Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy

Mallika Sastry, Ling Xu, Ivelin S. Georgiev, Carole A. Bewley, Gary J. Nabel, Peter D. Kwong

Research output: Contribution to journalArticle

Abstract

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 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 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.

Original languageEnglish (US)
Pages (from-to)197-207
Number of pages11
JournalJournal of Biomolecular NMR
Volume50
Issue number3
DOIs
StatePublished - Jul 2011
Externally publishedYes

Fingerprint

Nuclear magnetic resonance spectroscopy
HIV-1
Glycoproteins
Magnetic Resonance Spectroscopy
Isotopes
Proteins
Glycosylation
HIV Envelope Protein gp120
Biomolecular Nuclear Magnetic Resonance
Nuclear magnetic resonance
Affinity chromatography
Post Translational Protein Processing
Affinity Chromatography
Adenoviridae
Chemical shift
Noise
Epitopes
Antibodies

Keywords

  • Adenovirus vector
  • Eukaryotic expression
  • HIV-1 envelope
  • Isotope enrichment
  • N-linked glycosylation

ASJC Scopus subject areas

  • Spectroscopy
  • Biochemistry

Cite this

Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy. / Sastry, Mallika; Xu, Ling; Georgiev, Ivelin S.; Bewley, Carole A.; Nabel, Gary J.; Kwong, Peter D.

In: Journal of Biomolecular NMR, Vol. 50, No. 3, 07.2011, p. 197-207.

Research output: Contribution to journalArticle

Sastry, Mallika ; Xu, Ling ; Georgiev, Ivelin S. ; Bewley, Carole A. ; Nabel, Gary J. ; Kwong, Peter D. / Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy. In: Journal of Biomolecular NMR. 2011 ; Vol. 50, No. 3. pp. 197-207.
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