title: Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.
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.
authors.affiliation: 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.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Meola
authors.affiliation: 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.
authors.forename: Célia
authors.initials: C
authors.lastname: Deville
authors.affiliation: Département de virologie, Unité de virologie structurale, Institut Pasteur, 75015 Paris, France; CNRS URA3015, 75015 Paris, France.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Jeffers
authors.affiliation: Département de virologie, Unité de virologie structurale, Institut Pasteur, 75015 Paris, France; CNRS URA3015, 75015 Paris, France.
authors.forename: Pablo
authors.initials: P
authors.lastname: Guardado-Calvo
authors.affiliation: Département de virologie, Unité de virologie structurale, Institut Pasteur, 75015 Paris, France; CNRS URA3015, 75015 Paris, France.
authors.forename: Ieva
authors.initials: I
authors.lastname: Vasiliauskaite
authors.affiliation: 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.
authors.forename: Christina
authors.initials: C
authors.lastname: Sizun
authors.affiliation: Plateforme de production de protéines recombinantes, Institut Pasteur, 75015 Paris, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Girard-Blanc
authors.affiliation: Unité de spectrométrie de masse structurale et protéomique, Institut Pasteur, 75015 Paris, France; CNRS UMR 3528, Institut Pasteur, 75015 Paris, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Malosse
authors.affiliation: 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.
authors.forename: Carine
authors.initials: C
authors.lastname: van Heijenoort
authors.affiliation: Unité de spectrométrie de masse structurale et protéomique, Institut Pasteur, 75015 Paris, France; CNRS UMR 3528, Institut Pasteur, 75015 Paris, France.
authors.forename: Julia
authors.initials: J
authors.lastname: Chamot-Rooke
authors.affiliation: Département de virologie, Unité de virologie structurale, Institut Pasteur, 75015 Paris, France; CNRS URA3015, 75015 Paris, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Krey
authors.affiliation: 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.
authors.forename: Eric
authors.initials: E
authors.lastname: Guittet
authors.affiliation: Plateforme de production de protéines recombinantes, Institut Pasteur, 75015 Paris, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Pêtres
authors.affiliation: Département de virologie, Unité de virologie structurale, Institut Pasteur, 75015 Paris, France; CNRS URA3015, 75015 Paris, France.
authors.forename: Félix A
authors.initials: FA
authors.lastname: Rey
authors.affiliation: 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.
authors.forename: François
authors.initials: F
authors.lastname: Bontems
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D004330:Drosophila / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008279:Magnetic Resonance Imaging; D009590:Nitrogen Radioisotopes / Q000737:chemistry; D014176:Protein Biosynthesis; D061987:Sf9 Cells; D018411:Spodoptera; D014763:Viral Matrix Proteins / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D009590:Nitrogen Radioisotopes; D014763:Viral Matrix Proteins
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