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

		BioNMR > Educational resources > Journal club
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.

Webservers

NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

Preditor

TALOS

Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

PECAN

Flexibility from chemical shifts:

RCI

Interactions from chemical shifts:

HADDOCK

Chemical shifts re-referencing:

NMR model quality:

NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

Quality Control Check

NMR spectrum prediction:

FANDAS

MestReS

V-NMR

Flexibility from structure:

Backbone S2

Methyl S2

B-factor

Molecular dynamics:

Gromacs

Amber

Antechamber

Chemical shifts prediction:

From structure:

Shiftx2

Sparta+

Camshift

CH3shift- Methyl

ArShift- Aromatic

ShiftS

Proshift

PPM

CheShift-2- Cα

From sequence:

Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

CCPN

From NMR-STAR 3.1

Validate NMR-STAR 3.1

NMR sample preparation:

Protein disorder:

DisMeta

Protein solubility:

Isotope labeling:

Solid-state NMR:

Thread Tools

Search this Thread

Rate Thread

Display Modes

06-15-2011, 01:15 PM

nmrlearner

Senior Member

Join Date: Jan 2005

Posts: 23,732

Points: 193,617, Level: 100

Level up: 0%, 0 Points needed

Activity: 50.7%

Last Achievements

Award-Showcase

NMR Credits: 0

NMR Points: 193,617

Downloads: 0

Uploads: 0

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

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

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

J Biomol NMR. 2011 Jun 12;

Authors: Sastry M, Xu L, Georgiev IS, Bewley CA, Nabel GJ, Kwong PD

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.

PMID: 21667299 [PubMed - as supplied by publisher]

Source: PubMed

Did you find this post helpful?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins Publication year: 2011 Source: Journal of Magnetic Resonance, Volume 213, Issue 2, December 2011, Pages 423-441</br> Lewis E.Kay, MitsuhikoIkura, RolfTschudin, AdBax</br> Four new and complementary three-dimensional triple-resonance experiments are described for obtaining complete backboneH,C, andN resonance assignments of proteins uniformly enriched withC andN. The new methods all rely onH detection and use multiple magnetization transfers through well-resolved one-bondJcouplings. Therefore, the...	nmrlearner	Journal club	0	12-11-2011 07:57 AM
Probing the HIV gp120 envelope glycoprotein conformation by NMR. Probing the HIV gp120 envelope glycoprotein conformation by NMR. Probing the HIV gp120 envelope glycoprotein conformation by NMR. J Biol Chem. 2011 Jul 8;286(27):23975-81 Authors: Celigoy J, Ramirez B, Tao L, Rong L, Yan L, Feng YR, Quinnan GV, Broder CC, Caffrey M Abstract The HIV envelope glycoprotein gp120 plays a critical role in virus entry, and thus, its structure is of extreme interest for the development of novel therapeutics and vaccines. To date, high resolution structural information about gp120 in complex with gp41 has proven...	nmrlearner	Journal club	0	09-09-2011 06:42 PM
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy 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 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...	nmrlearner	Journal club	0	06-15-2011 02:31 AM
Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells Abstract The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed...	nmrlearner	Journal club	0	01-09-2011 12:46 PM
[NMR paper] Expression, purification, and isotope labeling of a gp120 V3 peptide and production o Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies. Related Articles Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies. Protein Expr Purif. 2002 Apr;24(3):374-83 Authors: Sharon M, Görlach M, Levy R, Hayek Y, Anglister J Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed...	nmrlearner	Journal club	0	11-24-2010 08:49 PM
[NMR paper] Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Related Articles Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat Struct Biol. 2001 Apr;8(4):334-8 Authors: Arora A, Abildgaard F, Bushweller JH, Tamm LK We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel...	nmrlearner	Journal club	0	11-19-2010 08:32 PM
Production of recombinant isotopically labelled peptide by fusion to an insoluble par Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin ?v?6 binding peptides for NMR. Related Articles Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin ?v?6 binding peptides for NMR. Mol Biosyst. 2010 Oct 18; Authors: Wagstaff JL, Howard MJ, Williamson RA The integrin ?v?6 is up-regulated in several cancers and has clinical potential for both tumour imaging and therapy. Peptide ligands have been developed...	nmrlearner	Journal club	0	10-19-2010 04:51 PM
[NMR paper] Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched proteins: a powerful new strategy for structure determination. Related Articles Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched proteins: a powerful new strategy for structure determination. Ciba Found Symp. 1991;161:108-19; discussion 119-35 Authors: Bax A, Ikura M, Kay LE, Barbato G, Spera S A procedure is described that affords complete 1H, 13C and 15N resonance assignment in proteins of up to about 25 kDa. The new...	nmrlearner	Journal club	0	08-21-2010 11:16 PM

« Previous Thread | Next Thread »

Posting Rules
You may not post new threads You may not post replies You may not post attachments You may not edit your posts BB code is On Smilies are On [IMG] code is On HTML code is On Trackbacks are Off Pingbacks are Off Refbacks are Off