Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications
| العنوان: | Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications |
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| المؤلفون: | Félix A. Rey, Thomas Krey, Julia Chamot-Rooke, Christian Malosse, Célia Deville, Christine Girard-Blanc, Pablo Guardado-Calvo, Eric Guittet, François Bontems, Ieva Vasiliauskaite, Scott A. Jeffers, Stéphane Petres, Christina Sizun, Annalisa Meola, Carine van Heijenoort |
| المساهمون: | Virologie Structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Production de Protéines Recombinantes et d'Anticorps (Plate-Forme), Institut Pasteur [Paris], Spectrométrie de Masse structurale et protéomique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP) |
| المصدر: | Journal of Structural Biology Journal of Structural Biology, Elsevier, 2014, 188 (1), pp.71-78. ⟨10.1016/j.jsb.2014.08.002⟩ Journal of Structural Biology, 2014, 188 (1), pp.71-78. ⟨10.1016/j.jsb.2014.08.002⟩ |
| بيانات النشر: | HAL CCSD, 2014. |
| سنة النشر: | 2014 |
| مصطلحات موضوعية: | NMR structural studies of eukaryotic proteins, Mutant, Sf9, Spodoptera, (15)N-labeling of proteins produced in Sf9 and S2 insect cells, Viral Matrix Proteins, Structural Biology, Sf9 Cells, Animals, Humans, [CHIM]Chemical Sciences, Amino Acids, Actin, chemistry.chemical_classification, Nitrogen Radioisotopes, biology, Schneider 2 cells, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Gene Expression Profiling, Viral glycoprotein domain characterization, Nuclear magnetic resonance spectroscopy, biology.organism_classification, Magnetic Resonance Imaging, Yeast, Amino acid, Non-polymerizable actin mutant, Biochemistry, chemistry, Protein Biosynthesis, Drosophila, Cellular and viral membrane fusion proteins |
| الوصف: | International audience; Nuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as (15)N, (13)C and (2)H. This is usually easy and inexpensive when the proteins are expressed in Escherichiacoli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a (15)N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a (15)N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure. |
| اللغة: | English |
| تدمد: | 1047-8477 1095-8657 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::61c35b59588d92465841fd7af2ae02cf https://hal.archives-ouvertes.fr/hal-01077147 |
| حقوق: | CLOSED |
| رقم الأكسشن: | edsair.doi.dedup.....61c35b59588d92465841fd7af2ae02cf |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10478477 10958657 |
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