Neutralizing nanobodies against vascular endothelial growth factor (VEGF) and its receptors are the most important types of medicine which are used in cancer therapy strategies. To increase the level of anti-VEGF (Nb42) nanobody accumulation and also to simplify its subsequent purification, we fused codon-optimized Nb42 nanobody to hydrophobin I (HFBI) stabilizing partner, and transiently expressed the fusion gene cassette in cucurbit plants using a zucchini yellow mosaic virus-based viral vector. The successful transcription of the target gene was confirmed with the use of RT-PCR and Real-time PCR analyses. According to the immunoblot assay results the expression level of Nb42-HFBI fusion protein, 22 μg/g fresh leaf tissue, was twofolds higher than the unfused nanobody. Furthermore, the endoplasmic reticulum (ER)-targeted Nb42-HFBI fusion protein was successfully purified from leaf extract using an aqueous-two phase system (ATPS) method. The use of HFBI fusion partner increased the expression level of the Nb42 recombinant protein while simultaneously facilitated its subsequent purification using an inexpensive non-chromatographic method known as ATPS. As a conclusion, the application of ER-targeted hydrophobin fusion strategy with the use of special features of plant viral vectors, can improve the value of plant-based transient expression platform for the production of important proteins with the industrial and therapeutic application.

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