NMR is a powerful tool for the structural and dynamic study of proteins. One of the necessary conditions for the study of these proteins is their isotopic labelling with 15N and 13C. One of the most widely used methods to obtain these labelled proteins is heterologous expression of the proteins in E. coli using 13C-D-glucose and 15NH4Cl as the sole nutrient sources. In recent years, the price of 13C-D-glucose has almost tripled, making it essential to develop labelling methods that are as cost effective as possible. In this work, different parameters were studied to achieve the most rational use of 13C-D-glucose, and an optimized method was developed to obtain labelled proteins with high labelling and low 13C-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth.
[NMR paper] Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies
Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies
The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using...
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12-01-2023 06:23 PM
[NMR paper] Biocatalytic reductive amination as a route to isotopically labelled amino acids suitable for analysis of large proteins by NMR
Biocatalytic reductive amination as a route to isotopically labelled amino acids suitable for analysis of large proteins by NMR
We demonstrate an atom-efficient and easy to use H(2)-driven biocatalytic platform for the enantioselective incorporation of ²H-atoms into amino acids. By combining the biocatalytic deuteration catalyst with amino acid dehydrogenase enzymes capable of reductive amination, we synthesised a library of multiply isotopically labelled amino acids from low-cost isotopic precursors, such as ²H(2)O and ^(15)NH(4)^(+). The chosen approach avoids the use of pre-labeled...
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11-16-2023 12:57 PM
[ASAP] Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917
Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917
https://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/0/bichaw.ahead-of-print/acs.biochem.8b00242/20180425/images/medium/bi-2018-00242b_0009.gif
Biochemistry
DOI: 10.1021/acs.biochem.8b00242
http://feeds.feedburner.com/~ff/acs/bichaw?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/bichaw/~4/xEMFb1XNf30
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04-26-2018 02:15 AM
[NMR paper] Synthesis of Isotopically Labelled All-Trans Retinals for DNP-Enhanced Solid State NMR Studies of Retinylidene Proteins.
Synthesis of Isotopically Labelled All-Trans Retinals for DNP-Enhanced Solid State NMR Studies of Retinylidene Proteins.
Related Articles Synthesis of Isotopically Labelled All-Trans Retinals for DNP-Enhanced Solid State NMR Studies of Retinylidene Proteins.
J Labelled Comp Radiopharm. 2017 Oct 28;:
Authors: Leeder AJ, Brown LJ, Becker-Baldus J, Mehler M, Glaubitz C, Brown RCD
Abstract
Three all-trans retinals containing multiple (13) C labels have been synthesised to enable DNP enhanced solid-state MAS NMR studies of novel...
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10-29-2017 02:06 PM
Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing
Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing
Abstract Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been...
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07-02-2012 06:18 AM
Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells
Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells
Abstract The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed...
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01-09-2011 12:46 PM
[NMR paper] Overexpression and purification of isotopically labeled Escherichia coli MutH for NMR
Overexpression and purification of isotopically labeled Escherichia coli MutH for NMR studies.
Related Articles Overexpression and purification of isotopically labeled Escherichia coli MutH for NMR studies.
Protein Expr Purif. 2003 Jun;29(2):252-8
Authors: Dutta A, Rao BJ, Chary KV
MutH is one of the enzymes involved in the methyl directed -GATC-based DNA repair system. We report a significantly optimized protocol to prepare isotopically (15N and/or 13C) labeled MutH in minimal medium with high yields for NMR studies. Under the various...
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11-24-2010 09:01 PM
Production of recombinant isotopically labelled peptide by fusion to an insoluble par
Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin ?v?6 binding peptides for NMR.
Related Articles Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin ?v?6 binding peptides for NMR.
Mol Biosyst. 2010 Oct 18;
Authors: Wagstaff JL, Howard MJ, Williamson RA
The integrin ?v?6 is up-regulated in several cancers and has clinical potential for both tumour imaging and therapy. Peptide ligands have been developed...
Counterintuitive method improves yields of isotopically labelled proteins expressed in flask-cultured Escherichia coli
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