Sequential protein expression and selective labeling for in-cell NMR in human cells

Luchinat, Enrico; Secci, Erica; Cencetti, Francesca; Bruni, Paola

doi:10.1016/j.bbagen.2015.12.023

Cited by 18 publications

(18 citation statements)

References 41 publications

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“…In most cases, the labeled medium can be recovered after protein expression, centrifuged to remove cell debris and residual DNA: PEI complex, and used again to prepare a second cell sample, which further reduces the cost per sample. For some applications, typically when some protein signals fall in a spectral region that is free from cellular background (for example, histidine ring N-1 H protons 5,24 ; isolated 1 H signals arising from the protein hydrophobic core 22,27 ), unlabeled samples can be prepared for 1 H-only in-cell NMR experiments, resulting in the cost per sample being greatly reduced. One can also use amino-acid-selective labeling schemes, by preparing a homemade cell growth medium following the composition of the commercial unlabeled medium and adding the isotopically labeled amino acid(s) according to the desired labeling scheme.…”

Section: Experimental Designmentioning

confidence: 99%

“…The protocol for protein overexpression in human HEK293T cells for in-cell NMR studies has been successfully applied to a number of systems 5,[22][23][24][25][26][27] . Several functional processes, including metal uptake 5,24 , disulfide-bond formation 5,22,24,26 and protein folding 22,26 , have been characterized for different proteins with different features and behaviors, and in different cellular compartments 23 .…”

Section: Applicationsmentioning

confidence: 99%

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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%

Section: Applicationsmentioning

confidence: 99%

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

2016

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“…Additionally, in organello NMR approaches are possible, in which protein expression is targeted to different cellular compartments by fusing specific targeting sequences to the protein of interest, such as a mitochondrial targeting sequence, as shown by our research group (Barbieri et al, 2014). Moreover, two or more proteins can be expressed, with only one selectively labelled, by controlling the timing of expression (Luchinat et al, 2016). The various approaches for in-cell NMR are summarized in Fig.…”

Section: Overview Of In-cell Nmr Approachesmentioning

confidence: 99%

In-cell NMR: a topical review

Luchinat

Banci

2017

IUCrJ

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112

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Classical structural biology approaches allow structural characterization of biological macromolecules in vitro, far from their physiological context. Nowadays, thanks to the wealth of structural data available and to technological and methodological advances, the interest of the research community is gradually shifting from pure structural determination towards the study of functional aspects of biomolecules. Therefore, a cellular structural approach is ideally needed to characterize biological molecules, such as proteins, in their native cellular environment and the functional processes that they are involved in. In-cell NMR is a new application of high-resolution nuclear magnetic resonance spectroscopy that allows structural and dynamical features of proteins and other macromolecules to be analyzed directly in living cells. Owing to its challenging nature, this methodology has shown slow, but steady, development over the past 15 years. To date, several in-cell NMR approaches have been successfully applied to both bacterial and eukaryotic cells, including several human cell lines, and important structural and functional aspects have been elucidated. In this topical review, the major advances of in-cell NMR are summarized, with a special focus on recent developments in eukaryotic and mammalian cells.

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“…An in‐organello NMR approach was also developed, in which protein expression was targeted to mitochondria via a specific targeting sequence . Furthermore, a protocol in which the timing of expression of two different proteins is controlled by coupling the silencing of a stably expressing gene with the transient expression of the second gene was set up, allowing the selective labelling of only one protein, which is critical to study protein‐protein interactions . These approaches start to become available to iNEXT users.…”

Section: Enabling Integrative Methodologies For Cellular Structural Bmentioning

confidence: 99%

iNEXT: a European facility network to stimulate translational structural biology

Boelens¹

2018

FEBS Letters

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Sequential protein expression and selective labeling for in-cell NMR in human cells

Cited by 18 publications

References 41 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

In-cell NMR: a topical review

iNEXT: a European facility network to stimulate translational structural biology

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