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.
Similar content being viewed by others
References
Drugmand, J. C., Schneider, Y. J., & Agathos, S. N. (2012). Insect cells as factories for biomanufacturing. Biotechnology Advances, 30(5), 1140–1157.
Kushnir, N., Streatfield, S. J., & Yusibov, V. (2012). Virus-like particles as a highly efficient vaccine platform: Diversity of targets and production systems and advances in clinical development. Vaccine, 31(1), 58–83.
Mena, J. A., & Kamen, A. A. (2011). Insect cell technology is a versatile and robust vaccine manufacturing platform. Expert Review of Vaccines, 10(7), 1063–1081.
Zhu, J. (2012). Mammalian cell protein expression for biopharmaceutical production. Biotechnology Advances, 30(5), 1158–1170.
Yusibov, V., et al. (2013). Hybrid viral vectors for vaccine and antibody production in plants. Current Pharmaceutical Design, 19(31), 5574–5586.
Verch, T., Yusibov, V., & Koprowski, H. (1998). Expression and assembly of a full-length monoclonal antibody in plants using a plant virus vector. Journal of Immunological Methods, 220(1–2), 69–75.
Gleba, Y., Klimyuk, V., & Marillonnet, S. (2007). Viral vectors for the expression of proteins in plants. Current Opinion in Biotechnology, 18(2), 134–141.
Roy, G., et al. (2011). Co-expression of multiple target proteins in plants from a tobacco mosaic virus vector using a combination of homologous and heterologous subgenomic promoters. Archives of Virology, 156(11), 2057–2061.
Dolja, V. V., Kreuze, J. F., & Valkonen, J. P. (2006). Comparative and functional genomics of closteroviruses. Virus Research, 117(1), 38–51.
Agranovsky, A. A., et al. (1994). Beet yellows closterovirus: Complete genome structure and identification of a leader papain-like thiol protease. Virology, 198(1), 311–324.
Peremyslov, V. V., Hagiwara, Y., & Dolja, V. V. (1998). Genes required for replication of the 15.5-kilobase RNA genome of a plant closterovirus. Journal of Virology, 72(7), 5870–5876.
Alzhanova, D. V., et al. (2000). Genetic analysis of the cell-to-cell movement of beet yellows closterovirus. Virology, 268(1), 192–200.
Alzhanova, D. V., et al. (2007). Virion tails of beet yellows virus: Coordinated assembly by three structural proteins. Virology, 359(1), 220–226.
Napuli, A. J., et al. (2003). The 64-kilodalton capsid protein homolog of beet yellows virus is required for assembly of virion tails. Journal of Virology, 77(4), 2377–2384.
Peremyslov, V. V., Pan, Y. W., & Dolja, V. V. (2004). Movement protein of a closterovirus is a type III integral transmembrane protein localized to the endoplasmic reticulum. Journal of Virology, 78(7), 3704–3709.
Prokhnevsky, A. I., et al. (2002). Interaction between long-distance transport factor and Hsp70-related movement protein of beet yellows virus. Journal of Virology, 76(21), 11003–11011.
Reed, J. C., et al. (2003). Suppressor of RNA silencing encoded by Beet yellows virus. Virology, 306(2), 203–209.
Peremyslov, V. V., Hagiwara, Y., & Dolja, V. V. (1999). HSP70 homolog functions in cell-to-cell movement of a plant virus. Proceedings of the National Academy of Sciences of the United States of America, 96(26), 14771–14776.
Hull, A. K., et al. (2005). Human-derived, plant-produced monoclonal antibody for the treatment of anthrax. Vaccine, 23(17–18), 2082–2086.
Zhu, H. Y., et al. (1998). Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member. The Journal of General Virology, 79(Pt 5), 1289–1298.
Karasev, A. V., et al. (1996). Organization of the 3′-terminal half of beet yellow stunt virus genome and implications for the evolution of closteroviruses. Virology, 221(1), 199–207.
Mett, V., et al. (2011). A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge. Human vaccines, 7, 183–190.
Brendel, V., Xing, L., & Zhu, W. (2004). Gene structure prediction from consensus spliced alignment of multiple ESTs matching the same genomic locus. Bioinformatics, 20(7), 1157–1169.
Mamedov, T., et al. (2012). Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F. Plant Biotechnology Journal, 10(7), 773–782.
Chapman, E. J., et al. (2004). Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes and Development, 18(10), 1179–1186.
