title: Cloning, expression, and purification of mouse heparanase.
abstract: Heparanase is an endoglucuronidase that plays an important role in tumor invasion and metastasis. A full-length heparanase gene was cloned from a mouse embryo cDNA library and determined to encode a protein of 535 amino acids that is 77% identical to human heparanase. The full-length mouse gene was stably expressed in NS0 myeloma cells. The recombinant mouse heparanase protein was purified to homogeneity from cell lysates by a combination of Con-A affinity chromatography, heparin affinity chromatography, and size exclusion chromatography. The purified protein consisted of a non-covalent heterodimer of 50- and 8-kDa polypeptides, similar to the human homolog. The protein was enzymatically active in assays using radiolabeled ECM and heparan sulfate as substrates. The maximum heparanase activity was observed at acidic conditions; however, significant activity was also detected at neutral pH. The enzymatic activity of mouse heparanase was blocked by known heparanase inhibitors.
authors.affiliation: ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA.
authors.forename: Hua-Quan
authors.initials: HQ
authors.lastname: Miao
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Navarro
authors.forename: Sheetal
authors.initials: S
authors.lastname: Patel
authors.forename: David
authors.initials: D
authors.lastname: Sargent
authors.forename: Henry
authors.initials: H
authors.lastname: Koo
authors.forename: Hong
authors.initials: H
authors.lastname: Wan
authors.forename: Anadellys
authors.initials: A
authors.lastname: Plata
authors.forename: Qinwei
authors.initials: Q
authors.lastname: Zhou
authors.forename: Dale
authors.initials: D
authors.lastname: Ludwig
authors.forename: Peter
authors.initials: P
authors.lastname: Bohlen
authors.forename: Paul
authors.initials: P
authors.lastname: Kussie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017385:Sequence Homology; D014407:Tumor Cells, Cultured; D000099175:Heparanase
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005966:Glucuronidase; D006497:Heparan Sulfate; D000099175:Heparanase