Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Gan, Qinglei; Fan, Chenguang

doi:10.1016/j.bbagen.2016.12.002

Cited by 27 publications

(23 citation statements)

References 41 publications

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“…Spectra were obtained in the positive ion, linear mode. The LC-MS/MS analyses were performed by Yale University Keck Proteomics Facility, following previous protocols [86, 87]. Proteins were digested in gel by trypsin, and analyzed by LC-MS/MS on an LTQ Orbitrap XL equipped with a nanoACQUITY UPLC system.…”

Section: Methodsmentioning

confidence: 99%

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

Venkat

Chen

Stahman

et al. 2018

Journal of Molecular Biology

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The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.

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Section: Methodsmentioning

confidence: 99%

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

Venkat

Chen

Stahman

et al. 2018

Journal of Molecular Biology

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“…24 Thirdly, the UAG codon could also be recognized by near-cognate tRNAs for the incorporation of canonical amino acids. 38, 39 While 2 mM Sep was added to the medium, the expression of the sfGFP variant increased significantly. The simultaneous incorporation of Sep and AcK was confirmed with full-length MS analyses (Figure 3b, S1-2).…”

Section: Resultsmentioning

confidence: 99%

Genetically Incorporating Two Distinct Post-translational Modifications into One Protein Simultaneously

et al. 2018

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Post-translational modifications (PTMs) play important roles in regulating a variety of biological processes. To facilitate PTM studies, the genetic code expansion strategy has been utilized to cotranslationally incorporate individual PTMs such as acetylation and phosphorylation into proteins at specific sites. However, recent studies have demonstrated that PTMs actually work together to regulate protein functions and structures. Thus, simultaneous incorporation of multiple distinct PTMs into one protein is highly desirable. In this study, we utilized the genetic incorporation systems of phosphoserine and acetyllysine to install both phosphorylation and acetylation into target proteins simultaneously in Escherichia coli. And we used this system to study the effect of coexisting acetylation and phosphorylation on malate dehydrogenase, demonstrating a practical application of this system in biochemical studies. Furthermore, we tested the mutual orthogonality of three widely used genetic incorporation systems, indicating the possibility of incorporating three distinct PTMs into one protein simultaneously.

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“…However, CFPS has no such limitations because all the translation components such as RFs and tRNAs can be modulated. Taking the advantage of CFPS, we have recently developed a method to increase the purity of pSer-containing proteins in the RF1-deficient background by reducing the amount of near-cognate tRNAs for the amber stop codon ( Gan and Fan, 2017 ) ( Figure 2 ). In this work, we first used an RF1-specific RNA aptamer to eliminate the RF1 activity in the CFPS system.…”

Section: Cell-free Protein Synthesis For Post-translational Modificatmentioning

confidence: 99%

The Application of Cell-Free Protein Synthesis in Genetic Code Expansion for Post-translational Modifications

et al. 2019

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The translation system is a sophisticated machinery that synthesizes proteins from 20 canonical amino acids. Recently, the repertoire of such composition has been expanded by the introduction of non-canonical amino acids (ncAAs) with the genetic code expansion strategy, which provides proteins with designed properties and structures for protein studies and engineering. Although the genetic code expansion strategy has been mostly implemented by using living cells as the host, a number of limits such as poor cellular uptake or solubility of specific ncAA substrates and the toxicity of target proteins have hindered the production of certain ncAA-modified proteins. To overcome those challenges, cell-free protein synthesis (CFPS) has been applied as it allows the precise control of reaction components. Several approaches have been recently developed to increase the purity and efficiency of ncAA incorporation in CFPS. Here, we summarized recent development of CFPS with an emphasis on its applications in generating site-specific protein post-translational modifications by the genetic code expansion strategy.

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Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Cited by 27 publications

References 41 publications

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

Genetically Incorporating Two Distinct Post-translational Modifications into One Protein Simultaneously

The Application of Cell-Free Protein Synthesis in Genetic Code Expansion for Post-translational Modifications

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