title: Purification of human alpha-L-fucosidase precursor expressed in Escherichia coli as a glutathione S-transferase fusion protein.
abstract: Alpha-L-fucosidase (FUC) is a glycosidase involved in the degradation of fucose-containing glycoconjugates. A cDNA representing the complete sequence of human FUC was inserted into the prokaryotic expression vector pGEX-2T. High levels of the glutathione S-transferase (GST) fusion protein were detected in Escherichia coli cells after induction with isopropyl thio-beta-D-galactopyranoside. The GST-FUC protein was mostly found as inclusion bodies and attempts to optimise its expression as a soluble form were unsuccessful. Nevertheless, the recombinant protein was purified by affinity chromatography on glutathione-sepharose and its fucosidase activity was characterised. After thrombin cleavage of the GST tag, the FUC precursor protein was purified by electro-elution.
authors.affiliation: Department of Biochemistry, Genetics and Immunology, Faculty of Sciences, University of Vigo, 36200, Vigo, Spain. adcarlos@uvigo.es
authors.forename: Alejandro
authors.initials: A
authors.lastname: de Carlos
authors.forename: Dolores
authors.initials: D
authors.lastname: Montenegro
authors.forename: Ana
authors.initials: A
authors.lastname: Alonso-Rodríguez
authors.forename: María
authors.initials: M
authors.lastname: Páez de la Cadena
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Rodríguez-Berrocal
authors.forename: Vicenta Soledad
authors.initials: VS
authors.lastname: Martínez-Zorzano
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D017931:DNA Primers; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics; D005644:alpha-L-Fucosidase / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D005644:alpha-L-Fucosidase