Abstract
Ectopically expressed intracellular recombinant antibodies, or intrabodies, are powerful tools to visualize proteins and study their function in fixed or living cells. However, many intrabodies are insoluble and aggregate in the reducing environment of the cytosol. To solve this problem, we describe an approach based on GFP-tagged intrabodies. In this protocol, the GFP is used both as a folding-reporter to select correctly folded intrabodies and as a fluorescent tag to localize the scFv and its associated antigen in eukaryotic cells. Starting from a scFv gene cloned in a retroviral vector, we describe retrovirus production, cell line transduction, and soluble intrabody characterization by microscopy and FACS analysis.
The original version of this chapter was revised. The erratum to this chapter is available at: DOI 10.1007/978-1-4939-6857-2_27
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-4939-6857-2_27
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
Lobato MN, Rabbitts TH (2003) Intracellular antibodies and challenges facing their use as therapeutic agents. Trends Mol Med 9:390–396
Visintin M, Meli GA, Cannistraci I, Cattaneo A (2004) Intracellular antibodies for proteomics. J Immunol Methods 290(1-2):135–153
Rinaldi AS, Freund G, Desplancq D, Sibler AP, Baltzinger M, Rochel N, Mély Y, Didier P, Weiss E (2013) The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells. Exp Cell Res 319(6):838–849
Cassimeris L, Guglielmi L, Denis V, Larroque C, Martineau P (2013) Specific In Vivo Labeling of Tyrosinated alpha-Tubulin and Measurement of Microtubule Dynamics Using a GFP Tagged, Cytoplasmically Expressed Recombinant Antibody. PLoS One 8:e59812
Philibert P, Stoessel A, Wang W, Sibler A, Bec N, Larroque C, Saven JG, Courtete J, Weiss E, Martineau P (2007) A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm. BMC Biotechnol 7:81
Robin G, Sato Y, Desplancq D, Rochel N, Weiss E, Martineau P (2014) Restricted diversity of antigen binding residues of antibodies revealed by computational alanine scanning of 227 antibody-antigen complexes. J Mol Biol 426:3729–3743
Freund G, Desplancq D, Stoessel A, Weinsanto R, Sibler AP, Robin G, Martineau P, Didier P, Wagner J, Weiss E (2014) Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells. J Mol Recognit 27:549–558
Auf der Maur A, Zahnd C, Fischer F, Spinelli S, Honegger A, Cambillau C, Escher D, Pluckthun A, Barberis A (2002) Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework. J Biol Chem 277:45075–45085
Tanaka T, Chung GTY, Forster A, Lobato MN, Rabbitts TH (2003) De novo production of diverse intracellular antibody libraries. Nucleic Acids Res 31:e23
Villani ME, Carli MD, Donini M, Traversini G, Lico C, Franconi R, Benvenuto E, Desiderio A (2008) Validation of a stable recombinant antibodies repertoire for the direct selection of functional intracellular reagents. J Immunol Methods 329:11–20
Waldo GS, Standish BM, Berendzen J, Terwilliger TC (1999) Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol 17(7):691–695
Guglielmi L, Denis V, Vezzio-Vie N, Bec N, Dariavach P, Larroque C, Martineau P (2011) Selection for intrabody solubility in mammalian cells using GFP fusions. Protein Eng Des Sel 24:873–881
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Renaud, E., Martineau, P., Guglielmi, L. (2017). Solubility Characterization and Imaging of Intrabodies Using GFP-Fusions. In: Tiller, T. (eds) Synthetic Antibodies. Methods in Molecular Biology, vol 1575. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6857-2_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6857-2_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6855-8
Online ISBN: 978-1-4939-6857-2
eBook Packages: Springer Protocols