In this report, the biological activity of the recombinant Arresten expressed in Nicotiana tabacum was studied. The gene coding for the tumor angiogenesis inhibitor Arresten was PCR-amplified from the plasmid pCA and its plant expression vector named pCAMBIAarr was constructed by inserting the Arresten cDNA fragment into the NcoI/BstEII sites of the plant binary expression vector pCAMBIA1301. Then pCAMBIAarr was transferred into Agrobacterium tumefacien LBA4404 by the freeze-thaw method. The adventitious shoots and regenerated plants of Nicotiana tabacum with hygromycinB-resistance were obtained via Agrobacterium-mediated leaf disk transformation method. Southern hybridization, RT-PCR and Western blotting analysis showed that the Arresten cDNA was integrated into the genome of some of the regenerated plants and the recombinant Arresten was expressed with a molecular size of 26 kD. Recombinant Arresten purified from transgenic tobacco leaves had an anti-proliferative effect on bovine endothelial cells. We speculate that biologically active recombinant Arresten can be produced by using plants as bioreactors.
Agrobacterium tumefaciens Angiogenesis Inhibitors