title: Bacterial expression and functional reconstitution of human heparanase.
abstract: Human heparanase is a heparan sulfate degrading enzyme located in the extracellular matrix playing a decisive role in angiogenesis and tumor metastasis. Translated as a 65 kDa inactive prae-form, the protein is processed into an 8 kDa and a 50 kDa subunit which form a non-covalently associated active heterodimer. We have expressed the two subunits separately in Escherichia coli which yielded active human heparanase upon reconstitution. The two purified subunits folded independently and secondary structure analysis by far-UV CD spectroscopy gave 33.1/11.1% α/β content for the 50 kDa subunit and 6.9/49% α/β content for the 8 kDa subunit. This heparanase expression system is easy and can be used for efficient screening for enzyme inhibitors.
authors.affiliation: Department of Pharmaceutical Sciences, University of Graz, Humboldtstrasse 46, 8010 Graz, Austria.
authors.forename: Sophie
authors.initials: S
authors.lastname: Winkler
authors.affiliation: Department of Pharmaceutical Sciences, University of Graz, Humboldtstrasse 46, 8010 Graz, Austria.
authors.forename: Daniela
authors.initials: D
authors.lastname: Schweiger
authors.affiliation: Department of Medical Oncology, Paterson Institute for Cancer Research, Manchester M204BX, United Kingdom.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wei
authors.affiliation: Department of Pharmaceutical Sciences, University of Graz, Humboldtstrasse 46, 8010 Graz, Austria.
authors.forename: Erich
authors.initials: E
authors.lastname: Rajkovic
authors.affiliation: Department of Pharmaceutical Sciences, University of Graz, Humboldtstrasse 46, 8010 Graz, Austria. Electronic address: andreas.kungl@uni-graz.at.
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Kungl
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015202:Protein Engineering; D000099175:Heparanase
chemical_list: D004791:Enzyme Inhibitors; D005966:Glucuronidase; D006497:Heparan Sulfate; D000099175:Heparanase
keywords: Angiogenesis; Extracellular matrix; Heparan sulfate; Human heparanase; Tumor metastasis