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Chromatographic Purification (chromatographic + purification)
Selected AbstractsPurification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Emerich Mihai Gazdag The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30,mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5,mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research. [source] Cell-free synthesis of 15N-labeled proteins for NMR studiesIUBMB LIFE, Issue 9 2005Kiyoshi Ozawa Abstract Modern cell-free in vitro protein synthesis systems present powerful tools for the synthesis of isotope-labeled proteins in high yields. The production of selectively 15 N-labeled proteins from 15 N-labeled amino acids is particularly economic and yields are often sufficient to analyze the proteins very quickly by two-dimensional NMR spectra recorded of the crude reaction mixture without concentration or chromatographic purification of the protein. We review methodological aspects of cell-free in vitro protein synthesis based on an Escherichia coli cell extract, in particular with regard to the production of 15 N-labeled proteins for analysis by NMR spectroscopy. IUBMB Life, 57: 615-622, 2005 [source] A simple and versatile protocol for the preparation of 1,3-functionalized heterocycles utilizing benzoylpyruvatesJOURNAL OF HETEROCYCLIC CHEMISTRY, Issue 4 2010Jens M. J. Nolsöe Acid-mediated condensation between benzoylpyruvates and various dinucleophiles in alcoholic solvent furnished the heterocyclic imprint in moderate to good yield. Combining a range of symmetric as well as nonsymmetric nitrogen/nitrogen or nitrogen/carbon centered dinucleophiles resulted in excellent regioselectivity. ,-Difunctionalized fused pyrimidines, pyridazines, and pyridines were produced in this manner. The protocol was designed to obviate chromatographic purification. J. Heterocyclic Chem., (2010). [source] On the purity of 2-[ortho -anilinyl]-1,3-benzoxazole derived from 2H -3,1-benzoxazine-2,4(1H)dione (isatoic anhydride) [1,2],JOURNAL OF HETEROCYCLIC CHEMISTRY, Issue 2 2010Karen M. Button The Lewis acid catalyzed synthesis and chromatographic purification of isatoic anhydride-derived 2-(2,-anilinyl)-1,3-benzoxazole (2) can result in the co-isolation of 2 and a light pink colored impurity (<5%). This latter species has been identified (NMR, single crystal X-ray diffraction, mp) as 2,-hydroxy-2-aminobenzanilide (3), which represents a predictable intermediate in the formation of 2. Compound 3 crystallizes in an orthorhombic crystal system of space group P212121 with four molecules in the unit cell (, = , = , = 90°; a = 6.715 (2) Å, b = 12.100 (4) Å, c = 13.321 (4) Å; V = 1082.2 (6) Å3). Pure 2 is characterized as a colorless, high-melting solid; unlike the dark colored oil that is isolated if 2 contains traces of 3. J. Heterocyclic Chem., (2010). [source] Facile synthesis of mono and dibenzo N,N'-disubstituted diaza 18-crown-6 derivativesJOURNAL OF HETEROCYCLIC CHEMISTRY, Issue 1 2001Giray Topal This study represents a facile synthesis of building blocks (1,3) of crown ethers and amine precoursers (4a-d). The study also involves synthesis of mono and dibenzo N, N -disubstituted diaza 18-crown-6 derivatives with high yield without chromatographic purification and high vacuum distillation. The complex ability of host the ethers was evaluated in terms of structural modification. [source] Analysis of carbohydrates and amino acids in vegetable waste waters by ion chromatographyPHYTOCHEMICAL ANALYSIS, Issue 2 2003Michele Arienzo Abstract High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gln, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed. Copyright © 2003 John Wiley & Sons, Ltd. [source] Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regressionBIOPOLYMERS, Issue 11 2008Yeqiu Wang Abstract The secondary structures of proteins (,-helical, ,-sheet, ,-turn, and random coil) in the solid state and when bound to polymer beads, containing immobilized phenyl and butyl ligands such as those as commonly employed in hydrophobic interaction chromatography, have been investigated using FTIR-ATR spectroscopy and partial least squares (PLS) methods. Proteins with known structural features were used as models, including 12 proteins in the solid state and 7 proteins adsorbed onto the hydrophobic surfaces. A strong PLS correlation was