Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles, including mammalian, yeast, insect, and plant cells, each of which has a distinct N-glycan diversification pathway. Yeast genetic engineering has enabled the overexpression of homogeneous high-mannose-type oligosaccharides with 13C labeling for NMR characterization of their conformational dynamics. The utility of stable isotope-assisted NMR spectroscopy has also been demonstrated using the Fc fragment of immunoglobulin G (IgG) as a model glycoprotein, providing useful information regarding intramolecular carbohydrateâ??protein interactions. Transverse relaxation optimization of intact IgG with a molecular mass of 150Â*kDa has been achieved by tailored deuteration of selected amino acid residues using a mammalian expression system. This offers a useful probe for the characterization of molecular interaction networks in multimolecular crowded systems typified by serum. Perspectives regarding the development of techniques for tailoring glycoform designs and isotope labeling of recombinant glycoproteins are also discussed.
Stable isotope labeling methods for DNA
Stable isotope labeling methods for DNA
Publication date: Available online 20 June 2016
Source:Progress in Nuclear Magnetic Resonance Spectroscopy</br>
Author(s): Frank H.T. Nelissen, Marco Tessari, Sybren S. Wijmenga, Hans A. Heus</br>
NMR is a powerful method for studying proteins and nucleic acids in solution. The study of nucleic acids by NMR is far more challenging than for proteins, which is mainly due to the limited number of building blocks and unfavorable spectral properties. For NMR studies of DNA molecules, (site specific) isotope enrichment is...
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06-21-2016 01:09 AM
[NMR paper] Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR.
Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR.
Related Articles Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR.
Methods Enzymol. 2015;565:193-212
Authors: Fan Y, Emami S, Munro R, Ladizhansky V, Brown LS
Abstract
Solid-state NMR (ssNMR) is a rapidly developing technique for exploring structure and dynamics of membrane proteins, but its progress is hampered by its low sensitivity. Despite the latest technological advances, routine ssNMR experiments still require...
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11-19-2015 05:22 PM
[NMR paper] Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems.
Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems.
Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems.
Protein Expr Purif. 2014 May 20;
Authors: Nars G, Saurel O, Bordes F, Saves I, Remaud-Siméon M, André I, Milon A, Marty A
Abstract
Extracellular lipase Lip2 from Yarrowia lipolytica is a promising biocatalyst with unusual structural features, as indicated by x-ray...
NMR-based stable isotope resolved metabolomics in systems biochemistry
NMR-based stable isotope resolved metabolomics in systems biochemistry
Abstract An important goal of metabolomics is to characterize the changes in metabolic networks in cells or various tissues of an organism in response to external perturbations or pathologies. The profiling of metabolites and their steady state concentrations does not directly provide information regarding the architecture and fluxes through metabolic networks. This requires tracer approaches. NMR is especially powerful as it can be used not only to identify and quantify metabolites in an unfractionated mixture such...
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03-03-2011 02:06 AM
Cell-free expression and stable isotope labelling strategies for membrane proteins
Cell-free expression and stable isotope labelling strategies for membrane proteins
Abstract Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems...
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01-09-2011 12:46 PM
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a m
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system.
Related Articles Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system.
Prog Nucl Magn Reson Spectrosc. 2010 May;56(4):346-59
Authors: Kato K, Yamaguchi Y, Arata Y
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10-19-2010 04:51 PM
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a m
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system
Publication year: 2010
Source: Progress in Nuclear Magnetic Resonance Spectroscopy, In Press, Accepted Manuscript, Available online 19 March 2010</br>
Koichi, Kato , Yoshiki, Yamaguchi , Yoji, Arata</br>
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Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
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