Abstract
Many antibody fragments, selected ex vivo by phage display, fail to form functional antigen-binding entities when expressed and used intracellularly (i.e., as intrabodies) because the interior of the cell poses significant challenges on the folding of antibodies. Such dropout can be avoided by employing intracellular selection methods like yeast or bacterial two hybrid systems. These involve four facile steps: construction of plasmids, transformation of microbial cells, intracellular expression of fusion proteins, and selection for reporter activity. Using E. coli as host instead of yeast offers the advantages of a faster growth and a higher transformation efficiency allowing to screen larger repertoires. This chapter describes the protocol, optimized to identify antigen-specific single domain antibodies (sdAbs), by bacterial two hybrid selection.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317
Barbas CF 3rd, Kang AS, Lerner RA et al (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci USA 88:7978–7982
Griffiths AD, Malmqvist M, Marks JD et al (1993) Human anti-self antibodies with high specificity from phage display libraries. EMBO J 12:725–734
Mattheakis LC, Bhatt RR, Dower WJ (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci USA 91:9022–9026
Hanes J, Plückthun A (1997) In vitro selection and evolution of functional proteins by ribosome display. Proc Natl Acad Sci USA 94:4937–4942
Zahnd C, Amstutz P, Pluckthun A (2007) Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat Methods 4:269–279
Lo AS, Zhu Q, Marasco WA (2008) Intracellular antibodies (intrabodies) and their therapeutic potential. In: Chernajovsky Y, Nissim A (eds) Therapeutic antibodies. Handbook of experimental pharmacology, vol 181. Springer, UK, pp 343–373
Messer A, Lynch SM, Butle DC (2009) Developing intrabodies for the therapeutic suppression of neurodegenerative pathology. Expert Opin Biol Ther 9:1189–1197
Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246
Visintin M, Settanni G, Maritan A et al (2002) The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies. J Mol Biol 317:73–83
Mossner E, Koch H, Pluckthun A (2001) Fast selection of antibodies without antigen purification: adaptation of the protein fragment complementation assay to select antigen-antibody pairs. J Mol Biol 308:115–122
Pelletier JN, Arndt KM, Pluckthun A et al (1999) An in vivo library-versus-library selection of optimized protein-protein interactions. Nat Biotechnol 17:683–690
Pelletier JN, Campbell-Valois FX, Michnick SW (1998) Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc Natl Acad Sci USA 95:12141–12146
Dove SL, Joung JK, Hochschild A (1997) Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature 386:627–630
Joung JK, Ramm EI, Pabo CO (2000) A bacterial two hybrid selection system for studying protein-DNA and protein-protein interactions. Proc Natl Acad Sci USA 97:7382–7387
Dove SL, Hochschild A (1998) Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target. Genes Dev 12:745–754
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Pellis, M., Muyldermans, S., Vincke, C. (2012). Bacterial Two Hybrid: A Versatile One-Step Intracellular Selection Method. In: Saerens, D., Muyldermans, S. (eds) Single Domain Antibodies. Methods in Molecular Biology, vol 911. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-968-6_9
Download citation
DOI: https://doi.org/10.1007/978-1-61779-968-6_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-967-9
Online ISBN: 978-1-61779-968-6
eBook Packages: Springer Protocols