Engineering Camelina sativa Seeds as a Green Bioreactor for the Production of Affordable Human Pro-insulin that Demonstrates Anti-diabetic Efficacy in Rats.

Abstract

The current production of recombinant insulin via fermenter-based platforms (Escherichia coli and yeast) could not fulfill its fast-growing commercial demands, thus leading to a great interest in its sustainable large-scale production at low cost using a plant-based system. In the present study, Agrobacterium tumefaciens-mediated nuclear stable genetic transformation of an industrial oilseed crop, Camelina sativa, to express pro-insulin (with three furin endoprotease cleavage sites) fused with cholera toxin B subunit (CTB) in their seeds was successfully achieved for the first time. The bar gene was used as a selectable marker for selecting transformants and producing herbicide-resistant camelina plants. The transformation process involved the infiltration of camelina inflorescences (at flower buds with partially opened flowers) with A. tumefaciens and harvesting the seeds (T₀) at maturity. The T₀ seeds were raised into the putative T₁ plants sprayed with Basta herbicide (0.03%, v/v), and the survived green transformed plants tested positive for pro-insulin and bar genes. A transformation frequency of 6.96% was obtained. The integration and copy number of the pro-insulin transgene and its expression at RNA and protein levels were confirmed in T₁ plants using Southern hybridization, semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqPCR), and quantitative real-time Time PCR (qPCR) and western blot analysis, respectively. Enzyme-linked immunosorbent Assay (ELISA) quantified the amount of expressed pro-insulin protein, and its anti-diabetic efficacy was validated in diabetic rats on oral feeding. Transgenic plants integrated the pro-insulin gene into their genomes and produced a maximum of 197 µg/100 mg of pro-insulin (0.804% of TSP) that had anti-diabetic efficacy in rats.