RESUMO
It has been known that VEGF(121) isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric protein comprising abrin-a (ABRaA) toxin A-chain and human VEGF(121). Expression of the ABRaA-VEGF(121) chimeric protein was carried out in E. coli strain BL21(DE3). ABRaA-VEGF(121) preparations were isolated from inclusion bodies, solubilized and purified by affinity and ion-exchanged chromatography (Ni-agarose and Q-Sepharose). Finaly, bacterial endotoxin was removed from the recombinant protein. Under non-reducing conditions, the recombinant protein migrates in polyacrylamide gel as two bands (about 84 kDa homodimer and about 42 kDa monomer). ABRaA-VEGF(121) is strongly cytotoxic towards PAE cells expressing VEGFR-2, as opposed to VEGFR-1 expressing or parental PAE cells. The latter are about 400 times less sensitive to the action of this fusion protein. The biological activity of the ABRaA domain forming part of the chimeric protein was assessed in vitro: ABRaA-VEGF(121) inhibited protein biosynthesis in a cell-free translation system. Preincubation of ABRaA-VEGF(121) with antibody neutralizing the biological activity of human VEGF abolished the cytotoxic effect of the chimeric protein in PAE/KDR cells. Experiments in vivo demonstrated that ABRaA-VEGF(121) inhibits growth of B16-F10 murine melanoma tumors.
