A team from Massachusetts Institute of Technology (MIT) has found that they can enhance nuclear magnetic resonance (NMR) spectroscopy in such a way as to facilitate the structure determination of proteins associated with a wide range of diseases.
Increasing the buffering capacity of minimal media leads to higher protein yield
Increasing the buffering capacity of minimal media leads to higher protein yield
Abstract
We describe a general and simple modification to the standard M9 minimal medium recipe that leads to an approximate twofold increase in the yield of heterologously expressed proteins in Escherichia coli BL21(DE3) bacteria. We monitored the growth of bacteria transformed with plasmids for three different test proteins in five minimal media with different concentrations of buffering salts and/or initial media pH. After purification of the over-expressed proteins,...
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01-07-2019 05:49 AM
[NMR paper] High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water.
High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water.
High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water.
Anal Chem. 2018 Mar 12;:
Authors: Szekely O, Olsen GL, Felli IC, Frydman L
Abstract
This study demonstrates the usefulness derived from relying on hyperpolarized water obtained by dissolution DNP, for site-resolved biophysical NMR studies of intrinsically disordered proteins. Thanks to the facile amide-solvent exchange experienced by protons in these proteins, 2D NMR...
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03-13-2018 07:39 PM
A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia Â* coli grown in shaker flasks
A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia Â* coli grown in shaker flasks
Abstract
We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E.Â*coli BL21(DE3) cells in shaker flasks that reduces D2O usage tenfold and d7-glucose usage by 30Â*%. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD600 of up to 10. Inducing the cells with isopropyl β-d-1-thiogalactopyranoside at an OD600 of 10,...
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11-19-2016 08:35 PM
[NMR paper] Robust High-Yield Methodologies for (2)H and (2)H/(15)N/(13)C Labeling of Proteins for Structural Investigations Using Neutron Scattering and NMR.
Robust High-Yield Methodologies for (2)H and (2)H/(15)N/(13)C Labeling of Proteins for Structural Investigations Using Neutron Scattering and NMR.
Related Articles Robust High-Yield Methodologies for (2)H and (2)H/(15)N/(13)C Labeling of Proteins for Structural Investigations Using Neutron Scattering and NMR.
Methods Enzymol. 2015;565:3-25
Authors: Duff AP, Wilde KL, Rekas A, Lake V, Holden PJ
Abstract
We have developed a method that has proven highly reliable for the deuteration and triple labeling ((2)H/(15)N/(13)C) of a...
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11-19-2015 05:22 PM
Enhanced-sensitivity NMR could reveal new clues to how proteins fold - Phys.Org
Enhanced-sensitivity NMR could reveal new clues to how proteins fold - Phys.Org
http://www.bionmr.com//t1.gstatic.com/images?q=tbn:ANd9GcRv5kcqepdG7qJM0UMWGlFFd6B7LANvsM9YhElwPbgnXWRAEWGTHs3JS_oQLgMHS8kZCRwpmbo
Phys.Org
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Enhanced-sensitivity NMR could reveal new clues to how proteins fold
Phys.Org
Until now, it has been difficult to fully characterize the different structures that proteins can take on in their natural environments. However, using a new technique known as sensitivity-enhanced nuclear magnetic resonance (NMR), MIT researchers have ...
...
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10-09-2015 04:49 PM
Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR Assignments
Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR Assignments
Publication date: Available online 6 August 2015
Source:Protein Expression and Purification</br>
Author(s): Broc R. Wenrich, Reilly E. Sonstrom, Riju A. Gupta, David Rovnyak</br>
Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or 13C’, etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the...
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08-07-2015 02:42 AM
Large, medically important class of proteins starts to yield its secrets - Eureka! Science News
Large, medically important class of proteins starts to yield its secrets - Eureka! Science News
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Eureka! Science News
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Large, medically important class of proteins starts to yield its secrets
Eureka! Science News
Such NMR structure studies were pioneered by Wüthrichand one of the recent innovations has been the use of "microcoils" to enable NMR studies of minute quantities of precious protein samples in solution. Wüthrich's lab, in collaboration with the ...
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07-13-2012 09:15 AM
High-yield Escherichia coli-based cell-free expression of human proteins
High-yield Escherichia coli-based cell-free expression of human proteins
Abstract Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease...
Enhanced NMR: Proteins yield
A team from Massachusetts Institute of Technology (MIT) has found that they can enhance nuclear magnetic resonance (NMR) spectroscopy in such a way as to facilitate the structure determination of proteins associated with a wide range of diseases.
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