Related ArticlesCombining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120.
Biotechniques. 2005 Sep;39(3):405-11
Authors: Desplancq D, Bernard C, Sibler AP, Kieffer B, Miguet L, Potier N, Van Dorsselaer A, Weiss E
The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.
Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment
Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment
Abstract Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with 15Nâ??1H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three...
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Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain
Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain
Abstract This report describes a novel method for overexpression of 13C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly 13C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast...
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06-27-2011 04:30 AM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
J Biomol NMR. 2011 Jan 19;
Authors: Fan Y, Shi L, Ladizhansky V, Brown LS
Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties...
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01-21-2011 01:22 AM
Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination
Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination
Abstract The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (ε- and ζ-SAIL Phe) and tyrosine (ε-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven...
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01-09-2011 12:46 PM
[NMR paper] New developments in isotope labeling strategies for protein solution NMR spectroscopy
New developments in isotope labeling strategies for protein solution NMR spectroscopy.
Related Articles New developments in isotope labeling strategies for protein solution NMR spectroscopy.
Curr Opin Struct Biol. 2000 Oct;10(5):585-92
Authors: Goto NK, Kay LE
The development of novel isotope labeling strategies for proteins has facilitated the study of the structure and dynamics of these molecules. In addition, the recent emergence of alternative methods of bacterial expression for obtaining isotopically labeled proteins permits the study of...
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11-19-2010 08:29 PM
[NMR paper] Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal doma
Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.nrc-cnrc.gc.ca-cisti-journals-rp-gifs-PubMed_logo_e.gif Related Articles Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.
Biochem Cell Biol. 1997;75(1):45-53
Authors: Fisher CA, Wang J, Francis GA, Sykes BD, Kay CM, Ryan RO
The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human...
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08-22-2010 03:31 PM
[NMR paper] Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal doma
Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.nrc-cnrc.gc.ca-cisti-journals-rp-gifs-PubMed_logo_e.gif Related Articles Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.
Biochem Cell Biol. 1997;75(1):45-53
Authors: Fisher CA, Wang J, Francis GA, Sykes BD, Kay CM, Ryan RO
The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human...
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08-22-2010 03:03 PM
[NMR paper] Hydrogen-1, carbon-13, and nitrogen-15 NMR spectroscopy of Anabaena 7120 flavodoxin:
Hydrogen-1, carbon-13, and nitrogen-15 NMR spectroscopy of Anabaena 7120 flavodoxin: assignment of beta-sheet and flavin binding site resonances and analysis of protein-flavin interactions.
Related Articles Hydrogen-1, carbon-13, and nitrogen-15 NMR spectroscopy of Anabaena 7120 flavodoxin: assignment of beta-sheet and flavin binding site resonances and analysis of protein-flavin interactions.
Biochemistry. 1990 Oct 16;29(41):9600-9
Authors: Stockman BJ, Krezel AM, Markley JL, Leonhardt KG, Straus NA
Sequence-specific 1H and 13C NMR...
Combining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120.
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