Construction and transformation of cassettes containing human proinsulin gene for transient expression in alfalfa plants

Currently, millions of people all over the world for escaping the lethal effects of diabetes require persistent injections of insulin. Thus, to develop a system with low cost and high production of insulin that can fit into the consumers, is essential. With evolving genetic engineering techniques, production of human insulin in bacteria and yeasts were feasible, but due to drawbacks of producing recombinant proteins in those organisms including high cost, the possibility of contamination with toxic proteins and costly purification steps, most research has focused on the production of recombinant proteins in plants. Therefore, this investigation was done to express transiently the human proinsulin in alfalfa leaves by two different constructs. Construct pLPC as a fusion gene B subunit heat liable toxin of E.coli - proinsulin - penetratin and constructs pEPC incorporation into a peptide containing extensin leader - Proinsulin - penetratin. These constructs were transferred into Agrobacterium strain LBA4404 and agroinfiltration. Gene expression in alfalfa leaves was attested by RT-PCR and specific primers. The production of active proinsulin was confirmed by medical diagnostic mono-bind kits. The amount of production of active proinsulin in agroinfiltrated alfalfa leaves with constructs pLPC and pEPC were estimated 76 and 71.6 nano-grams per gram of fresh alfalfa leaves respectively.