In Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E. coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage lambda-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5mM sample in 10mM phosphate, pH 6.05, 0.1M NaCl, recorded at 36 degrees C, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a=b=142.2A, c=192.1A, and diffracted beyond 2.7A resolution with synchrotron radiation.
Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.
Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.
Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.
Protein Expr Purif. 2011 Aug 31;
Authors: Chen W, Gamache E, Richardson D, Du Z, Wang C
Abstract
Alzheimer's disease (AD) is the most common type of dementia in elderly people. Senile plaques, a pathologic hallmark of AD, are composed of amyloid ? peptide (A?). A? aggregation produces...
nmrlearner
Journal club
0
09-13-2011 08:27 PM
Bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human APP binding protein LR11/SorLA for NMR studies.
Bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human APP binding protein LR11/SorLA for NMR studies.
Bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human APP binding protein LR11/SorLA for NMR studies.
Protein Expr Purif. 2011 Feb 11;
Authors: Wang X, Gill Jr RL, Zhu Q, Tian F
LR11 (SorLA) is a recently identified neuronal protein that interacts with amyloid precursor protein (APP), a central player...
[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...
nmrlearner
Journal club
0
11-24-2010 11:14 PM
[NMR paper] Purification, crystallization, NMR spectroscopy and biochemical analyses of alpha-phy
Purification, crystallization, NMR spectroscopy and biochemical analyses of alpha-phycoerythrocyanin peptides.
Related Articles Purification, crystallization, NMR spectroscopy and biochemical analyses of alpha-phycoerythrocyanin peptides.
Eur J Biochem. 2002 Oct;269(20):5046-55
Authors: Wiegand G, Parbel A, Seifert MH, Holak TA, Reuter W
The alpha-phycoerythrocyanin subunits of the different phycoerythrocyanin complexes of the phycobilisomes from the cyanobacterium Mastigocladus laminosus perform a remarkable photochemistry. Similar to...
nmrlearner
Journal club
0
11-24-2010 08:58 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
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...
nmrlearner
Journal club
0
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...
Expression, purification, crystallization, and NMR studies of the helicase interactio
Linear Mode
