This work was supported by the NIH (AI119813), the start-up from the University of Arkansas, and the award from Arkansas Biosciences Institute.

1. Site-Directed Mutagenesis of the Target Gene
Note: MDH is expressed under T7 promoter in the pCDF-1 vector with the CloDF13 origin and a copy number of 20 to 4034.
<ol>
	<li>Introduce the amber stop codon at the position 140 in the gene by primers (forward primer: GGTGTTTATGACTAGAACAAACTGTTCGGCG and reverse primer: GGCTTTTTTCAGCACTTCAGCAGCAATTGC), following the instruction of the site-directed mutagenesis kit.</li>
	<li>Amplify the template plasmid containing the gene of wild-type malate dehydratase, and insert the stop codon mutation by the polymerase chain reaction (PCR) reaction. In the reaction mixture, include 12.5 &#181;L of 2X DNA polymerase enzyme mix, 1.25 &#181;L of 10 &#181;M Forward primer, 1.25 &#181;L of 10 &#181;M Reverse primer, 1 &#181;L of template DNA (20 ng/&#181;L) (pCDF-1 plasmid containing the gene of wild-type MDH), and 9 &#181;L of nuclease-free water.
	<ol>
		<li>Use PCR reaction parameters as follows: Initial denaturation at 98 &#176;C for 30 s; 25 cycles of 10 s at 98 &#176;C, 30 s at 55 &#176;C, and 3 min at 72 &#176;C; final extension at 72 &#176;C for 3 min. After PCR, add the amplified material directly to the Kinase-Ligase-DpnI enzyme mix from the kit for 1 h at room temperature for circularization and template removal. 
		Note: The reaction mixture contains 1 &#181;L of PCR product, 5 &#181;L of 2X Reaction Buffer, 1 &#181;L of 10X Kinase-Ligase-DpnI enzyme mix, and 3 &#181;L of nuclease-free water.</li>
	</ol>
	</li>
	<li>Add 5 &#181;L of the reaction mix to the tube of 25 &#181;L thawed competent E. coli DH5&#945; cells from the kit. Carefully flick the tube to mix, and place the mixture on ice for 30 min. Heat shock the mixture at 42 &#176;C for 30 s, and place on ice for additional 5 min.
	<ol>
		<li>Pipette 600 &#181;L of room temperature Super Optimal broth with Catabolite repression (SOC) media from the kit into the mixture, incubate at 37 &#176;C for 60 min with shaking at 250 rpm, spread 100 &#181;L onto a lysogeny broth (LB) agar plate with the corresponding antibiotic, and incubate overnight at 37 &#176;C with shaking at 250 rpm.</li>
	</ol>
	</li>
	<li>Pick 4-6 single colonies into 6 mL fresh LB media with the corresponding antibiotic, and incubate at 37 &#176;C overnight with shaking at 250 rpm. Extract plasmids from each overnight culture by the plasmid purification kit, following the manufacturer&#39;s manual, then send plasmids for DNA sequencing according to the protocol of the service provider to confirm the stop codon mutation at correct positions.</li>
	<li>Store the strain with the correct sequence at -80 &#176;C by mixing 1 mL overnight culture and 300 &#181;L 100% DMSO.</li>
</ol>
2. Expression of the Acetylated Protein
<ol>
	<li>Insert the genes of optimized acetyllysyl-tRNA synthetase33 and optimized tRNAPyl 32 into the pTech plasmid. Place the tRNA synthetase gene under the constitutive lpp promoter. Place the tRNA gene under the constitutive proK promoter34.
	<ol>
		<li>Co-transform the expression vector34 containing the mutated TAG-containing gene of malate dehydrogenase, and the plasmid harboring the optimized acetyllysine incorporation system, into 25 &#181;L thawed competent E. coli BL21(DE3) cells by heat shock at 42 &#176;C for 10 s, and place on ice for additional 5 min.</li>
		<li>Pipette 600 &#181;L of room temperature SOC media into the mixture, incubate at 37 &#176;C for 60 min with shaking at 275 rpm, spread 100 &#181;L onto a plate with 100 &#181;g/mL streptomycin and 50 &#181;g/mL chloramphenicol, and incubate overnight at 37 &#176;C with shaking at 275 rpm.</li>
	</ol>
	</li>
	<li>Pick up a single colony from the plate, and inoculate into 15 mL fresh LB media with 100 &#181;g/mL streptomycin and 50 &#181;g/mL chloramphenicol in a 50 mL tube overnight at 37 &#176;C with shaking at the speed of 250 rpm. Transfer the 15 mL overnight culture to 300 mL fresh LB media with antibiotics in a 1 L flask, and incubate at 37 &#176;C with shaking at 250 rpm.</li>
	<li>Dissolve acetyllysine with water to make 100 mM stock solution, store at 4 &#176;C. Add 5 mM acetyllysine and 20 mM nicotinamide (inhibitor of deacetylases) to the growth media when absorbance reaches 0.5 at 600 nm.
	<ol>
		<li>Grow cells for an additional 1 h at 37 &#176;C, shaking at 250 rpm, then add 0.5 mM IPTG for protein expression, and grow cells at 25 &#176;C overnight, with shaking at 180 rpm. 
		NOTE: The expression conditions may need optimization for different proteins.</li>
	</ol>
