Antibody-affinity purification of novel alpha-L-fucosidase from mouse liver.
Ontology highlight
ABSTRACT:
SUBMITTER: Laury-Kleintop LD
PROVIDER: S-EPMC1148162 | biostudies-other | 1987 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:An alpha-fucosidase from the liver of the marine gastropod Charonia lampas was purified to homogeneity using a procedure that included cation-exchange and gel-filtration chromatography, chromatofocusing and a final series of affinity-chromatography steps which involved the following gel-immobilized ligands: N-(5-carboxy-1-pentyl)-1,5-dideoxy-1,5-imino-L-fucitol, N-(5-carboxy-1-pentyl)-2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol and thio-beta-D-galactoside. The enzyme was found to be a tetrameric glycoprotein with a native Mr of 208,000, and to exist in a number of isoforms displaying pI values in the range 6.0-6.4. Substrate-specificity studies using a number of fucosylated oligosaccharides of the lacto-N and lacto-N-neo series and a synthetic disaccharide confirmed that the enzyme catalyses the hydrolysis of a broad range of fucosidic linkages, and established the following hierarchy of susceptibility: Fuc alpha 2Gal beta 4Glc much much greater than Fuc alpha 6GlcNAc greater than Fuc alpha 2Gal beta 4GlcNAc greater than Gal beta 3(Fuc alpha 4)GlcNAC much much greater than Gal beta 4(Fuc alpha 3)GlcNAc. Similar relative rates of hydrolysis were also demonstrated using biantennary oligosaccharide alditols as substrates which contained fucose linked either alpha 3 or alpha 6 to the N-acetylglucosaminitol residue of the chitobiosyl core.
| S-EPMC1151565 | biostudies-other
Project description:alpha-l-Fucosidase was partially purified from the liver of a fucosidosis patient by column chromatography either on agarose-epsilon-aminohexanoylfucosamine or on concanavalin A-Sepharose, despite no apparent enzymic activity in the crude liver supernatant. Mixing studies indicated that the liver of the fucosidosis patient did not lack activators or contain inhibitors of alpha-l-fucosidase activity. The partially purified alpha-l-fucosidase from the liver of the fucosidosis patient exhibits a 4-5-fold-increased Michaelis constant for the 4-methylumbelliferyl substrate (700-750mum) and a greatly decreased thermostability at 55 degrees C compared with the normal liver enzyme. The pH-activity curve is similar to that for the normal enzyme between pH5 and 8, but quite dissimilar in the acid region (pH3.0-4.5): below pH4.5 the alpha-l-fucosidase shows no activity, whereas the normal enzyme retains considerable activity (>/=50% of maximal activity). Isoelectric focusing of the alpha-l-fucosidase revealed one major form with pI5.8 and other possible minor forms. No cross-reacting material was detected when the alpha-l-fucosidase was run in double-immunodiffusion experiments against the immunoglobulin-G fraction of anti-(alpha-l-fucosidase) antibodies, but the enzyme was immunoprecipitated by this immunoglobulin-G fraction. For at least the fucosidosis patient being studied here, all the data suggest retention of a thermolabile portion of normal alpha-l-fucosidase (with characteristic Michaelis constant and pH-activity curve) or production of a kinetically altered alpha-l-fucosidase with decreased catalytic activity but antigenic similarity to the normal enzyme.
| S-EPMC1162492 | biostudies-other
Project description:Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody-peptide interaction, opening opportunities for further improvements/modifications.
| S-EPMC2590912 | biostudies-literature
Project description:The subunit composition of human liver alpha-L-fucosidase was studied by using three different procedures for treating and electrophoresing the purified enzyme. Although the presence on only one subunit was found by conventional procedures using sodium dodecyl sulphate and urea, two non-identical subunits with mol. wts. of 59 000 and 54 000 were found when the alpha-L-fucosidase was reduced and S-carboxymethylated with iodoacetate followed by electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulphate and 8.0 M-urea.
| S-EPMC1186428 | biostudies-other
Project description:alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.
