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

In this work, we will demonstrate a facile protocol to produce homogeneously acetylated proteins at specific sites by using E.coli as the expression host. The reversible lysine acetylation of proteins is one of the most abundant post-translational modifications in nature. It plays important roles in a wide range of biological processes, including gene transcription, stress response, cellular differentiation, and metabolism.

Generating purely acetylated proteins at design positions is very useful for studying protein acetylation. However, it is difficult for most classic methods. This challenge has been solved by the genetic code expansion technique.

It utilizes an engineered pyrrolysyl-tRNA synthetase variate to charge tRNA-Pyl with acetyllysine, then utilizes the host translational machinery to suppress the UAG stop codon in the mRNA, and directs the incorporation of acetyllysine in the design position of the target protein. Firstly, the amber stop codon needs to be introduced into the corresponding position in the target gene by site-directed mutagenesis. Amplify the template plasmid containing the gene of the wild-type proteins, and insert the stop codon mutation from the primers by the PCR reaction.

After PCR, the amplified material is added directly to the kinase-ligase-Dpn1 enzyme mix for one hour at room temperature for circularization and template removal. Add five microliters of the mix to the tube of 25 microliters thawed competent E.coli cells. Carefully flick the tube to mix, and place the mixture on ice for 30 minutes.

Heat shock the mixture at 42 degrees Celsius for 30 seconds and place on ice for additional five minutes. Pipette 600 microliters of room temperature SOC media into the mixture. Incubate at 37 degrees Celsius for 60 minutes with shaking.

Spread 100 microliters onto the plate with the corresponding antibiotic, and incubate overnight at 37 degrees Celsius. The plasmid from the single colony is sent for sequencing. The second step is to express and purify site-specifically acetylated protein.

Pick up a single colony from the plate, which contains the mutated TAG-containing target gene and the acetyllysine incorporation system. Inoculate into 15 milliliters fresh LB media with corresponding antibiotics overnight at 37 degrees Celsius. Transfer the 15-milliliter overnight culture to 300 milliliter fresh LB media with antibiotics, and incubate at 37 degrees Celsius.

Add five-millimolar acetyllysine and 20-millimolar nicotinamide as the final concentrations in growth media when absorbance reaches 5 at 600 nanometers. Grow cells for an additional one hour at 37 degrees Celsius. Then add the inducer for protein expression, and grow cells at 25 degrees Celsius overnight.

Collect cells by centrifuging at 3, 000 g for 15 minutes. Discard the supernatant, and store cell pellets at negative 80 degrees. Thaw the frozen cell pellets on ice and resuspend with 15 milliliters of lysis buffer, five microliters of beta-mercaptoethanol, and one microliter of nuclease.

Cells are broken by sonication. Centrifuge crude extract at 20, 000 g for 25 minutes at four degrees. Filter the supernatant with a 45-micrometer membrane filter.

Load filtered sample to a column containing one milliliter of nickel NTA resin, equilibrated with 20 milliliters of lysis buffer. Wash the column with 20 milliliters of wash buffer. The target protein is eluded with two milliliters of elution buffer.

The purified protein is desalted by the PD-10 column by adding 2.5 milliliter sample, and collect desalted sample with 3.5 milliliter desalting buffer. The next step is to biochemically characterize purified acetylated protein. Denature proteins with the SDS sample buffer above 100 degrees for five minutes.

Centrifuge the mixture, load onto the SDS-PAGE gel, and run at 200 volts for 30 minutes. Wash the gel with distilled water and shake gently. To make the sandwich for western blotting, from cathode to anode is sponge, filter paper, transfer buffer, soaked SDS-PAGE gel, methanol-activated PVDF membrane, another filter paper, remove the bubbles in the sandwich, and another sponge.

Put the stack in the transfer tank. Run at a constant current of 350 milliamps for 45 minutes. Wash the PVDF membrane with 25 milliliters TBST buffer for five minutes with gentle shaking.

Block the membrane with 5%BSA and the TBST buffer for one hour at room temperature. Incubate the membrane with diluted HRP-conjugated acetyllysine antibody at room temperature for one hour or four degrees overnight with gentle shaking. Wash the membrane with 20 milliliters TBST buffer for five minutes with gentle shaking.

Repeat this step four times. Apply the chemiluminescence substrate to the membrane for one minute. Capture the signal with a CCD camera base imager.

The same volumes of elution fractions and protein purification were loaded on the SDS-PAGE gel. The yield of acetylated protein is about half of the wild-type protein, which is highly efficient, and the same expression strain without acetyllysine and growth media cannot produce proteins with detectable amount, indicating high purity of acetylated protein since there are no canonical amino acids incorporated at this specific position. Western blotting showed that there is no detectable acetylation in wild-type protein, indicating the low background of non-specific acetylation of this expression system, while acetylated protein had a clear band.

Tandem mass spectrometry confirmed that acetylation occurs at the correct site whose corresponding position in the gene was mutated to the stop codon. To get good yields and purity of acetylated proteins, the optimized acetyllysine incorporation system should be used for both high efficiency and high purity. BL21(DE3)cells should be used for low background and nonspecific acetylation to increase the purity.

The genetic incorporation system could be used in eukaryotic systems for broader applications.