title: Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells.
abstract: 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.
authors.affiliation: Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, Boulevard Sébastien Brant, Illkirch, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Freund
authors.forename: Dominique
authors.initials: D
authors.lastname: Desplancq
authors.forename: Audrey
authors.initials: A
authors.lastname: Stoessel
authors.forename: Robin
authors.initials: R
authors.lastname: Weinsanto
authors.forename: Annie-Paule
authors.initials: AP
authors.lastname: Sibler
authors.forename: Gautier
authors.initials: G
authors.lastname: Robin
authors.forename: Pierre
authors.initials: P
authors.lastname: Martineau
authors.forename: Pascal
authors.initials: P
authors.lastname: Didier
authors.forename: Jérôme
authors.initials: J
authors.lastname: Wagner
authors.forename: Etienne
authors.initials: E
authors.lastname: Weiss
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D045744:Cell Line, Tumor; D002470:Cell Survival; D003952:Diagnostic Imaging; D005453:Fluorescence; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000175:diagnosis*; D019151:Peptide Library; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D007202:Indicators and Reagents; D019151:Peptide Library; D018809:Proliferating Cell Nuclear Antigen; D057127:Single-Chain Antibodies
keywords: PCNA; bispecific molecules; imaging in living cells; in vitro affinity maturation; intrabodies; nuclear targeting; single-chain Fv