Development of recombinant human epidermal growth factor production in Nicotiana benthamiana

Abstract

Human epidermal growth factor (hEGF) is a short polypeptide that has gained clinical importance in the recent decade for wound healing. Currently, plant-based expression system is considered as an alternative viable platform for low-cost recombinant protein production. Hence, this study aims to produce hEGF in Nicotiana benthamiana plants via., transient expression using a geminiviral vector. The hEGF gene constructs were designed into six different constructs. The optimal expression was observed from the construct targeting the endoplasmic reticulum with C-terminal histidine tag at the yield level up to 15.695 µg/g LFW or 0.499% TSP. The plant-produced hEGF was purified by using Nickel affinity chromatography and confirmed its identity by SDS-PAGE and Western blot probed with anti-hEGF antibody. Furthermore, the preliminary study of the protein purification efficiency was found that the high extraction volume allows better hEGF recovery and extraction buffer pH 4 could largely remove host cell proteins (HCP), especially RuBisCO. However, ammonium sulfate precipitation is inapplicable for removing HCP from plant-produced hEGF. Furthermore, the study showed that no cytotoxic effects have been found in HaCaT cells from the plant-produced hEGF which is equivalent to commercial hEGF in HaCaT cells. Hence, this study supports that the potential of plant expression system offers a simple and cost-effective approach for the industrial-scale production of recombinant hEGF.