Abstract
Plant-based platforms are extensively use for the expression of recombinant proteins, including monoclonal antibodies (mAbs). Generally, immunoglobulins (Igs) are sorted to the apoplast, which is often afflicted with intense proteolysis. Here, we describe methods to transiently express mAbs sorted to central vacuole in Nicotiana benthamiana leaves and to characterize the obtained IgG. Central vacuole is an appropriate compartment for the efficient production of Abs, consequently vacuolar sorting should be considered as an alternative strategy to obtain high protein yields.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Marty F (1999) Plant vacuoles. Plant Cell 11:587–599
Vitale A, Raikhel NV (1999) What do proteins need to reach different vacuoles? Trends Plant Sci 4:149–155
Herman EM, Larkins BA (1999) Protein storage bodies and vacuoles. Plant Cell 11:601–613
Tamura K, Shimada T, Ono E, Tanaka Y, Nagatani A, Higashi S-i, Watanabe M, Nishimura M, Hara-Nishimura I (2003) Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. Plant J 35:545–555
Marin Viegas VS, Ocampo CG, Petruccelli S (2017) Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields. Bioengineered 8:203–211
Xiang L, Etxeberria E, Van Den Ende W (2013) Vacuolar protein sorting mechanisms in plants. FEBS J 280:979–993
De Marchis F, Bellucci M, Pompa A (2013) Traffic of human alpha-mannosidase in plant cells suggests the presence of a new endoplasmic reticulum-to-vacuole pathway without involving the Golgi complex. Plant Physiol 161:1769–1782
Ocampo CG, Lareu F, Marin Viegas VS, Mangano S, Loos A, Steinkellner H, Petruccelli S (2016) Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana. Plant Biotechnol J 14:2265–2275. https://doi.org/10.1111/pbi.12580
Denecke J, Botterman J, Deblaere R (1990) Protein secretion in plant cells can occur via a default pathway. Plant Cell 2:51–59
Matsuoka K, Neuhaus JM (1999) Cis-elements of protein transport to the plant vacuoles. J Exp Bot 50:165–174
Goulet C, Khalf M, Sainsbury F, D’Aoust MA, Michaud D (2012) A protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast. Plant Biotechnol J 10:83–94
Benchabane M, Goulet C, Rivard D, Faye L, Gomord V, Michaud D (2008) Preventing unintended proteolysis in plant protein biofactories. Plant Biotechnol J 6:633–648
Niemer M, Mehofer U, Torres Acosta JA, Verdianz M, Henkel T, Loos A, Strasser R, Maresch D, Rademacher T, Steinkellner H et al (2014) The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms. Biotechnol J 9:493–500
Castilho A, Steinkellner H (2012) Glyco-engineering in plants to produce human-like N-glycan structures. Biotechnol J 7:1088–1098
Fitchette AC, Cabanes-Macheteau M, Marvin L, Martin B, Satiat-Jeunemaitre B, Gomord V, Crooks K, Lerouge P, Faye L, Hawes C (1999) Biosynthesis and immunolocalization of Lewis a-containing N-glycans in the plant cell. Plant Physiol 121:333–344
Strasser R (2016) Plant protein glycosylation. Glycobiology 26:926–939
Gomord V, Fitchette A, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson C, Michaud D, Faye L (2010) Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol J 8:564–587
Lerouge P, Cabanes-Macheteau M, Rayon C, Fischette-Lainé AC, Gomord V, Faye L (1998) N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol 38:31–48
Misaki R, Sakai Y, Omasa T, Fujiyama K, Seki T (2011) N-terminal vacuolar sorting signal at the mouse antibody alters the N-linked glycosylation pattern in suspension-cultured tobacco BY2 cells. J Biosci Bioeng 112(5):476–484
Acknowledgment
This work was supported by the Agencia Nacional de Promoción CientÃfica y Tecnológica (ANPCyT) through the grants PICT Start Up 2015-0010 and PICT 2016-1722 and by Universidad Nacional de La Plata (grant 11X/754) to S.P. S.P. is a researcher from CONICET and Full Professor of the Facultad de Ciencias Exactas-UNLP; CGO is a fellow at CONICET.
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
Ocampo, C.G., Petruccelli, S. (2018). Vacuolar Targeting and Characterization of Recombinant Antibodies. In: Pereira, C. (eds) Plant Vacuolar Trafficking. Methods in Molecular Biology, vol 1789. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7856-4_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7856-4_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7855-7
Online ISBN: 978-1-4939-7856-4
eBook Packages: Springer Protocols