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 ≈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.