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

• DOI: 10.1002/jmr.2378
• Publication Date: 2014-07-07T02:53:45Z
• Authors: Guillaume Freund; Dominique Desplancq; Audrey Stoessel; Robin Weinsanto; Annie‐Paule Sibler; Gautier Robin; Pierre Martineau; Pascal Didier; Jérôme Wagner; Etienne Weiss
• Keywords: Diagnostic Imaging; 0301 basic medicine; Cell Survival; Molecular Sequence Data; Antibody Affinity; [SDV.CAN]Life Sciences [q-bio]/Cancer; bispecific molecules; Fluorescence; in vitro affinity maturation; 03 medical and health sciences; [SDV.CAN] Life Sciences [q-bio]/Cancer; Peptide Library; Cell Line, Tumor; Neoplasms; Proliferating Cell Nuclear Antigen; PCNA; imaging in living cells; Humans; Amino Acid Sequence; nuclear targeting; intrabodies; 3. Good health; single-chain Fv; Indicators and Reagents; Single-Chain Antibodies; Subcellular Fractions

Intrabodies, 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 © 2014 John Wiley & Sons, Ltd.