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-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

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

Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells

IMPACT: 1.868International audienceIntrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single-chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti-PCNA scFvs were screened in vitro after being tagged with dimeric glutathione-S-transferase. Anti-PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error-prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C-terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide-conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody-based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright (c) 2014 John Wiley & Sons, Ltd

Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells