A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm
Open Access
- 22 November 2007
- journal article
- Published by Springer Science and Business Media LLC in BMC Biotechnology
- Vol. 7 (1), 81
- https://doi.org/10.1186/1472-6750-7-81
Abstract
Background: Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.Results: We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the αβ tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressedin vivoin human cells since they were essentially localized in the nucleus.Conclusion: This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.Keywords
This publication has 65 references indexed in Scilit:
- Identification of a Universal VHH Framework to Graft Non-canonical Antigen-binding Loops of Camel Single-domain AntibodiesJournal of Molecular Biology, 2005
- A Structure-Based Database of Antibody Variable Domain DiversityJournal of Molecular Biology, 2005
- High-affinity Human Antibodies from Phage-displayed Synthetic Fab Libraries with a Single Framework ScaffoldJournal of Molecular Biology, 2004
- Phage-displayed Antibody Libraries of Synthetic Heavy Chain Complementarity Determining RegionsJournal of Molecular Biology, 2004
- The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodiesJournal of Molecular Biology, 2002
- Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodiesJournal of Molecular Biology, 2001
- A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffoldJournal of Molecular Biology, 2001
- The Protein Data BankNucleic Acids Research, 2000
- Antibody-antigen Interactions: Contact Analysis and Binding Site TopographyJournal of Molecular Biology, 1996
- Domain association in immunoglobulin molecules: The packing of variable domainsJournal of Molecular Biology, 1985