Lea Vojta, Lana Ljuma-Skupnjak, Ankica Budimir, Slobodan Vukičević and Hrvoje Fulgosi
Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
We report the production of hGM-CSF protein in leaves of two industrial cultivars of tobacco by using Agrobacterium-mediated transient expression. For overexpression, two binary destination vectors, p2GW7.0 and pH7WG2.0, were evaluated. The gene hGM-CSF was tagged on the 3’ end with the Hisand HA-epitopes and cloned into the destination vectors. Agrobacteria containing constructs GM-CSFHis- HA/pH7WG2.0 and GM-CSF-His-HA/p2GW7.0 were used for 5- and 10-minute-long vacuum infiltration of tobacco plants. The hGM-CSF accumulation was found to be dependent on the presence of wetting agent supplemented during vacuum-agroinfiltration, duration of vacuum-agroinfiltration, and the age of the leaves. We demonstrate that industrial tobacco cultivars, with very high biomass, are suitable platforms for rapid, low cost production of foreign proteins. Successful transient expression of the GM-CSF in industrial tobacco was achieved in less than three months, opening the possibility for future applications of this approach in rapid response production of various proteins of non-plant origin.