Expression of active human epidermal growth factor (hEGF) in tobacco plants by integrative and non-integrative systems

Human epidermal growth factor (hEGF) was expressed in Nicotiana plants using integrative and non-integrative systems. Nuclear transformation was carried out using genetic constructs that allow hEGF accumulation into the cytoplasmic or apoplastic spaces. Protein level did not exceed 0.00001% of total soluble protein in the cytoplasm, but reached values of up to 0.11% when hEGF was targeted to the apoplast. In addition, cytoplasmic and apoplastic hEGF versions were cloned into a viral vector derived from potato virus X. Transcripts from recombinant viruses were used to infect Nicotiana benthamiana and Nicotiana tabacum plants. While the recombinant protein was barely detectable in the case of the cytoplasmic version, it reached levels of up to 0.015% of total soluble proteins in plants infected with the apoplastic version. Extracts from transgenic plants exhibiting the highest hEGF accumulation and from plants infected with the viral apoplastic version were tested for biological activity in cumulus cells expansion assays. hEGF containing extracts showed biological activity comparable to commercial hEGF. Finally, radioreceptor binding assays showed that tobacco-expressed hEGF binds to its receptor with the same affinity as commercial hEGF. These data suggest that hEGF accumulation can be significantly increased by implementing adequate exporting strategies.