[NMR paper] Expression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies.
Related ArticlesExpression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies.
Methods Mol Biol. 2019;1947:31-55
Authors: Bumbak F, Bathgate RAD, Scott DJ, Gooley PR
Abstract
Escherichia coli (E. coli) is the most widely used expression host for recombinant proteins due to high expression yields and straightforward molecular cloning. Directed evolution of G protein-coupled receptors (GPCRs) has made several of these difficult to express membrane proteins amenable to prokaryotic expression. Here, we describe a protocol for near complete 13CH3-methionine labeling of a thermostable neurotensin receptor 1 (enNTS1) variant in E. coli for solution NMR-based dynamics studies. Our expression strategy utilizes methionine biosynthesis pathway inhibition forcing E. coli to incorporate exogenous methionine with 96% efficiency at expression levels of 2.6*mg enNTS1 per liter of expression culture containing 50*mg of 13CH3-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6*mg of functional Apo-state enNTS1.
Biosynthetic production of fully carbon-13 labeled retinal in E. coli for structural and functional studies of rhodopsins
Biosynthetic production of fully carbon-13 labeled retinal in E. coli for structural and functional studies of rhodopsins
Abstract
The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments, which commonly require isotopic labeling of retinal. Unfortunately, the de novo organic synthesis of an...
nmrlearner
Journal club
0
03-24-2019 10:41 PM
[NMR paper] Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies.
Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies.
Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies.
Biochim Biophys Acta. 2018 Mar 26;:
Authors: Bumbak F, Keen AC, Gunn NJ, Gooley PR, Bathgate RAD, Scott DJ
Abstract
Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by...
nmrlearner
Journal club
0
03-30-2018 06:40 PM
[NMR paper] Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II.
Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II.
Related Articles Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II.
Methods Enzymol. 2017;593:387-403
Authors: Yeliseev A, Gawrisch K
Abstract
Cannabinoid receptor type II (CB2) is an integral membrane protein with seven transmembrane helices that belongs to the large superfamily of rhodopsin-like G protein-coupled receptors. The CB2 is a part of the endocannabinoid system that plays a vital role in...
nmrlearner
Journal club
0
07-29-2017 10:35 AM
[NMR paper] A Facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.
A Facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.
Related Articles A Facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.
Protein Expr Purif. 2015 Jul 28;
Authors: Sharma SC, Armand T, Aurelia Ball K, Chen A, Pelton JG, Wemmer DE, Head-Gordon T
Abstract
Alzheimer's disease (AD) is a progressive neurodegenerative disease...
nmrlearner
Journal club
0
08-02-2015 08:22 PM
[NMR paper] Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.
Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.
Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.
Protein Expr Purif. 2014 Nov 12;
Authors: Chadwick AC, Jensen DR, Peterson FC, Volkman BF, Sahoo D
Abstract
Scavenger receptor class B type I (SR-BI), the high density lipoprotein (HDL) receptor, is important for the delivery of...
nmrlearner
Journal club
0
12-03-2014 04:05 PM
[NMR paper] Elucidation by NMR solution of neurotensin in small unilamellar vesicle environment: molecular surveys for neurotensin receptor recognition.
Elucidation by NMR solution of neurotensin in small unilamellar vesicle environment: molecular surveys for neurotensin receptor recognition.
Related Articles Elucidation by NMR solution of neurotensin in small unilamellar vesicle environment: molecular surveys for neurotensin receptor recognition.
J Biomol Struct Dyn. 2013;31(8):809-17
Authors: Da Costa G, Bondon A, Delalande O, Mouret L, Monti JP
Abstract
Neurotensin (NT) is a tridecapeptide, hormone in the periphery and neurotransmitter in the brain. We used high-resolution nuclear...
nmrlearner
Journal club
0
02-08-2014 05:45 PM
[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...
Expression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies.
Linear Mode
