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.
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 Wüthrich
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
Cell-free protein production and labeling protocol for NMR-based structural proteomic
Linear Mode
