Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system.
Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 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 and an anti-hexahistidine tag 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, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (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, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system 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. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.
Hou
Wei-Hong
WH
Laboratory for Cell Biology, Zhengzhou University, Zhengzhou 450052, China.
Fang
Tian
T
Chai
Yu-Rong
YR
Wang
Tian-Yun
TY
Wang
Jian-Min
JM
Xue
Le-Xun
LX
D020533:Angiogenesis Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002642:Chick Embryo; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D006131:Growth Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D006801:Humans; D018082:Kringles / Q000235:genetics* / Q000502:physiology; D051379:Mice; D041681:NIH 3T3 Cells; D010958:Plasminogen / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D006131:Growth Inhibitors; D011994:Recombinant Proteins; D010958:Plasminogen