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. (C) 2002 Elsevier Science B.V. All rights reserved.