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Development of a Single-Replicon miniBYV Vector for Co-expression of Heterologous Proteins

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Abstract

In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.

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Acknowledgments

The authors would like to thank Dr. Valerian Dolja of Oregon State University for the p35S-BYV-GFP plasmid and M. Levikova for assistance with ELISA. The authors are grateful to Dr. Stephen J. Streatfield for critical reading of the manuscript and Dr. Natasha Kushnir for editorial assistance.

Conflict of interest

The authors declare no conflict of interest.

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Author notes

  1. Tarlan Mamedov

    Present address: Department of Agricultural Biotechnology, Akdeniz University, Pınarbaşı Mh., 07985, Antalya, Turkey

  2. Rahila Rahimova

    Present address: Institut de Recherche en Cancerologie de Montpellier, 208 rue des Apothicaires, 34298, Montpellier Cedex 5, France

  3. Ananya Ghosh

    Present address: Siemens Healthcare Diagnostics Inc., 500 GBC Drive, Newark, DE, 19702, USA

  4. Vadim Mett

    Present address: Cleveland BioLabs, Inc., 73 High Street, Buffalo, NY, 14203, USA

Authors and Affiliations

  1. Fraunhofer USA Center for Molecular Biotechnology, 9 Innovation Way, Suite 200, Newark, DE, 19711, USA

    Alex Prokhnevsky, Tarlan Mamedov, Brett Leffet, Rahila Rahimova, Ananya Ghosh, Vadim Mett & Vidadi Yusibov

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  1. Alex Prokhnevsky
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Correspondence to Vidadi Yusibov.

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Prokhnevsky, A., Mamedov, T., Leffet, B. et al. Development of a Single-Replicon miniBYV Vector for Co-expression of Heterologous Proteins. Mol Biotechnol 57, 101–110 (2015). https://doi.org/10.1007/s12033-014-9806-5

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