	</li>
	<li>Collect cells by centrifuging at 3,000 x g at 4 &#176;C for 15 min, discard the supernatant, and wash cell pellets with the Phosphate-buffered saline (PBS) buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl). Collect washed cells at 10,000 x g at 4 &#176;C for 5 min, discard the supernatant, and store cell pellets at -80 &#176;C.</li>
</ol>
3. Purification of the Acetylated Protein
<ol>
	<li>Thaw the frozen cell pellets on ice, and re-suspend with 15 mL of lysis buffer (50 mM tris(hydroxymethyl)aminomethane (Tris) pH 7.8, 300 mM NaCl, 20 mM imidazole, and 20 mM nicotinamide), 5 &#181;L of &#946;-mercaptoethanol and 1 &#181;L Benzonase nuclease (250 units).</li>
	<li>Break cells by 40 kHz sonication at 70% power output with 10 cycles of 10 s short bursts, followed by intervals of 30 s for cooling to form crude extract. Centrifuge crude extract at 20,000 x g for 25 min at 4 &#176;C. Filter the supernatant with the 0.45 &#181;m membrane filter, and load into a column containing 1 mL of nickel-nitrilotriacetic acid (Ni-NTA) resin equilibrated with 20 mL of water and 20 mL of lysis buffer. 
	NOTE: Cells could also be broken by mild detergents, if sonication is not available.</li>
	<li>Wash the column with 20 mL of wash buffer (50 mM Tris pH 7.8, 300 mM NaCl, 50 mM imidazole, and 20 mM nicotinamide), and then elute with 2 mL of elution buffer (50 mM Tris pH 7.8, 300 mM NaCl, 150 mM imidazole, and 20 mM nicotinamide).</li>
	<li>Desalt the elution fraction with desalting buffer (25 mM Tris pH 7.8 and 10 mM NaCl) by the PD-10 column, following the manufacturer&#39;s manual. Measure the concentration of the eluted protein by following the instruction of the Bradford protein assay reagent. The desalted protein is ready for further experiments. 
	NOTE: Make 50% glycerol stock of the protein, and keep in -80 &#176;C for storage.</li>
</ol>
4. Biochemical Characterization of the Acetylated Protein
<ol>
	<li>SDS-PAGE and mass spectrometry analyses.
	<ol>
		<li>Denature proteins with the sodium dodecyl sulfate (SDS) sample buffer (5 &#181;L protein sample with 2 &#181;L 4X SDS sample buffer) in a 2 mL tube at 105 &#176;C for 5 min, centrifuge the mixture at 2000 x g for 10 s, load onto the 4-20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and run at 200 V for 30 min.</li>
		<li>Wash the gel with distilled water and shake gently for 5 min, repeating the process 3 times. Discard the water, and stain the gel with Coomassie blue stain for 1 h with gentle shaking. De-stain the gel with distilled water, shake gently for 30 min, and repeat this de-stain 3 times.</li>
		<li>Cut the band at 33 kDa on the Coomassie blue-stained SDS-PAGE gel, and send it to mass spectrometry facilities or companies to confirm the acetyllysine was incorporated at the designed position. 
		NOTE: The protocol of mass spectrometry analysis followed the previous experiment34.</li>
	</ol>
	</li>
	<li>Western Blotting
	<ol>
		<li>Run the SDS-PAGE gel with the same protocol in step 4.1. After the gel run, soak the gel with the transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, and 20% methanol) for 15 min.</li>
		<li>Activate a 0.2 &#181;m, 7 cm x 8.5 cm polyvinylidene difluoride (PVDF) membrane with methanol for 1 min, and rinse with transfer buffer before preparing the transfer sandwich. 
		NOTE: Methanol is hazardous in case of skin contact, eye contact, ingestion, or inhalation. Severe over-exposure can result in death.</li>
		<li>Make the transfer sandwich from cathode to anode (sponge, filter paper, SDS-PAGE gel, PVDF membrane, filter paper, and sponge). Put the stack in the transfer tank, run at constant current of 350 mA for 45 min. 
		NOTE: Transfer time may need optimization.</li>
		<li>Wash the PVDF membrane with 25 mL Tris-buffered saline, 0.1% Tween 20 (TBST) (137 mM NaCl, 20 mM Tris, 0.1% Tween-20, pH 7.6) buffer for 5 min with gentle shaking. Block the membrane with 5% Bovine serum albumin (BSA) in the TBST buffer for 1 h at room temperature.</li>
		<li>Incubate the membrane with HRP-conjugated acetyllysine-antibody with a final concentration of 1 &#181;g/mL diluted with 5% BSA in TBST at 4 &#176;C overnight with gentle shaking. 
		NOTE: For faster results, this step could be performed in room temperature for 1 h. The dilution of antibody may need optimization.</li>
		<li>Wash the membrane with 20 mL TBST buffer for 5 min with gentle shaking, repeating the step 4 times. Apply the chemiluminescence substrate to the membrane by following the manufacturer&#39;s instructions. Capture the signal with a charge-coupled device (CCD) camera-based imager.</li>
	</ol>
	</li>
</ol>

