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

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.