Expression of human interferon gamma in tobacco chloroplasts

Chloroplast genetic engineering enhances capacity of plants as a bioreactor for the production of recombinant proteins through increasing transgenes expression level and protein accumulation.In this feasibility study, the potential of high-yield expression of interferon gamma (IFNg), a valuable therapeutic human protein, is explored for tobacco chloroplasts.The interferon gamma gene (ifnG) and aminoglycoside 3 ' -adenylyltransferase (aadA) (as a selectable marker gene) were introduced into the inverted repeat (IR) of tobacco plastome as a dicistronic operon using a biolistic-mediated transformation method.The presence and expression of ifnG in the regenerated plants were confirmed in DNA, RNA, and protein levels by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis, respectively.Furthermore, quantification of expressed IFNg by Enzyme-linked immunosorbent assay (ELISA) showed that the level of expression in transplastomic plants was approximately 0.42% of the total soluble proteins (TSPs), which is much higher than its expression in nuclear transformed plants.Southern blot analysis was used to confirm homoplasmy in the T0 progeny.Overall, the results showed that the plastid transformation can be employed as an effective tool for large-scale production of pharmaceutical proteins.Moreover, dicistronic expression of ifnG and aadA genes in tobacco chloroplasts simplifies gene manipulation.