The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.
Protein Expr Purif. 2014 Mar 31;
Authors: Nespovitaya N, Barylyuk K, Eichmann C, Zenobi R, Riek R
Abstract
Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer polypeptide product lowered the yield of the target peptide and complicated its purification. The side product contained the N-terminal His6-tag together with the thioredoxin fusion partner and the specific enzymatic cleavage site-containing linker followed by an unknown peptide starting with the first 7 N-terminal amino acid residues of SS14, as revealed by the Edman degradation. The combination of DNA sequence analysis, the Edman degradation, and high-resolution mass spectrometry allowed to identify the amino acid sequence of the unknown peptide. The appearance of the side product was attributed to inefficient termination of mRNA translation. The stop codon and its downstream sequence optimization allowed eliminating the side product synthesis. The optimized expression system, purification protocol, and post-translational modification procedure yielded 1.5 mg of SS14 per liter of minimal medium. Nearly 99% incorporation of (13)C and (15)N isotopes was achieved, as demonstrated by high-resolution mass spectrometry.
PMID: 24698890 [PubMed - as supplied by publisher]
[NMR paper] Red wines attenuate TNF? production in human histiocytic lymphoma cell line: an NMR spectroscopy and chemometrics based study.
Red wines attenuate TNF? production in human histiocytic lymphoma cell line: an NMR spectroscopy and chemometrics based study.
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Food Chem. 2013 Dec 1;141(3):3124-30
Authors: Ali K, Iqbal M, Fortes AM, Pais MS, Korthout HA, Verpoorte R, Choi YH
Abstract
Nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses methods are applied to the metabolic profiling of different red wines...
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Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
J Biomol NMR. 2011 Jun 12;
Authors: Sastry M, Xu L, Georgiev IS, Bewley CA, Nabel GJ, Kwong PD
NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in (15)N, (13)C, or (2)H. The bacterial expression systems currently in use to...
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06-15-2011 01:15 PM
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy
Abstract NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in 15N, 13C, or 2H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an...
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06-15-2011 02:31 AM
[NMR paper] Recombinant locust apolipophorin III: characterization and NMR spectroscopy.
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Biochim Biophys Acta. 1998 Jul 31;1393(1):99-107
Authors: Weers PM, Wang J, Van der Horst DJ, Kay CM, Sykes BD, Ryan RO
Apolipophorin III (apoLp-III) from the locust Locusta migratoria is an exchangeable apolipoprotein that reversibly binds to lipoproteins. During lipid binding the protein has been proposed to undergo a major conformational change. To study the mechanism of...
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11-17-2010 11:15 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.
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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...
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10-19-2010 04:51 PM
[NMR paper] Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and
Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.
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Authors: Landrieu I, Casteels P, Odaert B, De Veylder L, Portetelle D, Lippens G, Van Montagu M, Inzé D
The CKS1At...
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08-21-2010 04:03 PM
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...
The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.
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