Purification of human alpha-L-fucosidase precursor expressed in Escherichia coli as a glutathione S-transferase fusion protein.
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.
de Carlos
Alejandro
A
Department of Biochemistry, Genetics and Immunology, Faculty of Sciences, University of Vigo, 36200, Vigo, Spain. adcarlos@uvigo.es
Montenegro
Dolores
D
Alonso-Rodríguez
Ana
A
Páez de la Cadena
María
M
Rodríguez-Berrocal
Francisco Javier
FJ
Martínez-Zorzano
Vicenta Soledad
VS
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*
D017931:DNA Primers; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D005644:alpha-L-Fucosidase