Related ArticlesExpression, 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 against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. This loop plays a crucial role in phenotypic determination, cytopathicity (syncytium induction), and coreceptor usage of HIV-1. The human monoclonal antibody 447-52D was found to neutralize a broad spectrum of HIV-1 strains. In order to solve the solution structure of the V3MN peptide bound to the 447-52D Fab fragment by NMR, large quantities of labeled peptide and a protocol for the purification of the Fab fragment were needed. An expression plasmid coding for the 23-residue V3 peptide of the HIV-1MN strain (V3MN peptide, YNKRKRIHIGPGRAFYTTKNIIG) linked to a derivative of the RNA-binding domain of hnRNCP1 was constructed. The fusion protein attached to the V3 peptide prevents its degradation. Using this system, U-15N, U-13C,15N, and U-13C,15N, 50% 2H labeled fusion protein molecules were expressed in Escherichia coli grown on rich Celtone medium with yields of about 240 mg/liter. The V3MN peptide was released by CNBr cleavage and purified by RP-HPLC, giving final yields of 6-13 mg/liter. This expression system is generally applicable for biosynthesis of V3-related peptides and was also used to prepare the V3JR-FL. The 447-52D Fab fragment was obtained by a short enzymatic papain cleavage of the whole antibody. Preliminary NMR spectra demonstrate that full structural analysis of the V3MN complexed to the 447-52D Fab is feasible. This system enables studies of the same epitope bound to different HIV-1 neutralizing antibodies.
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...
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06-15-2011 01:15 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...
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06-15-2011 02:31 AM
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
Protein Expr Purif. 2011 May 27;
Authors: Long F, Cho W, Ishii Y
Amyloid fibrils of Alzheimer's ?-amyloid peptide (A?) are a primary component of amyloid plaques, a hallmark of Alzheimer's disease (AD). Enormous attention has been given to the structural...
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06-07-2011 11:05 AM
[NMR paper] Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloi
Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR.
Related Articles Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR.
Protein Expr Purif. 2005 Jul;42(1):200-10
Authors: Sharpe S, Yau WM, Tycko R
Fibrillar protein aggregates contribute to the pathology of a number of disease states. To facilitate structural studies of these amyloid fibrils by solid-state NMR, efficient methods for the production of milligram...
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11-24-2010 11:14 PM
[NMR paper] Expression, purification, crystallization, and NMR studies of the helicase interactio
Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase.
Related Articles Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase.
Protein Expr Purif. 2004 Feb;33(2):304-10
Authors: Loscha K, Oakley AJ, Bancia B, Schaeffer PM, Prosselkov P, Otting G, Wilce MC, Dixon NE
In Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three...
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11-24-2010 09:25 PM
Expression, Purification, Detergent Screening and Solution NMR Backbone Assignment of
Expression, Purification, Detergent Screening and Solution NMR Backbone Assignment of the Human Potassium Channel Accessory Subunit MiRP1.
Expression, Purification, Detergent Screening and Solution NMR Backbone Assignment of the Human Potassium Channel Accessory Subunit MiRP1.
Protein Expr Purif. 2010 Nov 15;
Authors: Chen L, Lai C, Lai J, Tian C
MiRP1 (MinK related protein 1) is a membrane protein in the KCNE family. It can associate with and modulate various voltage gated potassium channels. Mutations in human MiRP1 have been found to cause many...
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11-20-2010 06:01 PM
[NMR paper] Design, high-level expression, purification and characterization of soluble fragments
Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies.
Related Articles Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies.
Protein Eng. 2001 Aug;14(8):573-82
Authors: Gesell JJ, Liu D, Madison VS, Hesson T, Wang YS, Weber PC, Wyss DF
RNA helicases represent...
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11-19-2010 08:44 PM
Enhanced production and isotope enrichment of recombinant glycoproteins produced in c
Abstract NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant...
Expression, purification, and isotope labeling of a gp120 V3 peptide and production o
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