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A., Rogulina, S., O'Donoghue, P., Rinehart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+amino+acid+composition+of+proteins+within+an+expanded+genetic+code.">Revealing the amino acid composition of proteins within an expanded genetic code.</a> Nucleic Acids Res. 43 (2), e8 (2015).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+method+for+genetic+incorporation+of+multiple+noncanonical+amino+acids+into+one+protein+in+Escherichia+coli.">A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli.</a> Mol Biosyst. 6 (4), 683-686 (2010).</li><li>Venkat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoding+thioacetyl-lysine+as+a+nondeacetylatable+analog+of+lysine+acetylation+in+Escherichia+coli.">Genetically encoding thioacetyl-lysine as a nondeacetylatable analog of lysine acetylation in Escherichia coli.</a> FEBS Open. , (2017).</li><li>Wan, W., Tharp, J. M., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysyl-tRNA+synthetase:+an+ordinary+enzyme+but+an+outstanding+genetic+code+expansion+tool.">Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.</a> Biochim Biophys Acta. 1844 (6), 1059-1070 (2014).</li><li>Gregoretti, I. V., Lee, Y. M., Goodson, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+evolution+of+the+histone+deacetylase+family:+functional+implications+of+phylogenetic+analysis.">Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis.</a> J Mol Biol. 338 (1), 17-31 (2004).</li><li>Weinert, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetyl-phosphate+is+a+critical+determinant+of+lysine+acetylation+in+E.+coli.">Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli.</a> Mol cell. 51 (2), 265-272 (2013).</li></ol>

The authors have nothing to disclose.

