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ISSN 1672-9145                                                Acta Biochim Biophys Sin 2004, 36(12): 845–850                                                  CN 31-1940/Q

Recombinant Mouse Canstatin Inhibits Chicken Embryo Chorioallantoic Membrane Angiogenesis and Endothelial Cell Proliferation¤

Wei-Hong HOU, Tian-Yun WANG, Bao-Mei YUAN, Yu-Rong CHAI, Yan-Long JIA, Fang TIAN, Jian-Min WANG, and Le-Xun XUE*

 

Laboratory for Cell Biology, Zhengzhou University, Zhengzhou 450052, China

 

Abstract        Human canstatin, a 24 kD fragment of the a2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

 

Key words        mouse canstatin; angiogenesis inhibitor; prokaryotic expression

 

 

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Received: September 22, 2004       Accepted: November 16, 2004

This work was supported by the grants from the Key Science and Technologies Program of Henan Province (No. 0122032500) and the National Natural Science Foundation of China (No. 30270031)

¤GenBank accession No. AY375463

This work was carried out in Henan Key Laboratory for Molecular Medicine

*Corresponding author: Tel, 86-371-6658332; Fax, 86-371-6997182; E-mail, [email protected]