Expression, purification, and micelle reconstitution of antimicrobial Piscidin 1 and Piscidin 3 for NMR studies.
Protein Expr Purif. 2014 Aug 12;
Authors: Chen W, Cotten ML
Abstract
Piscidin 1 and piscidin 3, which were discovered in the mast cells of hybrid striped sea bass, are homologous antimicrobial peptides that are active against drug-resistant bacteria. Piscidin 1, the more antimicrobial and hemolytic peptide, also has anti-HIV-1 and anti-cancer properties. To understand the reasons underlying the different biological activities of the two peptides and identify principles to design antimicrobial drugs with improved efficacy and lower toxicity, their atomic-level structures must be obtained under physiologically-relevant conditions. High-resolution backbone structures of both piscidins exist in the presence of hydrated phospholipid bilayers but full structures that include the side chains are missing. Here, the piscidins 1 and 3 genes were cloned into the TrpLE vector. The corresponding TrpLE-piscidin fusion partners were expressed in E. Coli and recovered from inclusion bodies. Following steps that included Ni-NTA chromatography, cyanogen bromide cleavage of the fusion proteins, and reverse-phase HPLC, purified piscidins 1 and 3 were recovered in very good yield and characterized by NMR. High quality (15)N-(1)H HSQC spectra of piscidins 1 and 3 bound to SDS micelles were collected, demonstrating the feasibility of producing and purifying the isotopically-labeled piscidin peptides required to determine their full structures by multidimensional NMR spectroscopy.
PMID: 25131859 [PubMed - as supplied by publisher]
[NMR paper] Preparation of uniformly isotope labeled KcsA for solid state NMR: Expression, purification, reconstitution into liposomes and functional assay.
Preparation of uniformly isotope labeled KcsA for solid state NMR: Expression, purification, reconstitution into liposomes and functional assay.
Preparation of uniformly isotope labeled KcsA for solid state NMR: Expression, purification, reconstitution into liposomes and functional assay.
Protein Expr Purif. 2013 Aug 1;
Authors: Bhate MP, Wylie BJ, Thompson A, Tian L, Nimigean C, McDermott AE
Abstract
We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled...
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08-07-2013 01:24 PM
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...
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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.
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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
[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...
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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...
Expression, purification, and micelle reconstitution of antimicrobial Piscidin 1 and Piscidin 3 for NMR studies.
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