Homoplasmic Stability and Cytoplasmic Inheritence of DARPin G3 Scaffold Protein in Generative and Vegetative Propagation of Transplastoic Tobacco Plants

Plastid engineering gives numerous benefits for the next generation of transgenic technology, consisting of the convenient use of transgene stacking and the production of high expression levels of recombinant proteins. Designed ankyrin repeat proteins (DARPin) are relatively small non-immunoglobulin scaffold proteins that bind to their specific target with high affinity. The G3 is a type of DARPin designed to bind to the HER2 tyrosine kinase receptor (human epidermal growth factor receptor 2). We previously developed a bioprocess for the production of DARPin G3 in tobacco chloroplasts as an imaging agent in HER2 over-expressed cancers. In this study, we analyzed the expression and homoplasmic stability of DARPin G3 gene in vegetative and generative T1 generation of transplastomic tobacco plants. The presence of DARPin G3 gene in the next generation of transplastomic plants was confirmed with specific primers by PCR analysis. Southern blot analysis confirmed the homoplasmic status of transplastomic plants. The western blot analysis confirmed the accumulation of the DARPin G3 in the chloroplasts of next generation of transplastomic plants. The DARPin G3 protein content was estimated around 33% by ELISA in chloroplast total soluble protein (TSP) of the transplastomic plants. Results confirmed that the DARPin G3 gene in vegetative and generative T1 generation of transplastomic tobacco plants was stably and highly expressed.