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Results for “Isotope Labeling”

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PubMed1996

Efficient production from Aspergillus niger of a heterologous protein and an individual protein domain, heavy isotope-labelled, for structure-function analysis.

Aspergillus niger has been used successfully to secrete proteins labelled with 13C and/or 15N to a specific activity of > 99% for high resolution NMR analysis. In the case of a heterologous protein, hen egg-white lysozyme, 15N single-labelled and 13C, 15N double-labelled forms were secreted at yields of 100-200 mg l-1 by optimising the type of carbon source used and the ratio of carbon to nitrogen. Another protein, the glucoamylase starch-binding domain from A. niger, was also produced as the 15N single-labelled form at 20-40 mg l-1.

Aspergillus niger↗
PubMed2005

Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies.

Antimicrobial peptide LL-37 plays an important role in human body's first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the incorporation of (15)N and other isotopes into the polypeptide for nuclear magnetic resonance (NMR) analysis. The DNA sequence encoding full-length LL-37 was chemically synthesized and cloned into the pET-32a(+) vector for protein expression in Escherichia coli strain BL21(DE3). The peptide was expressed directly as a His-tagged fusion protein without the inclusion of its precursor sequence. LL-37 was released from the fusion by formic acid cleavage at the AspPro dipeptide bond and separated from the carrier thioredoxin by affinity chromatography and reverse-phase HPLC. The peptide was identified by polyacrylamide gel electrophoresis and further confirmed by mass spectrometry and NMR spectroscopy. Antibacterial activity assays showed that the recombinant LL-37 purified from the bacterial source is as active as that from chemical synthesis. According to the antimicrobial peptide database (), 111 peptides contain a Met residue, but only 5 contain the AspPro pair, indicating a broader application of formic acid than cyanogen bromide in cleaving fusion proteins. The successful application to the expression of the 66-residue cytoplasmic tail of human MUC1 indicates that the system can be applied to other peptides as well.

Antigens, Neoplasm↗
PubMed1991

Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins.

Procedures are presented for the separation and determination of the isotopic enrichment of multiple human apolipoproteins labeled in vivo with a stable isotope amino acid. The isotopic enrichments of plasma lysine and plasma apolipoproteins were monitored for 16 days after a single intravenous dose of [4,4,5,5-2H4]lysine (5 mg/kg body weight). The use of a multiply deuterated amino acid enabled the measurement of isotopic enrichments above background over the entire 16-day time course in all proteins. Individual apolipoproteins were separated on a specially designed gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis system cast in a conventional slab gel apparatus which resolved apoB-100, apoE, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III-1, and apoC-III-2 on a single gel. After staining with Coomassie blue, proteins bands (containing 5 to 30 micrograms of individual apolipoprotein) were excised from the gel. Amino acids were recovered from hydrolyzed gel slices, derivatized, and analyzed by gas chromatography-mass spectrometry for determination of lysine isotopic enrichments. The utility of the method is demonstrated using examples of apolipoproteins B-100, A-I, A-II, C-I, C-II, and C-III from either total plasma d less than 1.21 g/ml lipoproteins or selected lipoprotein subfractions. Lysine isotopic enrichments of proteins were generally determined with a precision of better than 5%. The isotopic enrichment profiles were consistent with literature reports of apolipoprotein metabolic kinetics based on the use of radioiodinated apolipoproteins. The procedures outlined can be used to separate and measure the isotopic enrichment of virtually any apolipoprotein from any chosen lipoprotein fraction. Thus, these procedures should find wide application in the study of apolipoprotein metabolic kinetics.

Adult↗
PubMed1985

Measurements of exchange in the reaction catalysed by creatine kinase using 14C and 15N isotope labels and the NMR technique of saturation transfer.

