Evaluación preliminar de un sistema biológico eucariota (Nicotiana tabacum) escalable para la producción de insulina humana recombinante

Diabetes mellitus is a worldwide health problem, and its treatment represents economic pressure for patients and public health systems. Thus, it is important to develop alternatives to produce insulin, the main treatment for this disease, to reduce its price and increase its availability and effectiveness, which implies direct patient benefits. In this work, leaves of Nicotiana tabacumwere genetically modified to synthesize and release human insulin. For this purpose, the coding sequence was cloned in nuclear and plastidial expression cassettes for plant material. Its correct insertion was verified by PCR analysis of the genomic DNA of the derived clones. In addition, recombinant protein expression and release were quantified by ELISA. The concentration reached average levels of 800pmol/L in the supernatants of the pAF_ins construct derivatives, confirming the effectiveness of plastidial transformation. To preliminarily assess the bioactivity of the hormone, its ability to induce phosphorylation of its receptor in HepG2 cells was evaluated. The results showed increased phosphorylation after stimulation with commercially available recombinant insulin (positive control) and the recombinant molecule secreted by tobacco plants. No phosphorylation was observed upon the stimulation of cells with starvation medium (negative control) or wild-type plant extracts. The results show that N. tabacum is a viable and appropriate platform to produce recombinant human insulin in its functional configuration for therapeutic applications.