Genetically encoding thioacetyl‐lysine as a non‐deacetylatable analog of lysine acetylation in <i>Escherichia coli</i>

Venkat, Sumana; Nannapaneni, Dharma Theja; Gregory, Caroline; Gan, Qinglei; McIntosh, Matt; Fan, Chenguang

doi:10.1002/2211-5463.12320

Cited by 27 publications

(30 citation statements)

References 64 publications

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“…We showed that TAcK residues could be recognized by the AcK antibody and the effect of thioacetylation was similar to that of acetylation on the enzyme activities of malate dehydrogenase, indicating that TAcK could be an ideal mimic of AcK in acetylation studies. Furthermore, we confirmed that TAcK residues could resist the deacetylase [ 45 ]. This system will be particularly useful if long-lasting effects of acetylation need to be determined in living cells with the concern of endogenous deacetylases.…”

Section: Lysine Acetylation and Its Analogs By Genetic Code Expanssupporting

confidence: 62%

“…Later, by flexizyme-mediated tRNA aminoacylation, Xiong et al were able to incorporate thio-acetyllysine (TAcK) into histone H3 site-specifically with the cell-free translation system [ 44 ]. Recently, our group further engineered the AcKRS for recognition of TAcK and successfully incorporated TAcK into proteins in E. coli cells [ 45 ]. We showed that TAcK residues could be recognized by the AcK antibody and the effect of thioacetylation was similar to that of acetylation on the enzyme activities of malate dehydrogenase, indicating that TAcK could be an ideal mimic of AcK in acetylation studies.…”

Section: Lysine Acetylation and Its Analogs By Genetic Code Expansmentioning

confidence: 99%

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Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

et al. 2018

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Nowadays advanced mass spectrometry techniques make the identification of protein posttranslational modifications (PTMs) much easier than ever before. A series of proteomic studies have demonstrated that large numbers of proteins in cells are modified by phosphorylation, acetylation and many other types of PTMs. However, only limited studies have been performed to validate or characterize those identified modification targets, mostly because PTMs are very dynamic, undergoing large changes in different growth stages or conditions. To overcome this issue, the genetic code expansion strategy has been introduced into PTM studies to genetically incorporate modified amino acids directly into desired positions of target proteins. Without using modifying enzymes, the genetic code expansion strategy could generate homogeneously modified proteins, thus providing powerful tools for PTM studies. In this review, we summarized recent development of genetic code expansion in PTM studies for research groups in this field.

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Section: Lysine Acetylation and Its Analogs By Genetic Code Expanssupporting

confidence: 62%

Section: Lysine Acetylation and Its Analogs By Genetic Code Expansmentioning

confidence: 99%

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

et al. 2018

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“…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 acetyllysine 43 , thus this system could be a good alternative approach in this case.…”

Section: Discussionmentioning

confidence: 99%

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in <em>Escherichia Coli</em>

Venkat

Gregory

Meng³

et al. 2017

JoVE

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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.

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“…X is the growth rate in the exponential phase, which was automatically provided by Gen5 software designed for the BioTek microplate reader (Winooski, VT, USA). The monitoring of GFP expression by fluorescence followed previous studies [ 13 , 14 ].…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth

Chen

Venkat

Wilson

et al. 2018

Genes

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Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins.

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Genetically encoding thioacetyl‐lysine as a non‐deacetylatable analog of lysine acetylation in Escherichia coli

Cited by 27 publications

References 64 publications

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in <em>Escherichia Coli</em>

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth

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