A Gel‐Encapsulated Bioreactor System for NMR Studies of Protein–Protein Interactions in Living Mammalian Cells

Kubo, Shugo; Nishida, N.; Udagawa, Yuko; Takarada, Osamu; Ogino, Shuichi; Shimada, Ichio

doi:10.1002/ange.201207243

Cited by 25 publications

(18 citation statements)

References 16 publications

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“…3-mm Shigemi tubes can be used in 5-mm probes with a proper spinner/adaptor. If more cells are available, larger tubes (5 mm normal/Shigemi tubes) may be used to increase the signalto-noise ratio (S/N) of the experiments, as shown elsewhere 34,35 .…”

Section: Experimental Designmentioning

confidence: 99%

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

2016

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In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. (1)H and (1)H-(15)N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform (15)N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection.

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Section: Experimental Designmentioning

confidence: 99%

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

2016

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“…Therefore, much effort has been devoted to extending in-cell NMR studies in eukaryotic cells and better describes biological processes such as protein maturation, posttranslational modification, and redox reactions, which alter the structural and functional properties [10][11][12]. Isotope-labeled proteins are initially produced in E. coli and then introduced into eukaryotic cells either by microinjection to Xenopus laevis oocytes [13][14][15] or by endocytotic transport mediated by cell-penetrating peptides [11,16] or by diffusion through pore forming toxins and electroporation [17,18]. However, delivering isotope-labeled proteins into eukaryotic cells has been a major drawback of this technique hampering the direct observation of proteins, which are overexpressed and modified in their natural environment.…”

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confidence: 99%

Probing the interaction of a quercetin bioconjugate with Bcl‐2 in living human cancer cells with in‐cell NMR spectroscopy

Primikyri

Sayyad

Quilici³

et al. 2018

FEBS Letters

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In-cell NMR spectroscopy has emerged as a powerful technique for monitoring biomolecular interactions at an atomic level inside intact cells. However, current methodologies are inadequate at charting intracellular interactions of nonlabeled proteins and require their prior isotopic labeling. Herein, we describe for the first time the monitoring of the quercetin-alanine bioconjugate interaction with the nonlabeled antiapoptotic protein Bcl-2 inside living human cancer cells. STD and Tr-NOESY in-cell NMR methodologies were successfully applied in the investigation of the binding, which was further validated in vitro. In-cell NMR proved a very promising strategy for the real-time probing of the interaction profile of potential drugs with their therapeutic targets in native cellular environments and could, thus, open a new avenue in drug discovery.

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“…Efforts to increase sample viability have been made, and resulted in the design of bioreactors that can be fitted inside an NMR spectrometer. Such bioreactors have been reported for both bacteria (32) and human cells (33). In both designs, a flow is applied that replaces the spent medium with fresh medium, providing nutrients and stabilizing the external pH.…”

Section: Overview Of In-cell Nmr Approachesmentioning

confidence: 99%

A Unique Tool for Cellular Structural Biology: In-cell NMR

Luchinat

Banci

2016

Journal of Biological Chemistry

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Conventional structural and chemical biology approaches are applied to macromolecules extrapolated from their native context. When this is done, important structural and functional features of macromolecules, which depend on their native network of interactions within the cell, may be lost. In-cell nuclear magnetic resonance is a branch of biomolecular NMR spectroscopy that allows macromolecules to be analyzed in living cells, at the atomic level. In-cell NMR can be applied to several cellular systems to obtain biologically relevant structural and functional information. Here we summarize the existing approaches and focus on the applications to protein folding, interactions, and post-translational modifications.

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A Gel‐Encapsulated Bioreactor System for NMR Studies of Protein–Protein Interactions in Living Mammalian Cells

Cited by 25 publications

References 16 publications

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

Probing the interaction of a quercetin bioconjugate with Bcl‐2 in living human cancer cells with in‐cell NMR spectroscopy

A Unique Tool for Cellular Structural Biology: In-cell NMR

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