Abstract: Human endostatin cDNA was cloned from total RNA of human embryo liver tissue by RT-PCR. Endostatin DNA sequence encoded His2 and His4 were double changed to Leu2 and Val4 using site directed mutation technique. The mutant endostatin cDNA was inserted into the pET28b containing promoter T7. The recombinant plasmid transformed the E.coli BL21(DE3). Recombinant human endostatin was highly expressed as inclusion body when theexpression strain BL21-Mute was induced with IPTG. The result of SDS-PAGE analysis reveals that recombinant mutant endostatin accounts for up to 30% of soluble protein in E.coli. The mutant endostatin was purified and refolded farther, its purity was up to 98%. Recombinant mutant endostatin was inactive in inhibiting endothelial cell proliferation.

Key words: endostatin, cloning, mutant, E.coli