Related ArticlesSparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.
Glycobiology. 2020 Sep 08;:
Authors: Rogals MJ, Yang JY, Williams RV, Moremen KW, Amster IJ, Prestegard JH
Abstract
Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins requires enrichment with less abundant, NMR-active, isotopes such as 13C and 15N. This is routine for proteins that can be expressed in bacterial culture where low-cost isotopically enriched metabolic substrates can be used. However, it can be expensive for glycosylated proteins expressed in mammalian culture where more costly isotopically enriched amino acids are usually used. We describe a simple, relatively inexpensive procedure in which standard commercial media is supplemented with 13C-enriched glucose to achieve labeling of all glycans plus all alanines of the N-terminal domain of the highly glycosylated protein, CEACAM1. We demonstrate an ability to detect partially occupied N-glycan sites, sites less susceptible to processing by an endoglycosidase, and some unexpected truncation of the amino acid sequence. The labeling of both the protein (through alanines) and the glycans in a single culture requiring no additional technical expertise past standard mammalian expression requirements is anticipated to have several applications, including structural and functional screening of the many glycosylated proteins important to human health.
PMID: 32902634 [PubMed - as supplied by publisher]
[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...
nmrlearner
Journal club
0
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,...
nmrlearner
Journal club
0
02-28-2018 03:32 PM
NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13 C-methyl alanine
NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13 C-methyl alanine
Abstract
A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145Â*kDa dimer. It uses 13C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from 1Hâ??13C residual dipolar...
nmrlearner
Journal club
0
06-27-2017 03:27 AM
[NMR paper] Sparse labeling of proteins: Structural characterization from long range constraints
Sparse labeling of proteins: Structural characterization from long range constraints
Publication date: April 2014
Source:Journal of Magnetic Resonance, Volume 241</br>
Author(s): James H. Prestegard , David A. Agard , Kelley W. Moremen , Laura A. Lavery , Laura C. Morris , Kari Pederson</br>
Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In...
nmrlearner
Journal club
0
03-22-2014 01:28 AM
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Abstract In the last 15 years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope...
nmrlearner
Journal club
0
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
nmrlearner
Journal club
0
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>
More...
nmrlearner
Journal club
0
08-16-2010 03:50 AM
Enhanced production and isotope enrichment of recombinant glycoproteins produced in c
Abstract NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant...
Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.
Linear Mode
