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PubMed1997

High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins.

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilsson et al., 1987, Protein Eng. 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but with a need for reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with 15N.

Binding, Competitive↗
PubMed2005

Stable isotope labeling of entire Bacillus atrophaeus spores and vegetative cells using bioaerosol mass spectrometry.

Single vegetative cells and spores of Bacillus atrophaeus, formerly Bacillus subtilis var. niger, were analyzed using bioaerosol mass spectrometry. Key biomarkers were identified from organisms grown in 13C and 15N isotopically enriched media. Spore spectra contain peaks from dicipolinate and amino acids. The results indicate that compounds observed in the spectra correspond to material from the spore's core and not the exosporium. Standard compounds and mixtures were analyzed for comparison. The biomarkers for vegetative cells were clearly different from those of the spores, consisting mainly of phosphate clusters and amino acid fragments.

Aerosols↗
PubMed1992

Biosynthesis of bellenamine by Streptomyces nashvillensis using stable isotope labeled compounds.

The biosynthesis of bellenamine was studied by feeding 13C and 15N labeled precursors to the synthetic medium culture of Streptomyces nashvillensis MD743-GF4. The high degree of incorporation of D-[1-13C]beta-lysine indicated that it is a direct intermediate, while supplemented L-beta-lysine repressed the production of bellenamine. [2-13C]Glycine was well incorporated into the C-1' of the open-chain aldoaminal structure. All four nitrogens of bellenamine were derived from [15NH4]2SO4 present in the synthetic medium. In the addition of L-lysine and glycine, [15NH4]2SO4 was highly incorporated into CONH. The feeding experiments of 13C labeled acetates suggested that the D-beta-lysine moiety was derived from L-lysine by catalysis of a new 2,3-aminomutase, and L-lysine was biosynthesized from acetates via the TCA cycle and diaminopimelic acid pathway.

Acetates↗
PubMed1987

Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins.

A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on 15N (or 13C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-15N (or 13C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from 15N-bonded protons in the backbone and sidechains of uniformly 15N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-15N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of 15N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple 15N and 13C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl[15N]alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.

Deuterium↗
PubMed2003

Preparation of stable isotope-labeled 2-nitrobenzaldehyde derivatives of four metabolites of nitrofuran antibiotics and their comprehensive characterization by UV, MS, and NMR techniques.

A convenient method is presented for the preparation of the carbon-13-labeled 2-nitrobenzaldehyde derivatives of the nitrofuran metabolites 3-amino-2-oxazolidinone (AOZ), semicarbazide (SC), 1-aminohydantoin (AH), and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), with the purpose of using them as internal standards for the quantification of trace levels of nitrofuran residues by liquid chromatography-tandem mass spectrometry in foods of animal origin. The synthesis encompasses the nitration of [1,2,3,4,5,6-(13)C(6)]toluene prior to chromyl compound-mediated oxidation of the methyl group into the corresponding aldehyde. The four metabolites of nitrofuran antibiotics were derivatized independently with the resulting ring-labeled 2-nitrobenzaldehyde (NBA) to obtain the target compounds. Both the isotopically enriched and native substances were used to perform a comprehensive fragmentation study by electrospray ionization (ESI) collision-induced dissociation (CID) mass spectrometry (MS). Full characterization of the nitrofuran derivatives was accomplished with ultraviolet (UV) and exhaustive nuclear magnetic resonance (NMR) analysis. A major advantage of the described procedure is that it can be extended to the preparation of other carbon-13-labeled derivatives of metabolites of nitrofuran antibiotics.

Anti-Bacterial Agents↗
PubMed1986

Isotopic labeling of tyrosine, followed by 17O n.m.r.

A simple chemical procedure has been developed in order to introduce oxygen-17 labels into the carboxylic and phenolic sites of L-tyrosine. Detailed studies of the 17O n.m.r. chemical shift as a function of pH reveal an unusually large titration shift upon the deprotonation of the phenol group. This result suggests that 17O n.m.r. may contribute useful information about side chain properties in peptides and proteins.

Hydrogen-Ion Concentration↗
PubMed1979

Biosynthesis of the pyrimidine moiety of thiamin in Escherichia coli: incorporation of stable isotope-labeled glycines.

Methods are described for the cleavage, extraction, and subsequent gas chromatographic-mass spectrometric analysis of the pyrimidine moiety of thiamin as 2-methyl-4-amino-5-[(ethylthio)methyl]pyrimidine. The methods are of a general nature and can be applied to any system. Using these methods to evaluate the incorporation of 13C-, 15N-, and 2H-labeled glycines into the pyrimidine moiety of thiamin by Escherichia coli, we established that the nitrogen and carbon atoms of glycine are incorporated as a unit into the pyrimidine. 13C- and 15N-labeled glycines are incorporated at greater than 60% but deuterium from [2-(2)H2]glycine was incorporated at only 18%. A detailed analysis of the mass fragmentation pattern of the pyrimidine derivative has established that the glycine nitrogen atom supplies the N-1 of the pyrimidine and that the C-1 and C-2 of the glycine supplies the C-4 and C-6 of the pyrimidine, respectively. This evidence is consistent with the substitution of a C2 unit between the C-5 and C-4 of the 4-aminoimidazole ribonucleotide precursor during the biosynthesis of the pyrimidine moiety of thiamin in E. coli.

Carbon Isotopes↗
PubMed1975

[Concentration, purification, and isotopic labeling of sigma virus].

Sigma virus has been concentrated by means of polyethylene-glycol and diafiltration; the latter method is more efficient in preserving infectivity. When purified on sucrose gradient, sigma banded in a sharp peak without any polydispersion. We are now able to label sigma genome with 3H-uridine in Drosophila cell cultures.

Centrifugation, Density Gradient↗
PubMed2003

Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies.

The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V(L) and V(H), respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V(H) and V(L) were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V(L) and V(H) were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the V(H) and the V(L) chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V(L) of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible.

Amino Acid Sequence↗
PubMed1994

GC-MS determination of ratios of stable-isotope labelled to natural urea using [13C15N2]urea for studying urea kinetics in serum and as a means to validate routine methods for the quantitative assay of urea in dialysate.

A GC-MS determination of urea in serum or spent dialysate is described, using 13C15N2-labelled urea and assaying the area ratio of labelled to natural urea by mass fragmentographic monitoring of fragments m/e 153 and 156, after its eventual conversion into the trimethylsilylether-derivative of 2-hydroxypyrimidine. The procedure can be successfully applied in the follow-up of the disappearance of labelled urea in serum after intravenous injection in man, enabling kinetic parameters of urea to be established, e.g. for purposes of studying the effectiveness of dialysis procedures. Furthermore the method can be used for validation of routine methods for measuring urea in other fluids, in particular dialysate. Examples are given of both applications of the GC-MS method described.

Calibration↗