Cobalt-ion chelate affinity chromatography for the purification of brain neutral alpha-D-mannosidase and its separation from acid alpha-D-mannosidase.
Ontology highlight
ABSTRACT:
SUBMITTER: Mathur R
PROVIDER: S-EPMC1144168 | biostudies-other | 1984 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Getting insights on tissue or cell specific EV composition in pathophysiological conditions for developing therapeutics, biomarkers and diagnostic tools in modern medicine still remains a major bottleneck. Similarly, lack of standards for EV comparison also hampers progression in the field. Here, we therefore generated a tagged CD81 fusion protein highly enriched in EVs, enabling affinity isolation of EVs from complex matrices in a non-destructive and pure form without disturbing the here tested EV characteristics.
2024-11-22 | GSE251842 | GEO
Project description:Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.
| S-EPMC1172625 | biostudies-other
Project description:PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.
| S-EPMC4435810 | biostudies-literature
Project description:Human butyrylcholinesterase (HuBChE) is being developed as a therapeutic for protection from the toxicity of nerve agents. An enriched source of HuBChE is Cohn fraction IV-4 from pooled human plasma. For the past 40 years, purification of HuBChE has included affinity chromatography on procainamide-Sepharose. The present report supports a new affinity sorbent, Hupresin, for purification of HuBChE from Cohn fraction IV-4. Nine batches of 70-80 kg frozen Cohn fraction were extracted with water, filtered, and chromatographed on 30 L of Q-Ceramic ion exchange sorbent at pH 4.5. The 4% pure Q-eluent was pumped onto 4.2 L Hupresin, where contaminants were washed off with 0.3 M NaCl in 20 mM sodium phosphate pH 8.0, before 99% pure HuBChE was eluted with 0.1 M tetramethylammonium bromide. The average yield was 1.5 g of HuBChE from 80 kg Cohn paste. Recovery of HuBChE was reduced by 90% when the paste was stored at -20°C for 1 year, and reduced 100% when stored at 4°C for 24h. No reduction in HuBChE recovery occurred when paste was stored at -80°C for 3 months or 3 years. Hupresin and procainamide-Sepharose were equally effective at purifying HuBChE from Cohn fraction. HuBChE in Cohn fraction required 1000-fold purification to attain 99% purity, but 15,000-fold purification when the starting material was plasma. HuBChE (P06276) purified from Cohn fraction was a 340 kDa tetramer of 4 identical N-glycated subunits, stable for years in solution or as a lyophilized product.
| S-EPMC6326467 | biostudies-literature
Project description:Aflatoxins are mycotoxins that are produced by several species of filamentous fungi. In the European Union, the concentration limits for this group of mycotoxins in food and feed products are very low (on the order of parts per billion). Thus, relatively high amounts of these substances in their pure forms are required as reference standards. Chromatographic techniques based on solid stationary phases are generally used to purify these molecules; however, liquid-liquid chromatographic separations may be a promising alternative. Therefore, this study proposes a liquid-liquid chromatographic method for the separation of four aflatoxins and impurities. To optimise the method, numerous biphasic solvent systems (chloroform-, acetone- and acetic acid-based systems) were tested and evaluated in terms of their effectiveness at partitioning aflatoxins; the toluene/acetic acid/water (30:24:50, v/v/v/%) system was found to be the most efficient for application in centrifugal partition chromatographic instrument. Using liquid-liquid instrumental separation, the four aflatoxins, namely B1 (400 mg), B2 (34 mg), G1 (817 mg) and G2 (100 mg), were successfully isolated with 96.3%-98.2% purity from 4.5 L of Aspergillus parasiticus fermented material in a 250 mL centrifugal partition chromatography column. The identities and purities of the purified components were confirmed, and the performance parameters of each separation step and the whole procedure was determined. The developed method could be effectively used to purify aflatoxins for analytical applications.
