BioNMR
NMR aggregator & online community since 2003
BioNMR    
Learn or help to learn NMR - get free NMR books!
 

Go Back   BioNMR > Educational resources > Journal club
Advanced Search



Jobs Groups Conferences Literature Pulse sequences Software forums Programs Sample preps Web resources BioNMR issues


Webservers
NMR processing:
MDD
NMR assignment:
Backbone:
Autoassign
MARS
UNIO Match
PINE
Side-chains:
UNIO ATNOS-Ascan
NOEs:
UNIO ATNOS-Candid
UNIO Candid
ASDP
Structure from NMR restraints:
Ab initio:
GeNMR
Cyana
XPLOR-NIH
ASDP
UNIO ATNOS-Candid
UNIO Candid
Fragment-based:
BMRB CS-Rosetta
Rosetta-NMR (Robetta)
Template-based:
GeNMR
I-TASSER
Refinement:
Amber
Structure from chemical shifts:
Fragment-based:
WeNMR CS-Rosetta
BMRB CS-Rosetta
Homology-based:
CS23D
Simshift
Torsion angles from chemical shifts:
Preditor
TALOS
Promega- Proline
Secondary structure from chemical shifts:
CSI (via RCI server)
TALOS
MICS caps, β-turns
d2D
PECAN
Flexibility from chemical shifts:
RCI
Interactions from chemical shifts:
HADDOCK
Chemical shifts re-referencing:
Shiftcor
UNIO Shiftinspector
LACS
CheckShift
RefDB
NMR model quality:
NOEs, other restraints:
PROSESS
PSVS
RPF scores
iCing
Chemical shifts:
PROSESS
CheShift2
Vasco
iCing
RDCs:
DC
Anisofit
Pseudocontact shifts:
Anisofit
Protein geomtery:
Resolution-by-Proxy
PROSESS
What-If
iCing
PSVS
MolProbity
SAVES2 or SAVES4
Vadar
Prosa
ProQ
MetaMQAPII
PSQS
Eval123D
STAN
Ramachandran Plot
Rampage
ERRAT
Verify_3D
Harmony
Quality Control Check
NMR spectrum prediction:
FANDAS
MestReS
V-NMR
Flexibility from structure:
Backbone S2
Methyl S2
B-factor
Molecular dynamics:
Gromacs
Amber
Antechamber
Chemical shifts prediction:
From structure:
Shiftx2
Sparta+
Camshift
CH3shift- Methyl
ArShift- Aromatic
ShiftS
Proshift
PPM
CheShift-2- Cα
From sequence:
Shifty
Camcoil
Poulsen_rc_CS
Disordered proteins:
MAXOCC
Format conversion & validation:
CCPN
From NMR-STAR 3.1
Validate NMR-STAR 3.1
NMR sample preparation:
Protein disorder:
DisMeta
Protein solubility:
camLILA
ccSOL
Camfold
camGroEL
Zyggregator
Isotope labeling:
UPLABEL
Solid-state NMR:
sedNMR


Reply
Thread Tools Search this Thread Rate Thread Display Modes
  #1  
Unread 06-07-2016, 02:39 PM
nmrlearner's Avatar
Senior Member
 
Join Date: Jan 2005
Posts: 22,204
Points: 193,617, Level: 100
Points: 193,617, Level: 100 Points: 193,617, Level: 100 Points: 193,617, Level: 100
Level up: 0%, 0 Points needed
Level up: 0% Level up: 0% Level up: 0%
Activity: 50.7%
Activity: 50.7% Activity: 50.7% Activity: 50.7%
Last Achievements
Award-Showcase
NMR Credits: 0
NMR Points: 193,617
Downloads: 0
Uploads: 0
Default Highly efficient residue -selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

Highly efficient residue -selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

Abstract

We recently developed a practical protocol for preparing proteins bearing stereo-selectively 13C-methyl labeled leucines and valines, instead of the commonly used 13C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ?ilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ?ilvD?leuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.



Source: Journal of Biomolecular NMR
Reply With Quote


Did you find this post helpful? Yes | No

Reply
Similar Threads
Thread Thread Starter Forum Replies Last Post
Efficient segmental isotope labeling of multi-domain proteins using Sortase A
Efficient segmental isotope labeling of multi-domain proteins using Sortase A Abstract NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to...
nmrlearner Journal club 0 08-29-2015 09:18 PM
An economic approach to efficient isotope labeling in insect cells using homemade 15 N-, 13 C- and 2 H-labeled yeast extracts
An economic approach to efficient isotope labeling in insect cells using homemade 15 N-, 13 C- and 2 H-labeled yeast extracts Abstract Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein 15N and 13C labeling of up...
nmrlearner Journal club 0 06-12-2015 07:48 PM
[NMR paper] Efficient CO-CA transfer in highly deuterated proteins by band-selective homonuclear cross-polarization
Efficient CO-CA transfer in highly deuterated proteins by band-selective homonuclear cross-polarization Available online 14 March 2013 Publication year: 2013 Source:Journal of Magnetic Resonance</br> </br> Robust and efficient band-selective magnetization transfer between CO and CA spins can be achieved in highly deuterated solid proteins by dipolar-based homonuclear cross polarization. The approach is designed for moderate magic-angle spinning rates and high external magnetic fields where the isotropic chemical shift difference of CO and CA considerably exceeds the...
nmrlearner Journal club 0 03-16-2013 02:22 AM
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis. Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis. Protein Expr Purif. 2011 May 27; Authors: Long F, Cho W, Ishii Y Amyloid fibrils of Alzheimer's ?-amyloid peptide (A?) are a primary component of amyloid plaques, a hallmark of Alzheimer's disease (AD). Enormous attention has been given to the structural...
nmrlearner Journal club 0 06-07-2011 11:05 AM
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle. Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle. J Biomol NMR. 2010 Dec;48(4):179-92 Authors: Thakur CS, Sama JN, Jackson ME, Chen B, Dayie TK Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon...
nmrlearner Journal club 0 03-01-2011 12:14 PM
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle Abstract Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR...
nmrlearner Journal club 0 11-09-2010 03:17 PM
An economical method for producing stable-isotope labeled proteins by the E. coli cel
An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system Abstract Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein...
nmrlearner Proteins 0 11-07-2010 02:47 PM
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli Kit I. Tong, Masayuki Yamamoto and Toshiyuki Tanaka Journal of Biomolecular NMR; 2008; 42(1); pp 59-67 Abstract: A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo...
Brian Journal club 0 09-17-2008 10:20 PM


Thread Tools Search this Thread
Search this Thread:

Advanced Search
Display Modes Rate This Thread
Rate This Thread:

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is On
Trackbacks are Off
Pingbacks are Off
Refbacks are Off



BioNMR advertisements to pay for website hosting and domain registration. Nobody does it for us.



Powered by vBulletin® Version 3.7.3
Copyright ©2000 - 2022, Jelsoft Enterprises Ltd.
Copyright, BioNMR.com, 2003-2013
Search Engine Friendly URLs by vBSEO 3.6.0

All times are GMT. The time now is 02:16 AM.


Map