Genetic Code Expansion Method for Temporal Labeling of Endogenously Expressed Proteins

Schneider, Nils; Gäbelein, Christoph G.; Wiener, Jan; Georgiev, Tihomir; Gobet, Nicolas; Weber, Wilfried; Meier, Matthias

doi:10.1021/acschembio.8b00594

Cited by 5 publications

(3 citation statements)

References 18 publications

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“…This is often not the case for many proteins where only low affinity antibodies are available or none have so far been produced. While SCROL offers an alternative where the peptide tag is targeted by an antibody (Schneider et al, 2018), the SCROL cassette must be genomically incorporated to produce a stable cell line. (3) The increasing number of ncAAs allows for the development and optimization of techniques for specific purposes.…”

Section: Discussionmentioning

confidence: 99%

“…In an effort to further improve upon the specificity of FUNCAT-PLA, SCROL (Stop-Codon-Read-thrOugh-Label) was developed to study expression of a specific protein of interest (Schneider et al, 2018) (Figure 1C). A cassette containing the UAG amber codon, HA-tag, and an alternative stop codon (e.g., UGA opal codon) was genomically inserted at the 3'-end of the target protein using CRISPR/Cas9 in HEK293 cells.…”

Section: Protein Visualization Using Fluorescent Moleculesmentioning

confidence: 99%

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Using Genetic Code Expansion for Protein Biochemical Studies

Chung

Amikura

Söll

2020

Front. Bioeng. Biotechnol.

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Protein identification has gone beyond simply using protein/peptide tags and labeling canonical amino acids. Genetic code expansion has allowed residue-or site-specific incorporation of non-canonical amino acids into proteins. By taking advantage of the unique properties of non-canonical amino acids, we can identify spatiotemporal-specific protein states within living cells. Insertion of more than one non-canonical amino acid allows for selective labeling that can aid in the identification of weak or transient protein-protein interactions. This review will discuss recent studies applying genetic code expansion for protein labeling and identifying protein-protein interactions and offer considerations for future work in expanding genetic code expansion methods.

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Section: Discussionmentioning

confidence: 99%

Section: Protein Visualization Using Fluorescent Moleculesmentioning

confidence: 99%

Using Genetic Code Expansion for Protein Biochemical Studies

Chung

Amikura

Söll

2020

Front. Bioeng. Biotechnol.

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“…However, these techniques lack either covalent attachment or temporal control of target protein labeling, and hence, their use is limited to noncovalent associations with short, diffusion-limited visualization lifetimes. The ability to temporally control protein labeling would enable pulse-chase experiments for studying time-dependent processes such as response to a therapeutic agent or flux in metabolic reactions. − Temporally controlled covalent protein labeling would also prove useful in pull-down experiments, as the protein of interest (POI) could be selectively labeled and isolated from a complex system. , Thus, there remains a need for the development of a protein labeling strategy that can provide robust, stable, and temporally controlled labeling with low background signal.…”

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confidence: 99%

Fluorogenic Photoaffinity Labeling of Proteins in Living Cells

et al. 2019

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Genetically encoded fluorescent proteins or small-molecule probes that recognize specific protein binding partners can be used to label proteins to study their localization and function with fluorescence microscopy. However, these approaches are limited in signal-to-background resolution and the ability to temporally control labeling. Herein, we describe a covalent protein labeling technique using a fluorogenic malachite green probe functionalized with a photoreactive crosslinker. This enables a controlled covalent attachment to a genetically encodable fluorogen activating protein (FAP) with low background signal. We demonstrate covalent labeling of a protein in vitro as well as in live mammalian cells. This method is straightforward, displays high labeling specificity, and results in improved signal-to-background ratios in photoaffinity labeling of target proteins. Additionally, this probe provides temporal control over reactivity, enabling future applications in real-time monitoring of cellular events.

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Site‐Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover

et al. 2020

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Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio‐orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence‐based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse‐chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.

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Genetic Code Expansion Method for Temporal Labeling of Endogenously Expressed Proteins

Cited by 5 publications

References 18 publications

Using Genetic Code Expansion for Protein Biochemical Studies

Using Genetic Code Expansion for Protein Biochemical Studies

Fluorogenic Photoaffinity Labeling of Proteins in Living Cells

Site‐Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover

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