[Preparation of human recombinant kringle 1-5 and its bioactivity].
To investigate antiangiogenesis activity and effects on endothelial cell proliferation of human recombinant K1-5 expressed in E.coli BL21, the cDNA of human K1-5 obtained from a cloning vector pUC57K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E.coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed and purified to a purity of 96% by the nickel affinity chromatography. Refoled K1-5 protein was tested on chicken CAMs, and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, human recombinant K1-5 potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrated that human recombinant K1-5 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.
Hou
Wei-Hong
WH
Laboratory for Cell Biology, Zhengzhou University, Zhengzhou 450052, China. whhou@zzu.edu.cn
Chai
Yu-Rong
YR
Wang
Tian-Yun
TY
Wang
Jian-Min
JM
Jia
Yan-Long
YL
Xue
Le-Xun
LX
D020533:Angiogenesis Inhibitors / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D018082:Kringles / Q000235:genetics*; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology*
D020533:Angiogenesis Inhibitors; D011994:Recombinant Proteins; D010958:Plasminogen