Selection and characterization of Affibody ligands to the transcription factor c-Jun.
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 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 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 ligand, denoted Z(cJun518), was shown to interact with immobilized c-Jun protein with an apparent dissociation constant of 5 microM. 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 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 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 ligands directed against transcription factors and other oncogenic factors is discussed.
Lundberg
Emma
E
School of Biotechnology, Albanova University Center, Kungliga Tekniska Högskolan, Stockholm, Sweden.
Brismar
Hjalmar
H
Gräslund
Torbjörn
T
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D055767:Immobilized Proteins / Q000378:metabolism; D008024:Ligands; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D055767:Immobilized Proteins; D008024:Ligands; D019151:Peptide Library; D011506:Proteins; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D004247:DNA