Angiogenesis Inhibitor-Kringle5 Protein: Secretory Expression In Pichia Pastoris And Construction Of Mammal Expression Plasmid

OBJECT: To express angiogenesis inhibitor—K5 protein in Pichia pastoris , to establish purify process and bioassay methods, to construct a mammal expression plasmid and to analysis its bioactivity. METHODS: Transformed Pichia pastoris using electroporate method and selected high expression strains through G418—YPD solid medium and expression level. K5 protein was purified by anion exchange chromograph (DEAE Sepharose FF), followed by gel filtration (Sephacryl S-100). The apparent molecular weight was detected by 15% SDS-PAGE, and Isoelectric Point was tested by isoelectric focusing method. The biological activities were tested by the trails of inhibition of vascular endothelial cell proliferation, inhibition of endothelial cell transfer, inhibition of angiogensis of CAM and inhibition of transplanted tumor growth. The full DNA sequences of K5 protein were synthesized by two steps PCR, and PCR products were cloned into mammal expression plasmid pDV. Its activity of anti-tumor was studied in mouse tumor model. RESULTS: Some high level expression strains were obtained. The concentration of K5 protein was beyond 200mg/L in supernatant. Purification process was established. The purity of puritied k5 protein was beyond 95%. Some physical-chemical properties of K5 protein were analysised. In 15% SDS-PAGE, its apparent molecular weight was approximate 17. 5KD. About Isoelectric Point analysis by isoelectric focusing, its Isoelectric Point was at approximate pH5.2. The K5 protein could inhibit vascular endothelial cell transfer and proliferation, angiogensis of CAM and transplanted tumor growth. The mammal expression plasmid pDV —K5 plasmid could inhibit transplanted tumor growth in mouse model. CONCLUSION: K5 protein had been successfully expressed in Pichia pastoris and purified by two steps chromgraphic process. According the tests of in vivo and in vitro, the k5 protein was been proved it could inhibit endotheil cell transfer and proliferation, angiogensis of CAM and transplanted tumor growth. The full DNA sequences of K5 protein were obtained and cloned into mammal expression plasmid pDV. The primary study on its bioassay proved that it could inhibit transplanted tumor growth.