Publication date: Available online 28 August 2014 Source:Journal of Structural Biology
Author(s): Annalisa Meola , Célia Deville , Scott A. Jeffers , Pablo Guardado-Calvo , Ieva Vasiliauskaite , Christina Sizun , Christine Girard-Blanc , Christian Malosse , Carine van Heijenoort , Julia Chamot-Rooke , Thomas Krey , Eric Guittet , Stéphane Pêtres , Félix A. Rey , François Bontems
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 15N, 13C and 2H. This is usually easy and inexpensive when the proteins are expressed in E. coli, 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 15N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a 15N-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.
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
Abstract An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract,...
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Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells
Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells
Abstract The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed...
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01-09-2011 12:46 PM
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Abstract In the last 15 years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope...
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[NMR paper] Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (
Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/15N NMR in vitro assay.
Related Articles Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/15N NMR in vitro assay.
J Biotechnol. 2000 Apr 14;79(1):87-97
Authors: Doverskog M, Jacobsson U, Chapman BE, Kuchel PW, Häggström L
This is the second of two papers . where the general goal has been to determine and characterise the glutamine metabolism in Sf9...
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11-18-2010 09:15 PM
[NMR paper] Specific in vitro labeling of cells with a fluorine-19 probe encapsulated in antibody
Specific in vitro labeling of cells with a fluorine-19 probe encapsulated in antibody-targeted liposomes: a F-19 NMR spectroscopy study.
Related Articles Specific in vitro labeling of cells with a fluorine-19 probe encapsulated in antibody-targeted liposomes: a F-19 NMR spectroscopy study.
Magn Reson Med. 1993 Feb;29(2):252-5
Authors: Vion-Dury J, Machy P, Confort-Gouny S, Leserman L, Cozzone PJ
Liposomes containing dexamethasone phosphate (DMp) were covalently coupled to protein A and then incubated with murine L929 fibroblast and RDM4...
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08-21-2010 11:53 PM
[NMR paper] Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: applica
Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.
Related Articles Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.
Biochemistry. 1991 May 7;30(18):4491-4
Authors: Venters RA, Calderone TL, Spicer LD, Fierke CA
Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of glucose. We...
Robust and low cost uniform 15N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications
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