Expression And Purification Of RhPK-5 Protein In E.coli And Its Antiangiogenesis Activity

Human plasminogen kringle 5 (hPK-5) is a proteolytic fragment of plasminogen. Plasminogen contains 5 kringle domains; each kringle consists of 80 amino acids. The hPK-5 protein has been shown to inhibit proliferation of endothelial cells; moreover, its inhibitory activity is more potent than angiostatin. In addition hPK-5 also inhibits endothelial cell migration, an important process in angiogenesis. The protein exerts its effect on endothelial cells. Because of its high efficacy, cell type-selectivity, and short amino acid sequence, hPK-5 has considerable potential in the treatment of neovascular diseases and anti-cancer. The purpose of the present study is to explore the potential application of endogenous angiogeneic inhibitors by gene engineering in E. coli.The choice of an expression system for the high-level production of recombinant proteins depends on many factors. These include cell growth characteristics, expression levels, intracellular and extracellular expression, posttranslational modifications, and biological activity of the protein of interest, as well as regulatory issues in the production of therapeutic proteins. In addition, the selection of a particular expression system requires a cost breakdown in terms of process, design, and other economic considerations.Section I : Construction of pET22b(+)/hPK-5 vectorThe hPK-5 gene encoding 82 amino acid residues from C462 to C543 was recombined with the sequence of plasmid pET22b(+) for constructed a new expressed vector pET/hPK-5. Then it was transformed into E. coli BL21(DE3). The protein was expressed in E. coli BL21(DE3) by IPTG induction. Expression product was purified to almost homogeneity by Ni-chelating chromatography. Western blot analysis showed that rhPK-5 (recombinant human plasminogen kringle 5,rhPK-5) protein was recognized by mAb same as native hPK-5. The result suggested that we obtained correct gene sequence of hPK-5 and got the purified rhPK-5.Section II: Construction of pBV220/hPK-5 vectorFor obtaining high-level hPK-5 expression system, the hPK-5 gene was recombined with plasmid pBV220 to construct the vector of pBV/hPK-5. Then, it was transformed into E. coli JM109, B121(DE3), B121(DE3)-pLySs and DH5a respectively, and expressed hPK-5 induced in 42℃. The expression product constitutes about 25-32% of the total protein and exists in the form ofinclusion body. The hPK-5 protein was purified by affinity column chromatography. Western blot analysis demonstrates that it reacts positively with goat antibody.Protein production in E.coli can be increased significantly through the use of high-cell-density culture systems, which can be classified into five groups: pH, induction time, culture time, culture medium and medium volume. When the JM109 with pBV/hPK-5 cultured at a rotating shaker at 30癈 for 3 hours, pH 7.2,100mL medium in 500mL flasks, induced at 42 癈 for 6 hours, the recombinant protein of rhPK-5 showed good stability generating.Section HI: Purify & RefoldRecombinant hPK-5 protein was efficiently expressed in E.coli JM109 as inclusion bodies. After bacteria were smashed by ultrasound, TE buffer, 1%Ttiton X-100 and 2 M urea was used to efficiently extract inclusion bodies. The solubilization and renaturation conditions of hPK-5 were studied to develop a way to facilitate the purification of inclusion body. Highly purified inclusion body was obtained via several steps including washing three times with 5M urea buffers, washing 1 time with 3% DOC, solubilization in 8M urea for 12 hours and column chromatography. After denaturation with 8 M urea, hPK-5 was affinity-purified. Extracts from the induced E.coli were loaded onto Ni2+-nitrilotriacetic acid (Ni2+-NTA)-agarose and histidine-tagged proteins were selectively eluted to >98% purity under denaturation condition. Subsequent refolding step was optimized for a maximal recovery of biological active protein.The antiangiogenic activity of the hPK-5 was estimated using a chorioallantoic membrane (CAM) model of chicken embryos. Compared with the...