Abstract
This chapter provides a practical guide to the in planta transient production of bluetongue virus-like particles containing a fluorescent cargo protein. Bluetongue virus (BTV) particles are icosahedral, multishelled entities of a relatively large size. Heterologous expression of the four main structural proteins of BTV results in the assembly of empty virus-like particles which resemble the native virus externally, but are devoid of nucleic acid. The space within the particles is sufficient to allow incorporation of relatively large cargo proteins, such as green fluorescent protein (GFP), by genetic fusion to the structural protein VP3. The method described utilizes the pEAQ vectors for high-level transient expression of such particles in Nicotiana benthamiana.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Marsian J, Lomonossoff GP (2016) Molecular pharming—VLPs made in plants. Curr Opin Biotech 37:201–206. https://doi.org/10.1016/j.copbio.2015.12.007
Peyret H, Gehin A, Thuenemann EC, Blond D, El Turabi A, Beales L, Clarke D, Gilbert RJ, Fry EE, Stuart DI, Holmes K, Stonehouse NJ, Whelan M, Rosenberg W, Lomonossoff GP, Rowlands DJ (2015) Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins. PLoS One 10(4):e0120751. https://doi.org/10.1371/journal.pone.0120751
Aljabali AAA, Barclay JE, Lomonossoff GP, Evans DJ (2010) Virus templated metallic nanoparticles. Nanoscale 2(12):2596–2600. https://doi.org/10.1039/c0nr00525h
Wen AM, Shukla S, Saxena P, Aljabali AAA, Yildiz I, Dey S, Mealy JE, Yang AC, Evans DJ, Lomonossoff GP, Steinmetz NF (2012) Interior engineering of a viral nanoparticle and its tumor homing properties. Biomacromolecules 13(12):3990–4001. https://doi.org/10.1021/bm301278f
Rurup WF, Verbij F, Koay MST, Blum C, Subramaniam V, Cornelissen JJLM (2014) Predicting the loading of virus-like particles with fluorescent proteins. Biomacromolecules 15(2):558–563. https://doi.org/10.1021/bm4015792
Kar AK, Iwatani N, Roy P (2005) Assembly and intracellular localization of the bluetongue virus core protein VP3. J Virol 79(17):11487–11495. https://doi.org/10.1128/Jvi.79.17.11487-11495.2005
Thuenemann EC, Meyers AE, Verwey J, Rybicki EP, Lomonossoff GP (2013) A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles. Plant Biotechnol J 11(7):839–846. https://doi.org/10.1111/pbi.12076
Hewat EA, Booth TF, Loudon PT, Roy P (1992) 3-Dimensional reconstruction of baculovirus expressed bluetongue virus core-like particles by cryoelectron microscopy. Virology 189(1):10–20
Hewat EA, Booth TF, Roy P (1994) Structure of correctly self-assembled bluetongue virus-like particles. J Struct Biol 112(3):183–191
Sainsbury F, Thuenemann EC, Lomonossoff GP (2009) pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7(7):682–693. https://doi.org/10.1111/j.1467-7652.2009.00434.x
Sainsbury F, Lomonossoff GP (2008) Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol 148(3):1212–1218. https://doi.org/10.1104/pp.108.126284
Peyret H, Lomonossoff GP (2013) The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants. Plant Mol Biol 83(1–2):51–58. https://doi.org/10.1007/s11103-013-0036-1
Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai HF, Zhou XH, Chen Q (2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp (77). https://doi.org/10.3791/50521
Brillault L, Jutras PV, Dashti N, Thuenemann EC, Morgan G, Lomonossoff GP, Landsberg MJ, Sainsbury F (2017) Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery. ACS Nano 11 (4):3476–3484
Acknowledgments
This work was supported by the UK Biotechnological and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant “Understanding and Exploiting Plant and Microbial Secondary Metabolism” (BB/J004596/1), the BBSRC Synthetic Biology Research Centre ‘OpenPlant’ award (BB/L014130/1), and the John Innes Foundation. We thank Andrew Davis for photography.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Thuenemann, E.C., Lomonossoff, G.P. (2018). Delivering Cargo: Plant-Based Production of Bluetongue Virus Core-Like and Virus-Like Particles Containing Fluorescent Proteins. In: Wege, C., Lomonossoff, G. (eds) Virus-Derived Nanoparticles for Advanced Technologies. Methods in Molecular Biology, vol 1776. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7808-3_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7808-3_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7806-9
Online ISBN: 978-1-4939-7808-3
eBook Packages: Springer Protocols