Purification of human alpha-L-fucosidase precursor expressed in Escherichia coli as a glutathione S-transferase fusion protein

J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Mar 25;786(1-2):7-15. doi: 10.1016/s1570-0232(02)00721-3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Blotting, Western
  • Chromatography, Affinity
  • DNA Primers
  • Enzyme Precursors / genetics
  • Enzyme Precursors / isolation & purification*
  • Escherichia coli / genetics*
  • Glutathione Transferase / genetics*
  • Humans
  • Recombinant Fusion Proteins / genetics
  • alpha-L-Fucosidase / genetics
  • alpha-L-Fucosidase / isolation & purification*

Substances

  • DNA Primers
  • Enzyme Precursors
  • Recombinant Fusion Proteins
  • Glutathione Transferase
  • alpha-L-Fucosidase