Successful production of human epidermal growth factor in tobacco chloroplasts in a biologically active conformation

Human epidermal growth factor (hEGF) is an important therapeutic compound with multiple applications particularly in pharmaceutical industry. Human EGF has already been expressed in different expression systems, however, the production of hEGF with bioactivity in chloroplasts has not been successful so far. In this study, we expressed a 6 × His-tagged hEGF in tobacco chloroplasts in its native conformation for the potential of large-scale production of hEGF for industrial applications. Several transplastomic plant lines were obtained, which were screened by PCR (polymerase chain reaction) using primers specific to selectable gene aadA, hEGF- and GFP-coding sequences that were included in the chloroplast expression vector. The selected lines were confirmed to be homoplasmic by PCR verification and Southern blot analysis. Immunoblotting assays of homoplasmic lines using antibodies raised against hEGF confirmed the accumulation of hEGF in transplastomic plants and the ELISA results demonstrated the expression levels of hEGF were between 0.124% and 0.165% of the total soluble proteins (TSP), namely, 23.16-25.77 ng/g of the fresh weight. In terms of activity, the data from cell proliferation and elongation assays showed that the tobacco-derived recombinant hEGF was as bioactive as its commercial counterpart. To our knowledge, this is the first report of recombinant production of hEGF with native bioactivity form in the chloroplast stroma. Overall, our results demonstrate the potential of higher plant chloroplasts for the production of a human therapeutic, hEGF, in an active conformation.