期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 26, 期 3, 页码 425-431出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S1046-5928(02)00558-2
关键词
heparanase; heparan sulfate; gene expression; purification
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. (C) 2002 Elsevier Science (USA). All rights reserved.
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