title: Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
abstract: NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in (15)N, (13)C, or (2)H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched (15)N or (15)N/(13)C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the (15)N- and (15)N/(13)C-labeled gp120s after affinity chromatography were 45 and 44 mg/l, respectively, with an average of over 80% isotope incorporation. Recognition of the labeled gp120 by cognate antibodies that recognize complex epitopes showed affinities comparable to the unlabeled protein. NMR spectra, including (1)H-(15)N and (1)H-(13)C HSQCs, (15)N-edited NOESY-HSQC, and 3D HNCO, were of high quality, with signal-to-noise consistent with an efficient level of isotope incorporation, and with chemical shift dispersion indicative of a well-folded protein. The exceptional protein yields, good isotope incorporation, and ability to obtain well-folded post-translationally modified proteins make this mammalian system attractive for the production of isotopically enriched eukaryotic proteins for NMR spectroscopy.
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3027, USA.
authors.forename: Mallika
authors.initials: M
authors.lastname: Sastry
authors.forename: Ling
authors.initials: L
authors.lastname: Xu
authors.forename: Ivelin S
authors.initials: IS
authors.lastname: Georgiev
authors.forename: Carole A
authors.initials: CA
authors.lastname: Bewley
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Nabel
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D020349:Surface Plasmon Resonance
chemical_list: D015699:HIV Envelope Protein gp120