| S-EPMC6628226 | biostudies-literature
Project description:The lysosomal α-mannosidases are class II mannosidases that belong to glycoside hydrolase family 38 and play an important role in the degradation of asparagine-linked carbohydrates of glycoproteins. Based on peptide similarity to human and bovine lysosomal mannosidase (LM), recombinant α-mannosidase from Drosophila melanogaster (dLM408) was cloned and heterologously expressed in Pichia pastoris. The recombinant form of dLM408 designed for structural analysis lacks the transmembrane domain and was crystallized using standard vapour-diffusion and counter-diffusion techniques. The crystals grew as flat plates and as tetragonal bipyramids, respectively. The plate-shaped crystals exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to a minimum d-spacing of 3.5 Å.
| S-EPMC3412785 | biostudies-literature
Project description:Lentiviral vectors (LVs) are used in cell and gene therapies due to their ability to transduce both dividing and non-dividing cells while carrying a relatively large genetic payload and providing long-term gene expression via gene integration. Current cultivation methods produce titers of 105-107 transduction unit (TU)/mL; thus, it is necessary to concentrate LVs as well as remove process- and product-related impurities. In this work, we used a packaging cell line WinPac-RD-HV for LV production to simplify upstream processing. A direct capture method based on ion-exchange chromatography and cellulose nanofibers for LV concentration and purification was developed. This novel scalable stationary phase provides a high surface area that is accessible to LV and, therefore, has potential for high-capacity operation compared to traditional bead-based supports. We were able to concentrate LVs 100-fold while achieving a two-log removal of host cell protein and maintaining up to a 90% yield of functional vector.
| S-EPMC6804883 | biostudies-literature
Project description:BackgroundAffinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system.ResultsElution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml.ConclusionsAffinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.
| S-EPMC3127757 | biostudies-literature
Project description:Neutral alpha-D-mannosidase from monkey brain was purified by Co2+-chelate affinity chromatography and immunoadsorbent affinity chromatography. The purified enzyme, with subunit Mr 45,000, was essentially homogeneous with only traces of two contaminant proteins as revealed by SDS/polyacrylamide-gel electrophoresis and AgNO3 staining. The purified enzyme, on preincubation with Co2+ at 37 degrees C or 60 degrees C followed by assay, showed a time-dependent enhancement in activity. The enhanced activity of the enzyme persisted even after removal of the Co2+. Bacitracin could partially prevent the activation. An aminopeptidase activity that was stimulated by Co2+ both at 37 degrees C and at 60 degrees C was present in the purified enzyme. After preincubation of the enzyme with Co2+ there was evidence for the release of amino acids, as revealed by t.l.c., but the Mr determined by SDS/polyacrylamide-gel electrophoresis was not appreciably altered. It is suggested that a Co2+-stimulated thermostable aminopeptidase, inseparable from the neutral mannosidase, may be involved in the stimulation of neutral mannosidase activity during its preincubation with Co2+.
| S-EPMC1149359 | biostudies-other
Project description:Affinity adsorbents for bovine trypsin were prepared by covalently coupling p-(p'-amino-phenoxypropoxy)benzamidine to cellulose and to agarose. Trypsin binds to both adsorbents at pH6-8 and is released at low pH values or in the presence of n-butylamine hydrochloride. Pure beta-trypsin may be eluted from crude trypsin bound at pH8.0 to the cellulose adsorbent by stepwise elution with an acetate buffer, pH5.0. Both alpha- and beta-trypsin may be isolated by chromatography of crude trypsin on the agarose derivative in an acetate buffer, pH4.0. These two methods for purifying the trypsin are specific to the particular adsorbents. They are rapid and convenient in use. Both methods leave a mixture of the two enzymes bound to the adsorbent and release occurs only at low pH values. The effects of pH, composition and ionic strength of buffer and other variables on both purification methods are described. Affinity adsorbents of soya-bean trypsin inhibitor and of N-alpha-(N'-methyl-N'-sulphanilyl) sulphanilylagmatine bound to agarose were prepared, but were found to be of limited usefulness in the purification of trypsin.
| S-EPMC1168110 | biostudies-other