Chemical Synthesize, Expression, Purification And Characterization Of Human Plasminogen Kringle 5

Angiogenesis is a complex,multi-step process which occurs during normal physiology and in conjunction with pathological conditions.Inhibition of angiogenesis by treatment with angiostatin results in significant suppression of tumor grouth in both murine and human tumor models.Angiostatin contains the first four domains of plasminogen.the kringle5 of plasminogen which shares high sequence homology with angiostatin was found to inhibit endothelial cell proliferation and migration.Here,the results on the human plasminogen kringle5 is as follows:1 Chemical synthesize eight sequences of the Human Plasminogen Kringle 5 gene and construct the hPgn K₅ intact gene including 96 amino acid residues of hPgnGlu_[1] V⁴⁴⁹～P⁵⁴⁴ into E.coli expression vector.2 the hPgnK₅ including 83 amino acid residues of hPgnGlu_[1] C⁴⁶²ï½žP⁵⁴⁴ gene reformed by PCR and add EcoRI, BamHI Cleavage site to its 5', 3' terminal. Construct and express the hPgnK₅ protein in prokaryotic system and purified the hPgnK₅ protein. The protein shows its hPgnK.5 immunity in western blotting and inhibit the proliferation of endothelial cell in vitro, ED₅₀ is 5 μ g.3 eliminate the termination codon of hTNF α and add BamHI cleavage site to its 3' terminal. Construct and express the hTNF α -hPgnK₅ fusion protein in prokaryotic system. The fusion protein shows its hPgnK₅ immunity in Western blotting and inhibit the proliferation of tumor cell or endothelial cell in vitro. The characterization of the hTNF α -hPgnK₅ fusion protein with thrombin cleavage site is similar with the fusion protein without the thrombin cleavage site4 The purified hPgnK₅ protein inhibit the implant tumor growth of S180 or H22 in mouse,the inhibition rate is 53% or 55%,this result has not been reported previously..