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J Struct Biol. 2014 Oct;188(1):71-8. doi: 10.1016/j.jsb.2014.08.002. Epub 2014 Aug 28.

Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.

Author information

1
Laboratoire de chimie et biologie structurales, Institut de chimie des substances naturelles, CNRS UPR2301, Centre de recherche de Gif-sur-Yvette, 91190 Gif-sur-Yvette, France; Département de virologie, Unité de virologie structurale, Institut Pasteur, 75015 Paris, France; CNRS URA3015, 75015 Paris, France.
2
Laboratoire de chimie et biologie structurales, Institut de chimie des substances naturelles, CNRS UPR2301, Centre de recherche de Gif-sur-Yvette, 91190 Gif-sur-Yvette, France.
3
Département de virologie, Unité de virologie structurale, Institut Pasteur, 75015 Paris, France; CNRS URA3015, 75015 Paris, France.
4
Plateforme de production de protéines recombinantes, Institut Pasteur, 75015 Paris, France.
5
Unité de spectrométrie de masse structurale et protéomique, Institut Pasteur, 75015 Paris, France; CNRS UMR 3528, Institut Pasteur, 75015 Paris, France.
6
Laboratoire de chimie et biologie structurales, Institut de chimie des substances naturelles, CNRS UPR2301, Centre de recherche de Gif-sur-Yvette, 91190 Gif-sur-Yvette, France; Département de virologie, Unité de virologie structurale, Institut Pasteur, 75015 Paris, France; CNRS URA3015, 75015 Paris, France. Electronic address: francois.bontems@cnrs.fr.

Abstract

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.

KEYWORDS:

(15)N-labeling of proteins produced in Sf9 and S2 insect cells; Cellular and viral membrane fusion proteins; NMR structural studies of eukaryotic proteins; Non-polymerizable actin mutant; Viral glycoprotein domain characterization

PMID:
25172991
DOI:
10.1016/j.jsb.2014.08.002
[Indexed for MEDLINE]

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