Expression of Biologically Active Recombinant Arresten in Nicotiana tabacum

In this report, we studied the biological activity of the recombinant Arresten expressed in Nicotiana tabacum. 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 Nco I/BstE II 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 hygromycin B-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 antiproliferative effect on bovine endothelial cells. We speculate that biologically active recombinant Arresten can be produced by using plants as bioreactors.