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.

Automated summary

This study successfully targeted the accumulation of AntiVEGF recombinant protein to the plant apoplast, demonstrating higher accumulation compared to cytoplasm-targeted protein, possibly due to improper folding. The purified protein showed biological activity.