Investigating of the effect of subcellular targeting on Anti-VEGF recombinant nanobody accumulation in cucurbit plant

In the last three decades, molecular farming has emerged as an alternative and cost-effective strategy for the successful production of hundreds of large-scale pharmaceutical recombinant proteins using plants. Because tumor cells need angiogenesis to grow and invade, therefore, targeting the most important angiogenic factor (vascular endothelial growth factor, VEGF) using the plant-based recombinant anti-VEGF can be an effective approach to inhibit angiogenesis and cancer treatment. The low expression level of recombinant protein is one of the major obstacles for the commercialization of molecular pharming. Therefore, numerous techniques have been developed to enhance protein expression, including codon optimization of protein sequences, appropriate subcellular targeting; the use of strong promoters, and the testing of different plant-based expression systems. In this paper, we investigate the effect of apoplast and endoplasmic reticulum (ER) targeting on Anti-VEGF recombinant nanobody accumulation in cucurbit plant. for this purpose, different molecular analyzes (RT-PCR, Dotblot, SDS-PAGE, Westernblot and ELISA) were performed and the results showed that ER targeting increased protein expression about 6-fold compared to its expression in the apoplast. Therefore, the ER is a more suitable site for expression of the target recombinant protein and other recombinant proteins than for apoplast.