Publication date: Available online 23 December 2015 Source:Biochimica et Biophysica Acta (BBA) - General Subjects
Author(s): Enrico Luchinat, Erica Secci, Francesca Cencetti, Paola Bruni
Background In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein-protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells. Methods We established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR. Results SOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by 1H-15N heteronuclear NMR. Conclusions and General Significance We showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein-protein interactions by NMR in human cells. Graphical abstract
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Solid-state NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization.
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J Am Chem Soc. 2013 Apr 3;135(13):5105-10
Authors: Takahashi H, Ayala I, Bardet M, De Paëpe G, Simorre JP, Hediger S
Abstract
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Solid-State NMR on Bacterial Cells: Selective Cell Wall Signal Enhancement and Resolution Improvement using Dynamic Nuclear Polarization
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Solid-State NMR on Bacterial Cells: Selective Cell Wall Signal Enhancement and Resolution Improvement using Dynamic Nuclear Polarization
Takahashi, H., et al., Solid-State NMR on Bacterial Cells: Selective Cell Wall Signal Enhancement and Resolution Improvement using Dynamic Nuclear Polarization. J. Am. Chem. Soc., 2013.
http://dx.doi.org/10.1021/ja312501d
Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate...
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04-15-2013 08:52 AM
High-yield Escherichia coli-based cell-free expression of human proteins
High-yield Escherichia coli-based cell-free expression of human proteins
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03-17-2012 07:32 AM
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
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[NMR paper] Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for
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Authors: Kigawa T, Muto Y, Yokoyama S
For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity...
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Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative ferredoxin.
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Cell-free expression and labeling strategies for a new decade in solid-state NMR.
Cell-free expression and labeling strategies for a new decade in solid-state NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Cell-free expression and labeling strategies for a new decade in solid-state NMR.
N Biotechnol. 2010 Aug 3;
Authors: Abdine A, Verhoeven MA, Warschawski DE
Although solid-state NMR and cell-free expression have recently become standard methods in biology, the combination of the two is still at a very early stage of development. In this...