Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate

Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed from a subunit of 8 kDa and a subunit of 50 kDa. The two protein subunits are non-covalently associated. The cloning and expression of the two protein subunits in Escherichia coli, and their subsequent purification to homogeneity under native conditions result in the production of an active heparanase enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the non-reducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on heparanase, since the heparanase cleavage of fluorogenic tag would result in a measurable response. This article is protected by copyright. All rights reserved