A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 expressed <it>in vivo </it>in human cells since they were essentially localized in the nucleus.</p> <p>Conclusion</p> <p>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.</p

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm-0

<p><b>Copyright information:</b></p><p>Taken from "A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm"</p><p>http://www.biomedcentral.com/1472-6750/7/81</p><p>BMC Biotechnology 2007;7():81-81.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2241821.</p><p></p>rary (see additional file : Sequence of randomly picked clones)

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm-1

<p><b>Copyright information:</b></p><p>Taken from "A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm"</p><p>http://www.biomedcentral.com/1472-6750/7/81</p><p>BMC Biotechnology 2007;7():81-81.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2241821.</p><p></p>ed in under the control of the T7 promoter. Soluble extracts were prepared, separated by SDS-PAGE, and analyzed by Coomassie staining (a) or Western-blot (b) using 9E10 and an alkaline phosphatase conjugated anti-mouse IgG antibody (substrate BCIP/NBT). Each lane corresponded to 2 × 10cells. The arrow on the left indicates the position of the scFv

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm-2

<p><b>Copyright information:</b></p><p>Taken from "A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm"</p><p>http://www.biomedcentral.com/1472-6750/7/81</p><p>BMC Biotechnology 2007;7():81-81.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2241821.</p><p></p> positive and negative controls, respectively [5]. At 24 h post-transfection, cells were fixed and visualized under a fluorescent microscope with the fluorescein isothiocyanate filter set. The micrographs represent typical fields containing a similar number of cells in each case. Magnification: × 400

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm-4

<p><b>Copyright information:</b></p><p>Taken from "A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm"</p><p>http://www.biomedcentral.com/1472-6750/7/81</p><p>BMC Biotechnology 2007;7():81-81.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2241821.</p><p></p>es represent typical cells transfected with scFv13R4 and three representative anti-histones clones (2, 5 and 10). D, DAPI staining (blue) merged with the GFP signal