Homoplasmy stability of transplastomic tobacco plants (Nicotiana tabacum CV. Xhanti) containing human tissue-type plasminogen activator (K2S form) gene in T1 generation

Abstract

The rates of recombinant protein in nuclear-transformed plants are often less than 1% of total soluble proteins. As the plant plastid is highly polyploidy, plastid transformation can lead to high-level production of recombinant protein. In addition, plastid transformation has several other advantages such as prevention of gene escape that has a high importance in molecular farming. Tissue-type plasminogen activator (tPA) is an important protein that is used to treating clots in cardiovascular diseases. Thus, production of tPA protein in plant system was considered. The tPA (K2S form) gene was transferred to tobacco chloroplast genomes. In this study, we analyzed expression and stability of tPA gene in transplastomic tobacco plants in T1 generation. The presence of tPA gene in transplastomic plants was confirmed with specific primer by PCR analysis. Homoplasmy, gene expression, and protein assay were confirmed with southern blot, RT-PCR, western blotting and ELISA analysis. Results showed that the tPA gene in T1 generation of transplastomic tobacco plants is stable and is expressed. In addition, the maximum amount of tPA protein was estimated up to 0.13% of total soluble protein.