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 11-24-2010, 10:01 PM
nmrlearner's Avatar
Senior Member
 
Join Date: Jan 2005
Posts: 19,227
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 Cell-free protein production and labeling protocol for NMR-based structural proteomic

Cell-free protein production and labeling protocol for NMR-based structural proteomics.

Related Articles Cell-free protein production and labeling protocol for NMR-based structural proteomics.

Nat Methods. 2004 Nov;1(2):149-53

Authors: Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL

Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods. To illustrate this platform, we describe its application to a specific target (At3g01050.1) from Arabidopsis thaliana. After cloning the target gene into a specialized plasmid, we carry out a small-scale (50 mul) in vitro sequential transcription and translation trial to ascertain the level of protein production and solubility. Next, we prepare mRNA for use in a 4-ml semicontinuous cell-free translation reaction to incorporate (15)N-labeled amino acids into a protein sample that we purify and test for suitability for NMR structural analysis. We then repeat the cell-free approach with (13)C,(15)N-labeled amino acids to prepare a doubly labeled sample. The three-dimensional (3D) structure of At3g01050.1 shows that this protein is an unusual member of the beta-grasp protein family.

PMID: 15782178 [PubMed - indexed for MEDLINE]



Source: PubMed
Reply With Quote


Did you find this post helpful? Yes | No

Reply
Similar Threads
Thread Thread Starter Forum Replies Last Post
Erratum to: Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Erratum to: Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O Erratum to: Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O Content Type Journal Article Category Erratum Pages 1-1 DOI 10.1007/s10858-011-9562-9 Authors
nmrlearner Journal club 0 09-20-2011 05:02 AM
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O Abstract Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that...
nmrlearner Journal club 0 02-16-2011 09:34 PM
[NMR paper] Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions.
Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions. Related Articles Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions. J Biomol NMR. 2005 Jul;32(3):235-41 Authors: Ozawa K, Jergic S, Crowther JA, Thompson PR, Wijffels G, Otting G, Dixon NA Cell-free protein synthesis systems provide facile access to proteins in a nascent state that enables formation of soluble, native protein-protein complexes even if one of the protein components is prone...
nmrlearner Journal club 0 12-01-2010 06:56 PM
[NMR paper] Quantitation of protein expression in a cell-free system: Efficient detection of yiel
Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein. Related Articles Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein. J Biomol NMR. 2005 Jan;31(1):11-9 Authors: Neerathilingam M, Greene LH, Colebrooke SA, Campbell ID, Staunton D We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a...
nmrlearner Journal club 0 11-24-2010 11:14 PM
[NMR paper] Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for
Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. Related Articles Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. J Biomol NMR. 1995 Sep;6(2):129-34 Authors: Kigawa T, Muto Y, Yokoyama S For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity...
nmrlearner Journal club 0 08-22-2010 03:50 AM
Cell-free expression and labeling strategies for a new decade in solid-state NMR.
Cell-free expression and labeling strategies for a new decade in solid-state NMR. http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Cell-free expression and labeling strategies for a new decade in solid-state NMR. N Biotechnol. 2010 Aug 3; Authors: Abdine A, Verhoeven MA, Warschawski DE Although solid-state NMR and cell-free expression have recently become standard methods in biology, the combination of the two is still at a very early stage of development. In this...
nmrlearner Journal club 0 08-17-2010 03:36 AM
Cell-free protein synthesis of perdeuterated proteins for NMR studies
Cell-free protein synthesis of perdeuterated proteins for NMR studies Touraj Etezady-Esfarjani, Sebastian Hiller, Cristina Villalba and Kurt WŁthrich Journal of Biomolecular NMR; 2007; 39(3); pp 229-238 Abstract: Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins...
Deano Journal club 0 08-14-2008 10:01 PM
Wheat Germ Cell-Free Protein Production Workshop
Wheat Germ Cell-Free Protein Production Workshop PDF file The Center for Eukaryotic Structural Genomics (CESG) and the Nuclear Magnetic Resonance Facility at Madison (NMRFAM) are pleased to announce the first Wheat Germ Cell-Free Protein Production Workshop to be held from July 30 - August 4, 2006, at the Department of Biochemistry at the University of Wisconsin-Madison in Madison, Wisconsin, USA. To register for this workshop, complete the Registration Form on the next page and mail in with your NONREFUNDABLE
nmrlearner Conferences 0 05-23-2006 09:39 AM


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 - 2019, 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 01:46 PM.


Map