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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]