| S-EPMC1161289 | biostudies-other
Project description:?-L-Fucosidases are enzymes involved in metabolism of ?-L-fucosylated molecules, compounds with a fundamental role in different life essential processes including immune response, fertilization and development, but also in some serious pathological events. According to the CAZy database, these enzymes belong to families 29 and 95. Some of them are also reported to be able to catalyze transglycosylation reactions, during which ?-L-fucosylated molecules, representing compounds of interest especially for pharmaceutical industry, are formed.Activity-based screening of a genomic library was used to isolate the gene encoding a novel ?-L-fucosidase. The enzyme was expressed in E.coli and affinity chromatography was used for purification of His-tagged ?-L-fucosidase. Standard activity assay was used for enzyme characterization. Thin layer chromatography and mass spectrometry were used for transglycosylation reactions evaluation.Using a genomic library of Paenibacillus thiaminolyticus, constructed in E.coli DH5? cells, nucleotide sequence of a new ?-L-fucosidase isoenzyme was determined and submitted to the EMBL database (HE654122). However, no similarity with enzymes from CAZy database families 29 and 95 was detected. This enzyme was produced in form of histidine-tagged protein in E.coli BL21 (DE3) cells and purified by metaloaffinity chromatography. Hydrolytic and transglycosylation abilities of ?-L-fucosidase iso2 were tested using different acceptor molecules.In this study, new enzyme ?-L-fucosidase iso2 originating from Paenibacillus thiaminolyticus was described and prepared in recombinant form and its hydrolytic and transglycosylation properties were characterized. As a very low amino acid sequence similarity with known ?-L-fucosidases was found, following study could be important for different biochemical disciplines involving molecular modelling.
| S-EPMC4445282 | biostudies-literature
Project description:1. alpha-Cyano-4-hydroxycinnamate was coupled to Sepharose CL-4B activated with 1,2:3,4-bisepoxybutane. 2. The low-Km rat liver mitochondrial aldehyde dehydrogenase was specifically bound to this affinity medium, and could subsequently be eluted with alpha-cyano-4-hydroxycinnamate. 3. The enzyme purified in this manner had a subunit molecular mass of 55 kDa and a pI of approx. 6.5. A minor component of approx. 57 kDa was also present and had a significantly higher pI value; this may be the precursor for aldehyde dehydrogenase. 4. alpha-Cyanocinnamate and some related compounds were found to be uncompetitive inhibitors of the enzyme. 5. No cytosolic aldehyde dehydrogenase was bound to the affinity column, but a protein from a rat liver post-mitochondrial supernatant with a molecular mass of approx. 25 kDa was bound, and could be eluted subsequently with alpha-cyano-4-hydroxycinnamate.
| S-EPMC1138478 | biostudies-other
Project description:To investigate the cell type-specific expression features of dopaminergic neurons in ventral midbrain, we generated DAT-TRAP mice. We then performed gene expression profiling analysis using data obtained from RNA-seq of samples collected from 3- and 18-month old mice. OVX were also bred to overexpress human alpha-synuclein. Short and long-read sequencing were performed.
2023-10-01 | GSE215276 | GEO
Project description:The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359-394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1-0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.
| S-EPMC7926790 | biostudies-literature
Project description:Affinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to ligand denaturation and leaching from chromatographic support. Innovations in this area are aimed at developing robust and highly selective antibody ligands capable of withstanding harsh column sanitization conditions. We report the development and first-stage characterization of a selective short linear peptide ligand of the IgG Fc region capable of capturing all four IgG subclasses. The ligand was discovered through in vitro directed evolution. A focused phage-display library based on a previously identified peptide lead was subjected to a single-round screen against a pool of human IgG. The hits were identified with next-generation sequencing and ranked according to the enrichment ratio relative to their frequency in the pre-screened library. The top enriched peptide GSYWYNVWF displaying highest affinity for IgG was coupled to bromohydrin-activated agarose beads via a branched linker. The resulting affinity matrix was characterized with a dynamic binding capacity of approx. 43 mg/mL, on par with commercially employed protein A-based resin.
| S-EPMC8173005 | biostudies-literature