For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80Â*% can be achieved for 15N and 13C with yields comparable to expression in full media. For 2H,15N and 2H,13C,15N labeling, incorporation is only slightly lower with 75 and 73Â*%, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.
Auto-inducing media for uniform isotope labeling of proteins with 15 N, 13 C and 2 H
Auto-inducing media for uniform isotope labeling of proteins with 15 N, 13 C and 2 H
Abstract
Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with 15N, 13C and/or 2H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media....
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04-20-2015 10:04 PM
[NMR paper] Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.
Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.
Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.
J Struct Biol. 2014 Aug 27;
Authors: Meola A, Deville C, Jeffers SA, Guardado-Calvo P, Vasiliauskaite I, Sizun C, Girard-Blanc C, Malosse C, Heijenoort CV, Chamot-Rooke J, Krey T, Guittet E, Pêtres S, Rey FA, Bontems F
Abstract
Nuclear...
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09-01-2014 07:46 PM
Robust and low cost uniform 15N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications
Robust and low cost uniform 15N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications
Publication date: Available online 28 August 2014
Source:Journal of Structural Biology</br>
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</br>
Nuclear magnetic...
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08-29-2014 05:36 PM
High-Resolution HeteronuclearMultidimensional NMRof Proteins in Living Insect Cells Using a Baculovirus Protein ExpressionSystem
High-Resolution HeteronuclearMultidimensional NMRof Proteins in Living Insect Cells Using a Baculovirus Protein ExpressionSystem
Jumpei Hamatsu, Daniel O’Donovan, Takashi Tanaka, Takahiro Shirai, Yuichiro Hourai, Tsutomu Mikawa, Teppei Ikeya, Masaki Mishima, Wayne Boucher, Brian O. Smith, Ernest D. Laue, Masahiro Shirakawa and Yutaka Ito
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja310928u/aop/images/medium/ja-2012-10928u_0005.gif
Journal of the American Chemical Society
DOI: 10.1021/ja310928u...
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01-27-2013 11:41 PM
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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10-05-2011 08:57 PM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Abstract Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives,...
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Proteins
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01-22-2011 03:46 AM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
J Biomol NMR. 2011 Jan 19;
Authors: Fan Y, Shi L, Ladizhansky V, Brown LS
Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties...
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01-21-2011 01:22 AM
[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...
Affordable uniform isotope labeling with 2 H, 13 C and 15 N in insect cells
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