Plant Plastid Engineering

Wani, Shabir H.; Haider, Nadia; Kumar, Hitesh; Singh, N.B.

To create an edible vaccine, selected desired genes should be introduced into plants and then inducing these altered plants to manufacture the encoded proteins. Like conventional subunit vaccines, edible vaccines are composed of antigenic proteins and are devoid of pathogenic genes. Plastids of green plants as bioreactors for the production of vaccines and biopharmaceuticals are of great potential as indicated from a number of published studies [110] [111] [112] [113] [114] . The significance of using plants as production platforms for pharmaceuticals is due to the low production and delivery costs, easy scale-up and high safety standards regarding less risk of product contamination with human pathogen [27] . Keeping in view the high efficiency of plastids to express foreign genes, it is meaningful to explore this property of plastids for the production of proteinaceous pharmaceuticals, such as antigens, antibodies and antimicrobials. The candidate subunit vaccine against Clostridium tetani, causing tetanus was the first plastid-produced antigen that proved to be immunologically active in experimental animals [111] . In this initial attempt, fragment C of the tetanus toxin (TetC), a non-toxic protein fragment, was expressed from the tobacco plastid genome which resulted in high levels of antigen protein expression (30% of the plant's total soluble protein (TSP)). Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Significant development has been achieved towards the production of plastid-based vaccine for this infectious disease. Expression (14% of the plants TSP) of the pagA gene encoding the protective antigen (PA) from the tobacco plastid genome gave rise to stable antigen protein [112, 113] . The plastid-derived PA was equally effective in cytotoxicity assays as the bacterial protein produced in B. anthracis. The potential of plastid transformation as an alternative tool to produce high levels of HIV-1 Nef and p24 antigens in plant cells have been also demonstrated [115] . Different constructs were designed to express the p27 Nef protein either alone or as p24-Nef or Nef-p24 fusion proteins. All constructs were utilized to transform tobacco (cv. Petite Havana) plastids and the transplastomic lines. Analysis of p24-Nef and Nefp24 fusion proteins showed that both can be expressed to relatively high levels in plastids. As the best results in terms of protein expression levels were obtained with the p24-Nef fusion protein, the correspondent gene was cloned in a new expression vector. This construct was introduced into the tobacco and tomato plastid genomes. Transplastomic tobacco and tomato plants were analyzed and protein accumulation was found to be close to 40% of the leaf's total protein. Transcript and protein accumulation were analyzed in different ripening stage of tomato fruit and green tomatoes accumulated the fusion protein to 2.5% of the TSP [115] . Recently, a strategy for plastid production of antibiotics against pneumonia Streptococcus pneumonia has been outlined [116] . The authors describe it as a new technique for high level expression (to up to 30% of the plant's TSP) of antmicrobial proteins that are toxic to E. coli. It was also shown that the plastid-produced antibiotics efficiently kill pathogenic strains of Streptococcus pneumoniae, the causative agent of pneumonia, thus providing a promising strategy for the production of next-generation antibiotics in plants. In 2007