[NMR paper] Local deuteration enables NMR observation of methyl groups in proteins from eukaryotic and cell-free expression systems
Local deuteration enables NMR observation of methyl groups in proteins from eukaryotic and cell-free expression systems
Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple...
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03-27-2021 02:09 AM
Deuteration and selective labeling of alanine methyl groups of β 2 -adrenergic receptor expressed in a baculovirus-insect cell expression system
Deuteration and selective labeling of alanine methyl groups of β 2 -adrenergic receptor expressed in a baculovirus-insect cell expression system
Abstract
G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited....
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03-08-2018 01:24 PM
[NMR paper] Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
Related Articles Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
J Biomol NMR. 2018 Feb 28;:
Authors: Yanaka S, Yagi H, Yogo R, Yagi-Utsumi M, Kato K
Abstract
Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing...
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03-02-2018 03:20 PM
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
Abstract
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,...
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02-28-2018 03:32 PM
High-yield Escherichia coli-based cell-free expression of human proteins
High-yield Escherichia coli-based cell-free expression of human proteins
Abstract Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease...
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03-17-2012 07:32 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
[NMR paper] Methyl groups as probes for proteins and complexes in in-cell NMR experiments.
Methyl groups as probes for proteins and complexes in in-cell NMR experiments.
Related Articles Methyl groups as probes for proteins and complexes in in-cell NMR experiments.
J Am Chem Soc. 2004 Jun 9;126(22):7119-25
Authors: Serber Z, Straub W, Corsini L, Nomura AM, Shimba N, Craik CS, Ortiz de Montellano P, Dötsch V
Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes...
NMR observation of methyl groups in proteins from eukaryotic and cell-free expression systems - Phys.org
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