The genetic incorporation of noncanonical amino acids (ncAAs) is based on the suppression of an assigned codon, mostly the amber stop codon UAG36,37,38,39, by the ncAA-charged tRNA containing the corresponding anticodon. As is known, the UAG codon is recognized by the release factor-1 (RF1) in bacteria, and it can also be suppressed by near cognate tRNAs from hosts charged by canonical amino acids (cAAs) such as lysine and tyrosine40,41. So, the efficiency of ncAA incorporation at the UAG codon depends on the competition between ncAA-charged tRNAs and RF1, while the purity of ncAA incorporation relies on the competition between ncAA-charged tRNAs and cAA-charged near cognate tRNAs. Low yield and purity of the target acetylated protein may be caused by the low incorporation efficiency of the orthogonal pair introduced into the host cells. This problem could be solved by increasing the concentration of acetyllysine in the media, and using recently optimized acetyllysine incorporation systems, which increased the UAG codon suppression by 58 times32,33, both the efficiency and purity of acetyllysine incorporation will be improved. As shown in Figure 1 and comparing protein yields, the efficiency of acetyllysine incorporation was about 50%, and there was no detectable protein purified from cells harboring the AcK incorporation system and the mutant gene of MDH, but without AcK in growth media, which indicated the high purity of acetyllysine incorporation. Moreover, mass spectrometry analysis also did not show any cAAs at position 140 of MDH, indicating the homogeneity of the acetyllysine incorporation.
There are two main limitations of this approach. Firstly, because of the competition of acetyllysine-charged tRNA with both RF1 and cAA-charged near cognate tRNAs described above, currently, the maximum number of acetyllysine residues that can be simultaneously incorporated into a single protein is three33,42. Secondly, cells have other types of deacetylases, which resist nicotinamide and may deacetylate certain target proteins. So, those proteins may not reach 100% acetylation at specific sites. Recently, we have established a thio-acetyllysine incorporation system which can be used as a non-deacetylable analog of acetyllysine43, thus this system could be a good alternative approach in this case.
As mentioned before, the classic approach to biochemically characterize lysine acetylation is using site-directed mutagenesis. Glutamine is used as a mimic of acetyllysine, and arginine is utilized as a non-acetylated lysine mimic. However, both mimics are not real isosteres, and do not always yield the expected results30. The genetic code expansion strategy could generate homogeneously acetylated proteins at specific lysine residues, which is the most rigorous way to characterize acetylated proteins.
The genetic incorporation system for acetyllysine was derived from the pair of pyrrolysyl-tRNA synthetase variants, and their cognate tRNA from Methanosarcinaceae species, which is also known to be orthogonal in eukaryotes44. Previous studies have shown that this system could be applied in mammalian cells and certain animals for protein acetylation studies39, thus the present protocol could be expanded to mammalian cells and even animals for wider applications in medical research and industry. Furthermore, this protocol is also essentially the same protocol used to incorporate different kinds of ncAAs, necessitating a simple change to the orthogonal pair introduced into the host cells.
Lysine deacetylases (KDACs) remove the acetyl group from the acetylated lysine residue in proteins45. The sirtuin-type CobB is the only well-known deacetylase in E. coli, which can be inhibited by nicotinamide27. So, to prevent the deacetylation of acetylated protein generated during cell growth and protein purification, 20 to 50 mM nicotinamide should be added in both growth media and purification buffers. Once purified, acetylation of lysine residues is relatively stable due to the lack of deacetylase. Secondly, to lower the background of nonspecific acetylation at other lysine residues in the protein, the BL21 (DE3) strain was used as the expression strain, due to its significantly lower level of protein acetylation than commonly used K12-derived strains46. As shown in Figure 2, the wild-type MDH expressed from BL21(DE3) cells had no detectable acetylation by western blotting. This is another important factor to increase the purity of acetylation in the target protein.

Post-translational modifications (PTMs) of proteins occur after the translation process, and arise from covalent addition of functional groups to amino acid residues, playing important roles in almost all the biological processes, including gene transcription, stress response, cellular differentiation, and metabolism1,2,3. To date, about 400 distinctive PTMs have been identified4. The intricacy of the genome and the proteome is amplified to a great extent by protein PTMs, as they regulate protein activity and localization, and affect the interaction with other molecules such as proteins, nucleic acids, lipids, and cofactors5.
Protein acetylation has been at the forefront of PTMs studies in the last two decades6,7,8,9,10,11,12. Lysine acetylation was first discovered in histones more than 50 years ago13,14, has been well scrutinized, and is known to exist in more than 80 transcription factors, regulators, and various proteins15,16,17. Studies on protein acetylation have not only provided us with a deeper understanding of its regulatory mechanisms, but also guided treatments for a number of diseases caused by dysfunctional acetylation18,19,20,21,22,23. It was believed that lysine acetylation only happens in eukaryotes, but recent studies have shown that protein acetylation also plays key roles in bacterial physiology, including chemotaxis, acid resistance, activation, and stabilization of pathogenicity islands and other virulence related proteins24,25,26,27,28,29.
A commonly used method to biochemically characterize lysine acetylation is using site-directed mutagenesis. Glutamine is used as a mimic of acetyllysine because of its similar size and polarity. Arginine is utilized as a non-acetylated lysine mimic, since it preserves its positive charge under physiological conditions but cannot be acetylated. However, both mimics are not real isosteres and do not always yield the expected results30. The most rigorous approach is to generate homogeneously acetylated proteins at specific lysine residues, which is difficult or impossible for most classical methods due to the low stoichiometry of lysine acetylation in nature7,11. This challenge has been unraveled by the genetic code expansion strategy, which employs an engineered pyrrolysyl-tRNA synthetase variant from Methanosarcinaceae species to charge tRNAPyl with acetyllysine, utilizes the host translational machinery to suppress the UAG stop codon in the mRNA, and directs the incorporation of acetyllysine in the designed position of the target protein31. Recently, we have optimized this system with an improved EF-Tu-binding tRNA32 and an upgraded acetyllysyl-tRNA synthetase33. Furthermore, we have applied this enhanced incorporation system in acetylation studies of malate dehydrogenase34 and tyrosyl-tRNA synthetase35. Herein, we demonstrate the protocol for generating purely acetylated proteins from the molecular cloning to biochemical identification by using malate dehydrogenase (MDH), which we have extensively studied as a demonstrative example.