Kasschau, K. D., & Carrington, J. C. (2001). Long-distance movement and replication maintenance functions correlate with silencing suppression activity of potyviral HC-Pro. Virology, 285(1), 71–81.
Kasschau, K. D., et al. (2003). P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Developmental Cell, 4(2), 205–217.
Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76(9), 4350–4354.
Outchkourov, N., et al. (2007). Epitope analysis of the malaria surface antigen pfs48/45 identifies a subdomain that elicits transmission blocking antibodies. The Journal of Biological Chemistry, 282(23), 17148–17156.
Roeffen, W., et al. (2001). Plasmodium falciparum: Production and characterization of rat monoclonal antibodies specific for the sexual-stage Pfs48/45 antigen. Experimental Parasitology, 97(1), 45–49.
Chiba, M., et al. (2006). Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon. Virology, 346(1), 7–14.
Dolja, V. V., & Koonin, E. V. (2013). The closterovirus-derived gene expression and RNA interference vectors as tools for research and plant biotechnology. Frontiers in Microbiology, 4, 83.
Kurth, E. G., et al. (2012). Virus-derived gene expression and RNA interference vector for grapevine. Journal of Virology, 86(11), 6002–6009.
Giritch, A., et al. (2006). Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proceedings of the National Academy of Sciences of the United States of America, 103(40), 14701–14706.
Grohs, B. M., et al. (2010). Plant-produced trastuzumab inhibits the growth of HER2 positive cancer cells. Journal of Agricultural and Food Chemistry, 58(18), 10056–10063.
Roy, G., et al. (2010). A novel two-component Tobacco mosaic virus-based vector system for high-level expression of multiple therapeutic proteins including a human monoclonal antibody in plants. Virology, 405(1), 93–99.
Sainsbury, F., & Lomonossoff, G. P. (2008). Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiology, 148(3), 1212–1218.
Chen, Q., et al. (2011). Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Human vaccines, 7(3), 331–338.
Huang, Z., et al. (2010). High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnology and Bioengineering, 106(1), 9–17.
Kirkpatrick, R. B., et al. (1995). Heavy chain dimers as well as complete antibodies are efficiently formed and secreted from Drosophila via a BiP-mediated pathway. The Journal of Biological Chemistry, 270(34), 19800–19805.
Leitzgen, K., Knittler, M. R., & Haas, I. G. (1997). Assembly of immunoglobulin light chains as a prerequisite for secretion. A model for oligomerization-dependent subunit folding. The Journal of Biological Chemistry, 272(5), 3117–3123.
Garabagi, F., et al. (2012). Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana expression hosts. Plant Biotechnology Journal, 10(9), 1118–1128.
Culver, J. N., et al. (1993). Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes. Proceedings of the National Academy of Sciences of the United States of America, 90(5), 2055–2059.
Hagiwara, Y., Peremyslov, V. V., & Dolja, V. V. (1999). Regulation of closterovirus gene expression examined by insertion of a self-processing reporter and by northern hybridization. Journal of Virology, 73(10), 7988–7993.
Bosch, D., et al. (2013). N-glycosylation of plant-produced recombinant proteins. Current Pharmaceutical Design, 19(31), 5503–5512.
Gomord, V., et al. (2010). Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnology Journal, 8(5), 564–587.
Jacobs, P. P., & Callewaert, N. (2009). N-glycosylation engineering of biopharmaceutical expression systems. Current Molecular Medicine, 9(7), 774–800.
Milek, R. L., Stunnenberg, H. G., & Konings, R. N. (2000). Assembly and expression of a synthetic gene encoding the antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum in yeast. Vaccine, 18(14), 1402–1411.
Wujek, P., et al. (2004). N-glycosylation is crucial for folding, trafficking, and stability of human tripeptidyl-peptidase I. The Journal of biological chemistry, 279(13), 12827–12839.
Mamedov, T., & Yusibov, V. (2013). In vivo deglycosylation of recombinant proteins in plants by co-expression with bacterial PNGase F. Bioengineered, 4(5), 338–342.
Pushko, P., Pumpens, P., & Grens, E. (2013). Development of virus-like particle technology from small highly symmetric to large complex virus-like particle structures. Intervirology, 56(3), 141–165.
Dugdale, B., et al. (2013). In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants. The Plant Cell, 25(7), 2429–2443.
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-014-9806-5