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Enhanced expression and purification of anti-VEGF nanobody in cucurbit plants

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Abstract

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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Abbreviations

VEGF:

Vascular endothelial growth factor

HFBI:

Hydrophobin I

ER:

Endoplasmic reticulum

ATPS:

Aqueous-two phase system

MAbs:

Monoclonal antibodies

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Authors and Affiliations

  1. Department of Biotechnology and Plant Breeding, Ferdowsi University of Mashhad, Mashhad, Iran

    Mojgan Soleimanizadeh, Abdolreza Bagheri & Alireza Seifi

  2. Department of Biotechnology, Tarbiat Modares University, P.O. Box 14115-115, Tehran, Iran

    Mokhtar Jalali Javaran

  3. Venom and Biotherapeutics Molecules Lab, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Mahdi Behdani & Fatemeh Kazemi-Lomedasht

Authors
  1. Mojgan Soleimanizadeh
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  2. Abdolreza Bagheri
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  3. Mokhtar Jalali Javaran
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  4. Alireza Seifi
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  5. Mahdi Behdani
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  6. Fatemeh Kazemi-Lomedasht
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Correspondence to Mokhtar Jalali Javaran.

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Soleimanizadeh, M., Bagheri, A., Jalali Javaran, M. et al. Enhanced expression and purification of anti-VEGF nanobody in cucurbit plants. J. Plant Biochem. Biotechnol. 28, 263–270 (2019). https://doi.org/10.1007/s13562-018-0471-9

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  • DOI: https://doi.org/10.1007/s13562-018-0471-9

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