We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and 13C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of 2H, 13C and 15N using auto-induction or isopropyl-β-d-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.
[NMR paper] NMR studies of large proteins.
NMR studies of large proteins.
Related Articles NMR studies of large proteins.
J Mol Biol. 2017 Jul 17;:
Authors: Jiang Y, Kalodimos CG
Abstract
Recent breakthroughs in isotope-labeling and pulse sequence techniques have enabled the Nuclear Magnetic Resonance (NMR) characterization of large protein systems with molecular weights of hundreds of kDa. NMR studies of a great variety of large proteins have provided unique insights into the binding, dynamic, and allosteric mechanisms. Here we present a brief summary of these...
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07-22-2017 09:28 PM
NMR studies of large proteins
NMR studies of large proteins
Publication date: Available online 17 July 2017
Source:Journal of Molecular Biology</br>
Author(s): Yajun Jiang, Charalampos G. Kalodimos</br>
Recent breakthroughs in isotope-labeling and pulse sequence techniques have enabled the Nuclear Magnetic Resonance (NMR) characterization of large protein systems with molecular weights of hundreds of kDa. NMR studies of a great variety of large proteins have provided unique insights into the binding, dynamic, and allosteric mechanisms. Here we present a brief summary of these developments by...
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07-18-2017 07:52 AM
PhD Studentship – Applications of large-scale NMR, EPR and MRI simulations
From The DNP-NMR Blog:
PhD Studentship – Applications of large-scale NMR, EPR and MRI simulations
PhD Studentship – Applications of large-scale NMR, EPR and MRI simulations
PhD Supervisor: Ilya Kuprov
Application Deadline: 01 June 2016
Project Description:
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01-14-2016 07:24 AM
[NMR paper] Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.
Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.
Related Articles Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.
Protein Expr Purif. 2014 Mar 21;
Authors: Kroupa T, Prchal J, Doležal M, Ruml T, Hrabal R
Abstract
Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures orstudying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon(13)C and...
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03-26-2014 12:44 PM
Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements
Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements
Publication date: Available online 21 March 2014
Source:Protein Expression and Purification</br>
Author(s): Tomáš Kroupa , Jan Prchal , Michal Doležal , Tomáš Ruml , Richard Hrabal</br>
Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures orstudying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon13C and nitrogen15N. The recombinant expression of labeled proteins is simple...
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03-22-2014 01:28 AM
[NMR paper] A Cost-Effective Protocol for the Parallel Production of Libraries of (13)CH 3-Specifically Labeled Mutants for NMR Studies of High Molecular Weight Proteins.
A Cost-Effective Protocol for the Parallel Production of Libraries of (13)CH 3-Specifically Labeled Mutants for NMR Studies of High Molecular Weight Proteins.
Related Articles A Cost-Effective Protocol for the Parallel Production of Libraries of (13)CH 3-Specifically Labeled Mutants for NMR Studies of High Molecular Weight Proteins.
Methods Mol Biol. 2014;1091:229-44
Authors: Crublet E, Kerfah R, Mas G, Noirclerc-Savoye M, Lantez V, Vernet T, Boisbouvier J
Abstract
There is increasing interest in applying NMR spectroscopy to the study of...
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11-11-2013 01:30 AM
A cost-effective look at proteins - with a little help from robotics - Cordis News
http://www.bionmr.com//t3.gstatic.com/images?q=tbn:ANd9GcTypMhbcsoTBmkGzm1ZmzjTHj-b-AsNVL4y8L03Y0oBfj2cCZUDv2gZmGEzmim-0VUKjKStcwU
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A cost-effective look at proteins - with a little help from robotics
Cordis News
Until now, a number of methods have been used to study static protein structures, including X-ray crystallography and nuclear magnetic resonance. However, these methods are of no use for proteins that are on the move; analytical methods, computer ...
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A cost-effective look at proteins - with a little help from...
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09-14-2013 01:46 AM
[NMR paper] A simple and effective NMR cell for studies of encapsulated proteins dissolved in low
A simple and effective NMR cell for studies of encapsulated proteins dissolved in low viscosity solvents.
Related Articles A simple and effective NMR cell for studies of encapsulated proteins dissolved in low viscosity solvents.
J Biomol NMR. 2002 Aug;23(4):311-6
Authors: Flynn PF, Milton MJ, Babu CR, Wand AJ
Application of triple-resonance and isotope-edited-NOE methods to the study of increasingly larger macromolecules and their complexes remains a central goal of solution NMR spectroscopy. The slow reorientational motion of larger molecules...
Cost-effective large-scale expression of proteins for NMR studies
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