ArticleJournal of biomolecular NMR1998
A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: an application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C.
Article in Journal of biomolecular NMR, 1998. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed, 87 citations in OpenAlex.
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- Transcription is regulated by NusA:NusG interaction.Nucleic acids research · 2016Article
- Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions.Bioscience reports · 2015Article
- Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells.FEBS open bio · 2015Article
- Recombinant production of the amino terminal cytoplasmic region of dengue virus non-structural protein 4A for structural studies.PloS one · 2014Article
- An efficient method for the production of isotopically enriched cholesterol for NMR.Journal of lipid research · 2011Article
- Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.Journal of biomolecular NMR · 2010Article
- A general and efficient approach for NMR studies of peptide dynamics in class I MHC peptide binding grooves.Biochemistry · 2009Article
- Maximum entropy reconstruction of joint phi, psi-distribution with a coil-library prior: the backbone conformation of the peptide hormone motilin in aqueous solution from phi and psi-dependent J-couplings.Journal of biomolecular NMR · 2007Article
- Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR.Biophysical journal · 2006Article
- Direct determination of a membrane-peptide interface using the nuclear magnetic resonance cross-saturation method.Biophysical journal · 2005Article
- The E. coli NusA carboxy-terminal domains are structurally similar and show specific RNAP- and lambdaN interaction.Protein science : a publication of the Protein Society · 2005Article
- Signal assignments and chemical-shift structural analysis of uniformly 13C, 15N-labeled peptide, mastoparan-X, by multidimensional solid-state NMR under magic-angle spinning.Journal of biomolecular NMR · 2004Article
- SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins.Journal of structural and functional genomics · 2004Article
- Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans.Journal of biomolecular NMR · 2003Article
- A rapid method to attain isotope labeled small soluble peptides for NMR studies.Journal of biomolecular NMR · 2003Article
- Complete 1H, 13C, and 15N assignments of the N-terminal DNA binding domain of the TraR protein.Journal of biomolecular NMR · 2002Article
- The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet.Protein science : a publication of the Protein Society · 2001Article
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Authors and funding
4 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A new strategy is described for the production of peptides enriched with stable isotopes. Peptides of interest are expressed in Escherichia coli (E. coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin. This method yields as much as 30-100 mg/l of isotope-enriched fusion proteins in minimal media. A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni(2+)-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase. Since this enzyme is also expressed at a high level in E. coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases. In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies. This flexibility enables us to prepare peptides that are unstable in a soluble state in E. coli cells. As an example, the expression and the uniform stable isotope enrichment with 15N and/or 13C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our method. The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR.
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