Apoplastic Production of Recombinant AntiVEGF Protein Using Plant-Virus Transient Expression Vector.

Targeting of vascular endothelial growth factor (VEGF) using AntiVEGF can be a promising approach for angiogenesis inhibition and cancer therapy. In this study, we direct AntiVEGF recombinant protein accumulation to cucurbit plant apoplast using a suitable signal (Pr1b) sequence. After assembling the target gene construct and cloning into the expression vector, we infected the plants with the resulting pZYMV-AntiVEGF viral vector. Transcription of the target gene was confirmed with RT-PCR assays. The apoplast-targeted AntiVEGF recombinant protein was detected in infected plants by Dot-blot, western blot, and ELISA analysis. AntiVEGF protein accumulation in the apoplast resulted in levels of 1.2% of TSP (Total Soluble Protein) that demonstrated a two-order increase compared to the cytoplasm-targeted protein. After purification of AntiVEGF protein using aqueous two-phase system (ATPS), purified protein was analyzed with MTT assay. Our results reveal that production of biologically active and correctly processed apoplast-targeted AntiVEGF recombinant protein is possible in plant apoplast. The low level of cytoplasm-targeted AntiVEGF recombinant protein might result from the degradation of improperly folded protein.