Selection and characterization of Affibody (R) ligands to the transcription factor c-Jun

摘要

c-Jun is a highly oncogenic transcription factor involved in the development of different types of cancer. In the present study we have generated c-Jun-binding-affinity proteins from a phage-displayed library of so-called 'Affibody (R) ligands', developed by combinatorial engineering of a non-immunoglobulin-based scaffold protein. Homodimeric c-Jun protein was recombinantly produced in Escherichia coli and, prior to selection, the quality of the target protein was investigated by binding analyses, which indicated specific binding to a double-stranded DNA hairpin construct containing a c-Jun response element, but not to a control sequence. Isolated Affibody (R) variants from the phage selection were expressed in E. coli, purified by affinity chromatography and their interaction with c-Jun was analysed. In biosensor analyses, one Affibody (R) ligand, denoted Z(cJun518) was shown to interact with immobilized c-Jun protein with an apparent dissociation constant of 5 mu M. By constructing a head-to-tail homodimeric version of Z(cJun518), its apparent affinity for c-Jun could be increased threefold, suggesting co-operativity effects in the binding to the immobilized c-Jun protein. Further characterization of the Z(cJun518) Affibody (R) molecule demonstrated, in both affinity-capture and Western-blotting experiments, its ability to interact selectively with c-Jun, even when the c-Jun target was present in a complex protein background consisting of a bacterial cell lysate. Z(cJun518) could also be used to stain the c-Jun-overexpressing cell line C8161 visualized by confocal fluorescence microscopy. Results from competition experiments indicated that the binding epitope on c-Jun for the Z(cJun518) Affibody (R) molecule was separate from the binding sites of both a polyclonal antibody raised against the unstructured N-terminal domain and a double-stranded DNA hairpin containing a c-Jun response element. The potential intracellular use of Affibody (R) ligands directed against transcription factors and other oncogenic factors is discussed.