Purification of human alpha-L-fucosidase precursor expressed in Escherichia coli as a glutathione S-transferase fusion protein |
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Authors: | de Carlos Alejandro Montenegro Dolores Alonso-Rodríguez Ana Páez de la Cadena María Rodríguez-Berrocal Francisco Javier Martínez-Zorzano Vicenta Soledad |
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Affiliation: | Department of Biochemistry, Genetics and Immunology, Faculty of Sciences, University of Vigo, 36200, Vigo, Spain. adcarlos@uvigo.es |
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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. |
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