caa a22 4500 445325828 CHVBK 20180317142720.0 cr unu---uuuuu 170323e20110701xx s 000 0 eng 10.1007/s10858-011-9506-4 doi (NATIONALLICENCE)springer-10.1007/s10858-011-9506-4 Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy [Elektronische Daten] [Mallika Sastry, Ling Xu, Ivelin Georgiev, Carole Bewley, Gary Nabel, Peter Kwong] NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in 15N, 13C, or 2H. 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 15N or 15N/13C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the 15N- and 15N/13C-labeled gp120s after affinity chromatography were 45 and 44mg/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 1H-15N and 1H-13C HSQCs, 15N-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. The Author(s), 2011 Adenovirus vector nationallicence Eukaryotic expression nationallicence HIV-1 envelope nationallicence Isotope enrichment nationallicence N-linked glycosylation nationallicence Sastry Mallika Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut Xu Ling Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut Georgiev Ivelin Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut Bewley Carole Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892, Bethesda, MD, USA aut Nabel Gary Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut Kwong Peter Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut Journal of Biomolecular NMR Springer Netherlands 50/3(2011-07-01), 197-207 0925-2738 50:3<197 2011 50 10858 https://doi.org/10.1007/s10858-011-9506-4 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9506-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sastry Mallika Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut NATIONALLICENCE 700 1- Xu Ling Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut NATIONALLICENCE 700 1- Georgiev Ivelin Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut NATIONALLICENCE 700 1- Bewley Carole Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892, Bethesda, MD, USA aut NATIONALLICENCE 700 1- Nabel Gary Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut NATIONALLICENCE 700 1- Kwong Peter Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, 20892-3027, Bethesda, MD, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 50/3(2011-07-01), 197-207 0925-2738 50:3<197 2011 50 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer