Chloroplast-based overexpression of Pharmaceuticals to develop Cost-effective Therapeutics

Abstract

Interferon, one of the very powerful components of the immune system, are cytokines in nature, produced and secreted by virtually all eukaryotic cells in response to stimulation by certain viruses, bacteria, antigens and mitogens; they mediate their biological activity by binding to specific receptors on the cell surface and modify cell differentiation and exhibit immunoregulatory activity. Interferon mainly classified as alpha, beta and gamma, based on their common genomic location, structural similarities and competition for binding to the same receptor. The development of recombinant interferon embodied profound hope for the cure of many debilitating and potentially fatal diseases. Various recombinant interferons currently dominate the annual market and are approved to treat many chronic viral diseases as well as a variety of cancers and different types of leukemia. The only type of interferon produced commercially is alpha. The production of interferon alpha from microbial to mammalian expression system, have certain precincts in terms of cost, scalability, safety and authenticity. Modern biotechnology exploits transgenic crops to get large quantities of complex proteins in a cost-effective way. In order to overcome several challenges from biosafety point of view, the chloroplast transformation strategy is one of the best approaches since plastids are strictly maternally inherited in most of the cultivated species. Further, chloroplasts allow hyper expression of recombinant DNA with correct folding of expressed protein due to the presence of chaperonin proteins. Hence transplastomic technology is the best choice, now days, to overexpress pharmaceuticals cost-effectively. In the present study the interferon alpha genes were synthesized by using complex set of oligos. After sequence confirmation of the synthesized genes, the histidine residues along with the thrombin protease site were engineered upstream to the synthetic interferon alpha 5 and 2 genes. The recombinant fragments were then tethered with chloroplast light inducible promoter, rbcL followed by sequential cloning to develop chloroplast transformation vector to target the cassettes into the inverted repeat region of plastome through two events of homologous recombination. Since chloroplast and E. coli transcriptional and translational machineries have similarities to express recombinant genes, therefore the chloroplast specific expression cassettes were first expressed in E. coli and selection was performed on spectinomycin because the vector carries selectable marker gene aadA that encodes aminoglycoside-3`-adenyltransferase and confers resistance to both spectinomycin and streptomycin. The expression of interferon genes was assessed and a reasonable level of recombinant protein was recorded. Encouraged from these experiments with E. coli cells harboring chloroplast transformation vector, sterile tobacco leaves were bombarded using biolistic gun. The putative transgenic plants, obtained as a result of antibiotic selection of chopped bombarded leaves, were subjected to different round of selection and regeneration for homoplasmicity. The spectinomycin-resistant shoots were routinely analyzed for gene presence through PCR and the expression of introduced synthetic genes was recorded using ELISA technique. It was experienced that mature leaves contained high levels interferon compared to young and senescence leaves.