Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Methods Mol Biol. 2014;1118:169-87
Authors: Ozawa K, Qi R
Abstract
Cell-free protein synthesis (CFPS) offers a fast and inexpensive approach to selectively label proteins with isotopes that can then be detected by nuclear magnetic resonance (NMR) spectroscopy directly in the translation mixture. We describe a PCR-based approach for production of protein-coding circularized DNA templates that can be expressed in Escherichia coli extract in CFPS dialysis system. This approach typically yields target protein concentrations close to 1 mg/mL, which is sufficient for subsequent analysis by 2D (1)H,(15)N-NMR. Furthermore, this PCR-based technique also enables parallel preparation of mutant proteins in a high-throughput mode, enabling rapid assignments of NMR signals. This chapter describes the general CFPS protocol that we used to rapidly assign residue-specific cross peaks from 2D (1)H,(15 N-NMR) spectra obtained from 12 Ile/Ala substituted mutants of the 40 kDa protein complex, ?CTS:?c16.
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...
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Site-specific free energy changes in proteins upon ligand binding by NMR: Ca(2+) -displacement by Ln(3+) in a Ca(2+) -binding protein from Entamoeba histolytica.
Site-specific free energy changes in proteins upon ligand binding by NMR: Ca(2+) -displacement by Ln(3+) in a Ca(2+) -binding protein from Entamoeba histolytica.
Site-specific free energy changes in proteins upon ligand binding by NMR: Ca(2+) -displacement by Ln(3+) in a Ca(2+) -binding protein from Entamoeba histolytica.
Chem Biol Drug Des. 2011 Jan 14;
Authors: Chandra K, Mustafi SM, Muthukumar S, Chary KV
The study of protein-ligand interaction has been of a great interest in contemporary structural biology. The understanding of the nature...
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01-18-2011 10:22 PM
[NMR paper] Cell-free synthesis of 15N-labeled proteins for NMR studies.
Cell-free synthesis of 15N-labeled proteins for NMR studies.
Related Articles Cell-free synthesis of 15N-labeled proteins for NMR studies.
IUBMB Life. 2005 Sep;57(9):615-22
Authors: Ozawa K, Dixon NE, Otting G
Modern cell-free in vitro protein synthesis systems present powerful tools for the synthesis of isotope-labeled proteins in high yields. The production of selectively 15 N-labeled proteins from 15 N-labeled amino acids is particularly economic and yields are often sufficient to analyze the proteins very quickly by two-dimensional NMR...
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[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...
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[NMR paper] Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals to
Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with amino acid residues.
Related Articles Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with amino acid residues.
Magn Reson Chem. 2004 Feb;42(2):218-30
Authors: Saitô H, Mikami J, Yamaguchi S, Tanio M, Kira A, Arakawa T, Yamamoto K, Tuzi S
We have so...
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[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...
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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...
Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
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