Cloning, expression, and purification of mouse heparanase.
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.
Miao
Hua-Quan
HQ
ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA.
Navarro
Elizabeth
E
Patel
Sheetal
S
Sargent
David
D
Koo
Henry
H
Wan
Hong
H
Plata
Anadellys
A
Zhou
Qinwei
Q
Ludwig
Dale
D
Bohlen
Peter
P
Kussie
Paul
P
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
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005966:Glucuronidase; D006497:Heparan Sulfate; D000099175:Heparanase