<table><tbody><tr><td>Bradford protein assay</td><td>Bio-Rad</td><td>5000006</td><td>Protein concentration</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747</td><td>SDS sample buffer</td></tr><tr><td>Coomassie G-250 Stain</td><td>Bio-Rad</td><td>1610786</td><td>SDS-PAGE gel staining</td></tr><tr><td>4-20% SDS-PAGE ready gel</td><td>Bio-Rad</td><td>4561093</td><td>Protein determination</td></tr><tr><td>Ac-K-100 (HRP Conjugate)</td><td>Cell Signaling</td><td>6952</td><td>Antibody</td></tr><tr><td>IPTG</td><td>CHEM-IMPEX</td><td>194</td><td>Expression inducer</td></tr><tr><td>N&lt;sup&gt;&amp;epsilon;&lt;/sup&gt;-Acetyl-L-lysine</td><td>CHEM-IMPEX</td><td>5364</td><td>Noncanonical amino acid</td></tr><tr><td>PD-10 desalting column</td><td>GE Healthcare</td><td>17085101</td><td>Desalting</td></tr><tr><td>Q5 Site-Directed Mutagenesis Kit</td><td>NEB</td><td>E0554</td><td>Introducing the stop codon</td></tr><tr><td>BL21 (DE3) cells</td><td>NEB</td><td>C2527</td><td>Expressing strain</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>QIAGEN</td><td>27106</td><td>Extracting plasmids</td></tr><tr><td>Ni-NTA resin</td><td>QIAGEN</td><td>30210</td><td>Affinity purification resin</td></tr><tr><td>nicotinamide</td><td>Sigma-Aldrich</td><td>N3376</td><td>Deacetylase inhibitor</td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td>Reducing agent</td></tr><tr><td>BugBuster Protein Extraction Reagent</td><td>Sigma-Aldrich</td><td>70584</td><td>Breaking cells</td></tr><tr><td>Benzonase nuclease</td><td>Sigma-Aldrich</td><td>E1014</td><td>DNase</td></tr><tr><td>ECL Western Blotting Substrate</td><td>ThermoFisher</td><td>32106</td><td>Chemiluminescence</td></tr><tr><td>Premixed LB Broth</td><td>VWR</td><td>97064</td><td>Cell growth medium</td></tr><tr><td>Bovine serum albumin</td><td>VWR</td><td>97061-416</td><td>western blots blocking</td></tr></tbody></table>

a facile protocol to generate site-specifically acetylated proteins in escherichia coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

