Transgenic Expression and Identification of Recombinant Human Proinsulin in Peanut

Abstract

The increased incidence of diabetes, coupled with the introduction of alternative insulin delivery methods that rely on higher doses, is expected to result in a substantial escalation in the future demand for affordable insulin. Plant-based systems offer a safe and economical method for producing pharmaceutical proteins. We used peanut (Arachis hypogaea L.) as bio-reactors to express biosafe, stable proinsulin. We designed two proinsulin analogues (FAIA and LAIA) with substitutions in their amino acid sequences. The fast-acting insulin analogue (FAIA) contains a Gly inserted between Cys19 and Gly20, as well as a Pro28Asp substitution, in the B chain. The long-acting insulin analogue (LAIA) contains a Gly inserted between Cys19 and Gly20 and two Arg residues inserted into the terminus of the B chain, as well as an Asn21Gly substitution in the A chain. Four plasmids were constructed: pROKII-Flag-FAIA, pROKII-Flag-LAIA, pCAMBIA2301-Oleosin-FAIA and pCAMBIA2301-Oleosin-LAIA. These plasmids were transferred into peanut to produce recombinant proinsulin. Western blot and GUS staining analysis indicated that some transgenic peanut successfully expressed exogenous proinsulin. Peanut seeds can act as insulin storage sites, which is the foundation for further production of recombinant proinsulin from peanut seeds.