Abstract
As a complement to the intracellular antibody capture method to isolate intracellular single domain antibody fragments (iDabs) from high diverse libraries, we describe here a simple mammalian two-hybrid (M2H) protocol using a “bait-prey hybrid single plasmid” to assess those interfering iDabs that will block protein–protein interactions of a target with its natural partner proteins. This rapid method identifies interfering iDabs in one step and improves the reproducibility of the results between experiments and samples (e.g., different single domain antibody clones) compared to traditional M2H. This method yields functional, interfering iDabs and can be applied to any interfering molecule for use as a research tool or template for clinical inhibitor production.
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References
Tanaka T, Lobato MN, Rabbitts TH (2003) Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies. J Mol Biol 331:1109–1120
Colby DW, Chu Y, Cassady JP, Duennwald M, Zazulak H, Webster JM, Messer A, Lindquist S, Ingram VM, Wittrup KD (2004) Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. Proc Natl Acad Sci U S A 101:17616–17621
Tanaka T, Sewell H, Waters S, Philips S, Rabbitts TH (2011) Single domain intracellular antibodies from diverse libraries: emphasizing dual functions of LMO2 protein interactions using a VH single domain. J Biol Chem 286:3707–3716
Tse E, Lobato MN, Forster A, Tanaka T, Chung GTY, Rabbitts TH (2002) Intracellular antibody capture technology: application to selection of single chain Fv recognising the BCR-ABL oncogenic protein. J Mol Biol 317:85–94
Tanaka T, Rabbitts TH (2010) Protocol for the selection of single-domain antibody fragments by third generation intracellular antibody capture. Nat Protoc 5:67–92
Tanaka T, Williams RL, Rabbitts TH (2007) Tumour prevention by a single antibody domain inhibiting binding of signal transduction molecules to activated RAS. EMBO J 26:3250–3259
Bartel PL, Chien C, Sternglanz R, Fields S (1993) Using the two-hybrid system to detect protein-protein interaction. In: Hartley DA (ed) Cellular interaction in development: a practical approach. IRL Press, Oxford, pp 153–179
Visintin M, Tse E, Axelson H, Rabbitts TH, Cattaneo A (1999) Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc Natl Acad Sci USA 96:11723–11728
Vojtek AB, Hollenberg SM, Cooper JA (1993) Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74:205–214
Sambrook J, Russell PW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Engebrecht JBR, Kaderbhai MA (1992) Escherichia coli, plasmids, and bacteriophages. Wiley, New York
Sadowski I, Bell B, Broad P, Hollis M (1992) GAL4 fusion vectors for expression in yeast or mammalian cells. Gene 118:137–141
Tucker KL, Beard C, Dausmann J, Jackson-Grusby L, Laird PW, Lei H, Li E, Jaenisch R (1996) Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev 10:1008–1020
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Tanaka, T., Rabbitts, T.H. (2012). Selection of Functional Single Domain Antibody Fragments for Interfering with Protein–Protein Interactions Inside Cells: A “One Plasmid” Mammalian Two-Hybrid System. 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_11
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DOI: https://doi.org/10.1007/978-1-61779-968-6_11
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-61779-968-6
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