achieved between predictions derived from the experimental data for 4 proteins adsorbed onto the phenyl-modified beads and reference data obtained from the X-ray crystallographic structures with r2 values of 0.9974, 0.9864, 0.9924, and 0.9743 for ,-helical, ,-sheet, ,-turn, and random coiled structures, respectively. On the other hand, proteins adsorbed onto the butyl sorbent underwent greater secondary structural changes compared to the phenyl sorbent as evidenced from the poorer PLS r2 values (r2 are 0.9658, 0.9106, 0.9571, and 0.9340). The results thus indicate that the secondary structures for these proteins were more affected by the butyl sorbent, whereas the secondary structure remains relatively unchanged for the proteins adsorbed onto the phenyl sorbent. This study has important ramifications for understanding the nature of protein secondary structural changes following adsorption onto hydrophobic sorbent surfaces. This knowledge could also enable the development of useful protocols for enhancing the chromatographic purification of proteins in their native bioactive states. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 895,905, 2008. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Natural Antigenic Peptides from Squamous Cell Carcinoma Recognized by Autologous HLA-DR8,restricted CD4+ T CellsCANCER SCIENCE, Issue 8 2002Hiroaki Kondo A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC,20, was effectively lysed by HLA-DRB1·08032 (HLA-DRS)-restricted autologous CD4+ T cell line, TcOSC,20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse-phase high-performance liquid chromatography (RP-HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano-liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA-DR8 binding motif, and TcOSC,20 showed a rather strong cytotoxic response to one of the synthetic pep tides, namely, amino acid residues 321,336 of human a-enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells. [source] Simple Preparation of Dimeric Cinchona Alkaloid Derivatives on Polystyrene Supports and a Highly Enantioselective Catalytic Heterogeneous Dimerization of KetenesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 9 2010Ravindra Abstract A convenient route for the covalent immobilization of quinidine and hydroquinidine pyridazine ethers on insoluble polystyrene supports is described, which avoids the need of chromatographic purifications at any stage. The use of the heterogeneized alkaloid derivatives in the asymmetric organocatalytic dimerization of ketenes afforded high enantioselectivity values (90,97% ee) in the course of 20 reaction cycles. [source] Antiproliferative effect of flavonoids and sesquiterpenoids from Achillea millefolium s.l. on cultured human tumour cell linesPHYTOTHERAPY RESEARCH, Issue 5 2009Boglárka Csupor-Löffler Abstract The antiproliferative activities of n -hexane, chloroform, aqueous-methanol and aqueous extracts of the aerial parts of the Achillea millefolium aggregate on three human tumour cell lines were investigated by means of MTT assays. The chloroform-soluble extract exerted high tumour cell proliferation inhibitory activities on HeLa and MCF-7 cells, and a moderate effect on A431 cells; accordingly, it was subjected to detailed bioactivity-guided fractionation. As a result of the multistep chromatographic purifications (VLC, CPC, PLC, gel filtration), five flavonoids (apigenin, luteolin, centaureidin, casticin and artemetin) and five sesquiterpenoids (paulitin, isopaulitin, psilostachyin C, desacetylmatricarin and sintenin) were isolated and identified by spectroscopic methods. The antiproliferative assay demonstrated that centaureidin is the most effective constituent of the aerial parts of yarrow: high cell growth inhibitory activities were observed especially on HeLa (IC50 0.0819 µm) and MCF-7 (IC50 0.1250 µm) cells. Casticin and paulitin were also highly effective against all three tumour cell lines (IC50 1.286,4.76 µm), while apigenin, luteolin and isopaulitin proved to be moderately active (IC50 6.95,32.88 µm). Artemetin, psilostachyin C, desacetylmatricarin and sintenin did not display antiproliferative effects against these cell lines. This is the first report on the occurrence of seco -pseudoguaianolides (paulitin, isopaulitin and psilostachyin C) in the Achillea genus. Copyright © 2008 John Wiley & Sons, Ltd. [source] | |||||||||||||