Expression In Escherichia Coli And Pichia Pastoris, Purification And Biological Activities Of Human Plasminogen Kringle 5

Antiangiogenesis therapy is one of the most promising strategies for cancer treatment. Human plasminogen Kringle 5 is a novel angiogenesis inhibitor. But its in vivo antitumor potency remains to be investigated, and the expression system to produce adequate amounts of the recombinant protein for in vivo use is also a problem.Firstly, a gene coding for hK5 was artificially synthesized according to the codon usage bias of Pichia pastoris. The gene was subcloned into Escherichia coli expression vector pThioHisA and expressed at high level as a fusion protein of thioredoxin. Recombinant hKS with native N-terminus was generated by enterokinase cleavage and affinity chromatography purification.Secondly, gene of hKS was placed under the control of AOX1 promoter in an integrative Pichia expression vector carrying eight copies of the expression cassette. Through electroporation transformation of GS115 and G418 selection, a strain of Pichia pastoris capable of secreting hKS into the medium at a level up to 150mg/L was constructed. The protein was purified to almost homogeneity by DEAE-Sepharose ion exchange and Sephadex G-7S gel filtration chromatographies.Lastly, the activities of recombinant hK5s from E.coli and Pichia pastoris were investigated. Recombinant hK5s were capable of inhibiting the growth of Ecv304 cells (human umbilical vein endothelial cells-derived cell line) in a dose-dependent manner and showed obvious antiangiogenic activity on a chorioallantoic membrane model (CAM) of chicken embryos. Furthermore, the hK5 inhibited the growth of tumor on a model of T739 mice significantly.This is the first description of generating hKS by Ecoli and Pichia pastoris throgh recombinant DNA techniques and characterizing its antitumor...