[The cloning expression, purification and activity of human angiostatin K (1-3) gene].

OBJECTIVE: To clone the sequence of human Angiostatin cDNA and obtain the protein of recombinant angiostatin for further development. METHODS: Fresh human liver tissue was used for the extraction of total RNA and amplified the Angiostatin cDNA through RT-PCR method . After the recombinant plasmid pET30a-Angiostatin was constructed and confirmed, it was transduced into Rosetta (DE3). Then the transformation was used for fermentation and induced expression. The protein was identified by Western Blot and purified by Ni-NTA affinity chromatography. RESULTS: The sequence of human Angiostatin cDNA was identical with genebank. Angiostatin (K1-3) was expressed and purified. The target protein made up 30% of the total bacterial protein. The purity is above 90%. CONCLUSION: Angiostatin K (1-3) can be reached using fusion vector pET30a. The product have the biological activity.

[Fusion expression and identification of angiostatin and endostatin in E. coli BL21(DE3)].

Angiostatin(AS) and endostatin(ES) are both potent endogenous angiogenesis inhibitors, and the combination of AS and ES has been shown to have synergistic antiangiogenic effects. Here we report the fusion protein AS-ES expressed in E. coli which has antiangiogenic effects. At first, AS and ES genes were cloned respectively through RT-PCR, then fusion gene was made through gene splicing ,finally pET-42 (b)/AS-ES expression plasmid was constructed and transduced in E. coli BL21 (DE3). Target protein was in form of inclusion body,the rate of expression was about 14%, and MW about 65KD. Western blotting assay showed expressed protein had specific immune reaction to both the antibodies of AS and ES. The expressed protein which was refolded and purified through heparin affinity chromatography had antiangiogenic effect to vessels on chicken embryo chorioallantoic membrane. The results show that fusion protein AS-ES was expressed successfully in E. coli, and the expressed protein,which was renatured and purified, had immuno-reactivity to anti-AS and anti-ES in Western blotting and angiogenesis inhibition activity.

Generation and role of angiostatin in human platelets.

Platelets regulate new blood vessel growth, because they contain a number of angiogenesis promoters and inhibitors. Additionally, platelets contain matrix metalloproteinases (MMPs), which when released mediate platelet adhesion and aggregation, and plasminogen, a fibrinolytic system enzyme that serves to limit blood clot formation. Enzymatic cleavage of plasminogen by MMPs generates angiostatin, an angiogenesis inhibitor. Therefore, we examined whether platelets generate angiostatin during aggregation in vitro. Platelets were isolated from healthy human donors and then aggregated with collagen, thrombin, or HT-1080 fibrosarcoma cells. Angiostatin was detected by Western blot analysis in the platelet releasates of all blood donors irrespective of the aggregating agent used. Platelet pellet homogenates showed the presence of angiostatin in all donors, which was released upon aggregation. Furthermore, platelet-derived angiostatin was isolated and purified by lysine-Sepharose affinity chromatography from collagen-aggregated platelet releasates. Bioassay of platelet-derived angiostatin showed that it inhibited the formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in an in vitro angiogenesis model. Inhibition of angiostatin in platelet releasates promoted the formation of capillary structures by HUV-EC-Cs. We conclude that healthy human platelets contain angiostatin, which is released in active form during platelet aggregation, and platelet-derived angiostatin has the capacity to inhibit angiogenesis.