Premium
Selection and characterization of Affibody® ligands to the transcription factor c‐Jun
Author(s) -
Lundberg Emma,
Brismar Hjalmar,
Gräslund Torbjörn
Publication year - 2009
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20070178
Subject(s) - microbiology and biotechnology , phage display , chemistry , peptide library , binding site , recombinant dna , escherichia coli , dissociation constant , c jun , target protein , biology , transcription factor , biochemistry , peptide sequence , peptide , gene , receptor
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 μM. 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.