An improved expression protocol is proposed for amino acid type-specific [13C], [15N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449â??456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.
An economic approach to efficient isotope labeling in insect cells using homemade 15 N-, 13 C- and 2 H-labeled yeast extracts
An economic approach to efficient isotope labeling in insect cells using homemade 15 N-, 13 C- and 2 H-labeled yeast extracts
Abstract
Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein 15N and 13C labeling of up...
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06-12-2015 07:48 PM
Affordable uniform isotope labeling with 2 H, 13 C and 15 N in insect cells
Affordable uniform isotope labeling with 2 H, 13 C and 15 N in insect cells
Abstract
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...
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04-30-2015 09:13 PM
[NMR paper] Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--media.wiley.com-assets-2250-98-WileyOnlineLibrary-Button_120x27px_FullText.gif Related Articles Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
Chembiochem. 2013 Jan 30;
Authors: Michel E, Skrisovska L, Wüthrich K, Allain FH
Abstract
Current solution NMR techniques enable structural investigations of proteins in molecular particles with sizes...
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02-03-2013 10:19 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
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
[NMR paper] Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for
Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies.
Related Articles Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies.
FEBS Lett. 2004 Jun 18;568(1-3):117-21
Authors: Mason AJ, Siarheyeva A, Haase W, Lorch M, van Veen H, Glaubitz C
The ABC transporter LmrA in Lactococcus lactis confers resistance to a wide range of antibiotics and cytotoxic drugs and is a functional homologue of P-glycoprotein. Recently, solid-state NMR...
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11-24-2010 09:51 PM
[NMR paper] Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for
Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
Related Articles Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
J Biomol NMR. 1995 Sep;6(2):129-34
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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08-22-2010 03:50 AM
[NMR paper] Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled ba
Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: potential for NMR structure determination of large proteins.
Related Articles Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: potential for NMR structure determination of large proteins.
J Biomol NMR. 1999 May;14(1):79-83
Authors: Kelly MJ, Krieger C, Ball LJ, Yu Y, Richter G, Schmieder P, Bacher A, Oschkinat H
NMR investigations of larger macromolecules (> 20...
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08-21-2010 04:03 PM
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
Kit I. Tong, Masayuki Yamamoto and Toshiyuki Tanaka
Journal of Biomolecular NMR; 2008; 42(1); pp 59-67
Abstract:
A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo...
An improved protocol for amino acid type-selective isotope labeling in insect cells
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