Expression of recombinant interferon α-2a in tobacco chloroplasts using micro projectile bombardment
Chloroplast-based expression of pharmaceuticals provides cost-effective benefits to the consumer. In order to establish the transplastomic biopharmaceuticals, the interferon α-2a gene along with aadA gene was flanked by the tobacco chloroplast inverted repeat region for two events of homologous recombination. Chloroplast transformation was accomplished upon bombardment of fully expanded 4 to 6 weeks-old tobacco leaves using helium gun. Green shoots regenerated from single antibiotic resistant cells were subjected to further rounds of selection and regeneration to develop homoplasmic clones. The molecular analysis of the antibiotic-resistant plants confirms the presence of interferon alpha-2a as well as aadA genes in the plastid genome. Moreover, the interferon alpha-2a protein was purified by using Ni-NTA purification columns. The presence of a fragment of 20 kDa size on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography (HPLC) chromatogram confirms the expression of IFNA2a in the transgenic tobacco chloroplasts.
Key words: Interferon, therapeutic, synthetic gene, tobacco, transplastomic strategy, biolistic method.