The yield of acetylated MDH protein was 15 mg per 1 L culture, while that of wild-type MDH was 31 mg per 1 L culture. Purified proteins were analyzed by SDS-PAGE as shown in Figure 1. The wild-type MDH was used as a positive control34. The protein purified from cells harboring the acetyllysine (AcK) incorporation system and the mutant mdh gene, but without AcK in growth media, was used as a negative control. Lysine acetylation of purified proteins was detected by western blotting using the acetyllysine-antibody as shown in Figure 2. The acetylation of the lysine residue 140 in the malate dehydrogenase was confirmed by tandem mass spectrometry analysis as shown in Figure 3.
The protein sequence of MDH protein (The fragment for tandem MS analysis is in bold):
MKVAVLGAAGGIGQALALLLKTQLPSGSELSLYDIAPVTPGVAVDLSHIPTAVKIKGFSGEDAT 
PALEGADVVLISAGVARKPGMDRSDLFNVNAGIVKNLVQQVAKTCPKACIGIITNPVNTTVAIAA 
EVLKKAGVYDKNKLFGVTTLDIIRSNTFVAELKGKQPGEVEVPVIGGHSGVTILPLLSQVPGV 
SFTEQEVADLTKRIQNAGTEVVEAKAGGGSATLSMGQAAARFGLSLVRALQGEQGVVECAY 
VEGDGQYARFFSQPLLLGKNGVEERKSIGTLSAFEQNALEGMLDTLKKDIALGEEFVNK
The protein sequence of optimized acetyllysyl-tRNA synthetase33:
MDKKPLDVLISATGLWMSRTGTLHKIKHYEISRSKIYIEMACGDHLVVNNSRSCRPARAFRYHKY 
RKTCKRCRVSDEDINNFLTRSTEGKTSVKVKVVSEPKVKKAMPKSVSRAPKPLENPVSAKAST 
DTSRSVPSPAKSTPNSPVPTSASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRR 
KKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKN 
FCLRPMMAPNLLNYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRE 
NLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGF 
GLERLLKVKHDFKNIKRAARSESYYNGISTNL
The gene sequence of optimized tRNAPyl 32:
GGAAACGTGATCATGTAGATCGAATGGACTCTAAATCCGTTCAGTGGGGTTAGATTCCCC 
ACGTTTCCGCCA
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57061/57061fig1.jpg" /> 
Figure 1: The Coomassie blue-stained SDS-PAGE gel of purified full-length MDH and its AcK-containing variant. The same volumes of elution fractions were loaded onto the SDS-PAGE gel. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57061/57061fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57061/57061fig2.jpg" /> 
Figure 2: The western blotting of purified wild-type MDH and its AcK-containing variant. The same volumes of elution fractions were loaded. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57061/57061fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57061/57061fig3.jpg" /> 
Figure 3: LC-MS/MS analysis of AcK-containing MDH variant. The tandem mass spectrum of the peptide (residues 135-142) AGVYDKACNK from purified acetylated MDH variant. KAC denotes AcK incorporation. The partial sequence of the peptide containing the AcK can be read from the annotated b or y ion series. Matched peaks were in red. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57061/57061fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli at JoVE.com

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

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

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

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

a-facile-protocol-to-generate-site-specifically-acetylated-proteins

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.8K Views.  University of Arkansas. Uitbreiding van de genetische code dient als een krachtig hulpmiddel om te bestuderen van een breed scala aan biologische processen, met inbegrip van eiwit acetylation. Hier tonen we een facile protocol misbruiken van deze techniek voor het genereren van homogeen geacetyleerd eiwitten op specifieke sites in Escherichia coli cellen.

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

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Biochemistry

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

Chemistry

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Bioengineering

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Process Evaluation and Kinetics of Recombinant Chitin Deacetylase Expression in E. coli Rosetta pLysS Cells Using a Statistical Technique

In recent years, the greener route of the deacetylation of chitin to chitosan using the enzyme chitin deacetylase has gained importance. Enzymatically converted chitosan with emulating characteristics has a broad range of applications, particularly in the biomedical field. Several recombinant chitin deacetylases from various environmental sources have been reported, but there are no studies on process optimization for the production of these recombinant chitin deacetylases. The present study used the central composite design of response surface methodology to maximize the recombinant bacterial chitin deacetylase (BaCDA) production in E. coli Rosetta pLysS. The optimized process conditions were 0.061% glucose concentration, 1% lactose concentration, an incubation temperature of 22 &#176;C, an agitation speed at 128 rpm, and 30 h of fermentation. At optimized conditions, the expression due to lactose induction was initiated after 16 h of fermentation. The maximum expression, biomass, and BaCDA activity were recorded 14 h post-induction. At the optimized condition, the BaCDA activity of expressed BaCDA was increased ~2.39-fold. The process optimization reduced the total fermentation cycle by 22 h and expression time by 10 h post-induction. This is the first study to report the process optimization of recombinant chitin deacetylase expression using a central composite design and its kinetic profiling. Adapting these optimal growth conditions could result in cost-effective, large-scale production of the lesser-explored moneran deacetylase, embarking on a greener route for biomedical-grade chitosan production.

Process Evaluation and Kinetics of Recombinant Chitin Deacetylase Expression in E. coli Rosetta pLysS Cells Using a Statistical Technique

In the current protocol, a statistical technique, central composite design (CCD), was applied to optimize the process conditions for the expression of recombinant bacterial chitin deacetylase (BaCDA) in E. coli Rosetta pLysS cells. The employment of CCD resulted in a ~2.39-fold increase in the expression and activity of BaCDA.

In recent years, the greener route of the deacetylation of chitin to chitosan using the enzyme chitin deacetylase has gained importance. Enzymatically ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Biology

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

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

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