of cells exhibited sfGFP expression after 24 h of Klac supplementation, increasing to ~37% after 48 h (Figure 3B). Fluorescence intensity also increased with longer incubation time. Collectively, these results suggest that Klac can be efficiently and specifically incorporated into proteins in both E. coli and mammalian cells. Therefore, our approach provides a convenient tool for protein lactylation study.
Protein sequence of hENO1-89Klac:
MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGASTGIYEALELR 
DNDKTRYMGKGVSKAVEHINKTIAPALVSKKLNVTEQEK(lac)IDKLMIEM 
DGTENKSKFGANAILGVSLAVCKAGAVEKGVPLYRHIADLAGNSEVILPV 
PAFNVINGGSHAGNKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNV 
IKEKYGKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKVVIGM 
DVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLYKSFIKDYPVVSIE 
DPFDQDDWGAWQKFTASAGIQVVGDDLTVTNPKRIAKAVNEKSCNCLL 
LKVNQIGSVTESLQACKLAQANGWGVMVSHRSGETEDTFIADLVVGL 
CTGQIKTGAPCRSERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAKLE 
HHHHHH
Protein sequence of sfGFP-149Klac:
MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK 
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPE 
GYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL 
GHKLEYNFNSHK(lac)VYITADKQKNGIKANFKIRHNVEDGSVQLA 
DHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFV 
TAAGITHGMDELYKGSHHHHHH
<img alt="Coomassie blue and immunoblot analysis of protein expression; molecular weight markers visible." class="xfigimg" src="/files/ftp_upload/70209/70209fig1.jpg" data-altupdatedat="1779163243000" data-alt="Figure 1"> 
Figure 1: Genetic encoding of Klac into hENO1 in E. coli. (A) The Coomassie blue-stained SDS-PAGE gel of purified full-length hENO1 and hENO1-89Klac. The triangle indicates full-length hENO1 expression. (B, C) Immunoblotting analysis of purified wild-type hENO1 and hENO1-89Klac, detected against anti-His, anti-L-Lactyl Lysine. <a href="https://www.jove.com/files/ftp_upload/70209/70209fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mass spectrometry spectra, protein sequence, peptide fragmentation; mass/charge, amino acid analysis." class="xfigimg" src="/files/ftp_upload/70209/70209fig2.jpg" data-altupdatedat="1779163243000" data-alt="Figure 2"> 
Figure 2: Mass spectrometry analysis of purified hENO1-89Klac. (A) ESI-TOF MS spectrum of hENO1-89Klac. (B) Tandem MS spectrum of purified hENO1-89Klac. <a href="https://www.jove.com/files/ftp_upload/70209/70209fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell fluorescence microscopy and flow cytometry showing GFP expression with Klac treatment, 24-48h." class="xfigimg" src="/files/ftp_upload/70209/70209fig3.jpg" data-altupdatedat="1779163243000" data-alt="Figure 3"> 
Figure 3: Genetic encoding of Klac into sfGFP in HEK293T cells. (A) Representative fluorescence images of HEK293T cells co-transfected with KlacRS and sfGFP-149TAG reporter (Scale bar, 50 µm). (B) FACS analysis of HEK293T cells co-transfected with KlacRS and sfGFP-149TAG reporter. <a href="https://www.jove.com/files/ftp_upload/70209/70209fig3large.jpg" target="_blank">Please click here to view a larger version of this figure</a>.

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.

site-specific-lysine-lactylation-via-genetic-code-expansion-e-coli

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 ...

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.

A Streamlined Protocol for Site-Specific Protein Lactylation in E. coli and Mammalian Cells

Louis Luk

Proteins are important biomolecules to sustain life-they perform a wide array of critical&nbsp;roles in natural organisms and biological&nbsp;systems. Over the previous decades, advances in technology have allowed&nbsp;us to modify proteins in&nbsp;very&nbsp;targeted ways, which opens up an even larger range of tailored functions for specific desired applications.&nbsp;Therefore, approaches for the selective modification of proteins are indispensable in life science and biomedical research. This Methods Collection will highlight video articles documenting innovative and gold-standard methods, and will cover topics in: enzymatic approaches for protein modifications, chemical approaches (including bioorthogonal reactions) for protein modifications, chemical genetic approaches for protein modulation, and site-specific protein modification for biological applications.

JoVE Chemistry surveys a broad scope of chemical disciplines. This collection includes fundamental and applied research that investigates molecular interactions, properties, and structure spanning in the following core divisions: analytical, organic, organometallic, inorganic, bioinorganic, and biochemistry. This section also includes the design and preparation of materials and small molecules for advanced or unique applications.

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

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