31P-NMR measurements of saturation transfer have been used to measure exchange between the gamma-phosphate of ATP and phosphocreatine and between the beta-phosphate of ATP and the beta-phosphate of ADP in the reaction catalysed by creatine kinase in vitro. The calculated exchange fluxes have been compared with measurements of 15N label exchange between creatine and phosphocreatine and 14C label exchange between ATP and ADP. At pH 8.0 the fluxes between phosphocreatine and the gamma-phosphate of ATP and between the beta-phosphates of ATP and ADP, measured by saturation transfer, were the same and equal, within experimental error, to the fluxes between creatine and phosphocreatine, measured by 15N label exchange, and between ADP and ATP, measured by 14C label exchange. At pH 7.0 the flux between phosphocreatine and the gamma-phosphate of ATP, measured by saturation transfer, was equal, within experimental error, to the flux between creatine and phosphocreatine, measured by 15N label exchange. However, at low ADP concentrations (less than 0.2 mM), the flux between ATP and ADP measured by saturation transfer was significantly less than that between phosphocreatine and ATP and, more importantly, less than the ADP-ATP exchange flux measured by 14C label exchange. The saturation transfer and isotope exchange measurements at pH 7.0 have shown that it is valid to equate saturation transfer measurements of exchange between phosphocreatine and ATP in vivo with the potential for net chemical flux through the reaction. The observed discrepancy at pH 7.0 between the 14C and saturation transfer measurements of ATP----ADP exchange can be explained if there is significant loss of saturation in an intermediate in the exchange reaction. Under these conditions analysis of the exchange according to two-site exchange model is invalid. In magnetisation transfer measurements of exchange in other enzyme catalysed reactions, the possible presence of a kinetically significant intermediate and therefore the validity of data analysis using a two-site exchange model should be considered.

Adenosine Diphosphate↗
PubMed1992

Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VIII. Isotope labeling of ginsenoside Rb2.

To clarify the pharmacokinetics of absorption, distribution and excretion of ginsenoside Rb2 (Rb2), one of the major saponins of the root of Panax ginseng, following oral administration to rats, a tritium (3H) labeling of Rb2 was examined. The C-12 position of Rb2 was labeled with 3H-sodium borohydride (3H-NaBH4) and 12-3H Rb2 and 12-3H-epi Rb2 was synthesized. This method of specific position labeling of Rb2 may be applicable to other ginsenosides. In the near future, the pharmacokinetics of Rb2 in rats may be clarified with 3H labeled Rb2.

Animals↗
PubMed2002

One-pot two-step enzymatic coupling of pyrimidine bases to 2-deoxy-D-ribose-5-phosphate. A new strategy in the synthesis of stable isotope labeled deoxynucleosides.

The enzymatic synthesis of thymidine from 2-deoxy-D-ribose-5-phosphate is achieved, in a one-pot two-step reaction using phosphoribomutase (PRM) and commercially available thymidine phosphorylase (TP). In the first step the sugar-5-phosphate is enzymatically rearranged to alpha-2-deoxy-D-ribose-1-phosphate. Highly active PRM is easily obtained from genetically modified overproducing E. coli cells (12,000 units/84 mg protein) and is used without further purification. In the second step thymine is coupled to the sugar-1-phosphate. The thermodynamically unfavorable equilibrium is shifted to the product by addition of MnCl(2) to precipitate inorganic phosphate. In this way the overall yield of the beta-anomeric pure nucleoside increases from 14 to 60%. In contrast to uracil, cytosine is not accepted by TP as a substrate. Therefore, 2'-deoxy-cytidine is obtained by functional group transformations of the enzymatically prepared 2'-deoxy-uridine. The method has been demonstrated by the synthesis of [2',5'-(13)C(2)]- and [1',2',5'-(13)C(3)]thymidine as well as [1',2',5'-(13)C(3)]2'-deoxyuridine and [3',4'-(13)C(2)]2'-deoxycytidine. In addition the nucleoside bases thymine and uracil are tetralabeled at the (1,3-(15)N(2),2,4-(13)C(2))-atomic positions. All compounds are prepared without any scrambling or dilution of the labeled material and are thus obtained with a very high isotope enrichment (96-99%). In combination with the methods that have been developed earlier it is concluded that each of the (13)C- and (15)N-positions and combination of positions of the pyrimidine deoxynucleosides can be efficiently labeled starting from commercially available and highly (13)C- or (15)N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia.

Catalysis↗
PubMed1989

Reductive methods for isotopic labeling of antibiotics.

Methods for the reductive methylation of the amino groups of eight different antibiotics using 3HCOH or H14COH are presented. The reductive labeling of an additional seven antibiotics by NaB3H4 is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented.

Aminoglycosides↗