ArticleJournal of biomolecular NMR2011
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O.
Article in Journal of biomolecular NMR, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 50 citations in OpenAlex.
- Exploring the dynamics and structure of PpiB in living Escherichia coli cells using electron paramagnetic resonance spectroscopy.Protein science : a publication of the Protein Society · 2024Article
- Deuteration of proteins boosted by cell lysates: high-resolution amide and HMagnetic resonance (Gottingen, Germany) · 2024Article
- E. coli "Stablelabel" S30 lysate for optimized cell-free NMR sample preparation.Journal of biomolecular NMR · 2023Article
- Cell-free synthesis of proteins with selectivelyMagnetic resonance (Gottingen, Germany) · 2023Article
- Specific isotopic labelling and reverse labelling for protein NMR spectroscopy: using metabolic precursors in sample preparation.Biochemical Society transactions · 2022Review
- Cell-Free Expression and Photo-Crosslinking of the Human Neuropeptide YFrontiers in pharmacology · 2019Article
- Modern Technologies of Solution Nuclear Magnetic Resonance Spectroscopy for Three-dimensional Structure Determination of Proteins Open Avenues for Life Scientists.Computational and structural biotechnology journal · 2017Review
- Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.Biochemistry · 2016Article
- Selective (15)N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy.Journal of biomolecular NMR · 2014Article
- Engine out of the chassis: cell-free protein synthesis and its uses.FEBS letters · 2014Review
- Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets.Journal of biomolecular NMR · 2013Article
- Advances in NMR structures of integral membrane proteins.Current opinion in structural biology · 2013Review
- Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, epsilon, θ and β reveals a highly flexible arrangement of the proofreading domain.Nucleic acids research · 2013Article
- Cell-free protein synthesis: the state of the art.Biotechnology letters · 2013Review
- Biosynthetically directed ²H labelling for stereospecific resonance assignments of glycine methylene groups.Journal of biomolecular NMR · 2013Article
- Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition.Journal of biomolecular NMR · 2011Article
Corrections and comments
- Erratum issued
Authors and funding
4 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that reduction of E. coli S30 extracts with NaBH(4) presents a simple and inexpensive way to achieve cleaner selective isotope labelling in cell-free protein synthesis reactions. The purpose of the NaBH(4) is to inactivate all pyridoxal-phosphate (PLP) dependent enzymes by irreversible reduction of the Schiff bases formed between PLP and lysine side chains of the enzymes or amino groups of free amino acids. The reduced S30 extracts retain their activity of protein synthesis, can be stored as well as conventional S30 extracts and effectively suppress conversions between different amino acids. In addition, inactivation of PLP-dependent enzymes greatly stabilizes hydrogens bound to α-carbons against exchange with water, minimizing the loss of α-deuterons during cell-free production of proteins from perdeuterated amino acids in H(2)O solution. This allows the production of highly perdeuterated proteins that contain protons at all exchangeable positions, without having to back-exchange labile deuterons for protons as required for proteins that have been synthesized in D(2)O.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.