A simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks
A simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks
A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val 1H/13C methyl protonated proteins from 100Â*ml cultures in M9â??++â??/D2O medium induced at high (OD600â??~â??10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of 2H/15N/13C and 1H/13C labeled proteins, yields comparable quantities of protein from 100Â*ml cell culture to those obtained using a conventional 1 L culture with M9/D2O medium, while using three-fold less α-ketoisovaleric (1,2,3,4-13C4; 3,4â?²,4â?²,4â?²-d4) and α-ketobutyric (13C4; 3,3-d2) acid precursors.
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] Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
J Biomol NMR. 2016 Jun 2;
Authors: Monneau YR, Ishida Y, Rossi P, Saio T, Tzeng SR, Inouye M, Kalodimos CG
Abstract
A simple and cost effective method to independently and stereo-specifically incorporate -methyls in Leu and Val in proteins is presented. Recombinant proteins...
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06-04-2016 11:08 AM
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications
Abstract
A simple and cost effective method to independently and stereo-specifically incorporate -methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The...
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06-03-2016 04:52 PM
[NMR paper] CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
Related Articles CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
J Biomol NMR. 2015 Nov 13;
Authors: Kerfah R, Hamelin O, Boisbouvier J, Marion D
Abstract
A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic...
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11-15-2015 07:55 PM
CH 3 -specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample
CH 3 -specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample
Abstract
A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back 13Câ??13C TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu)...
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11-14-2015 03:37 PM
Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies
Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies
Publication date: Available online 6 January 2015
Source:Journal of Magnetic Resonance</br>
Author(s): Hannes Klaus Fasshuber , Jean-Philippe Demers , Veniamin Chevelkov , Karin Giller , Stefan Becker , Adam Lange</br>
Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two...
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01-07-2015 11:26 AM
Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-pro S and valine-pro S methyl groups for the detection of long range NOEs
Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-pro S and valine-pro S methyl groups for the detection of long range NOEs
Abstract
Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L...
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11-28-2014 11:37 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...
A simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks
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