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Purification to homogeneity of Charonia lampas alpha-fucosidase by using sequential ligand-affinity chromatography.

ABSTRACT: 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.

SUBMITTER: Butters TD

PROVIDER: S-EPMC1151565 | biostudies-other | 1991 Oct

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Affinity 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. RESULTS: Elution 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. CONCLUSIONS: Affinity 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.

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Purification to homogeneity of Charonia lampas alpha-fucosidase by using sequential ligand-affinity chromatography.

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