Lundberg Emma, Brismar Hjalmar, Gräslund Torbjörn
School of Biotechnology, Albanova University Center, Kungliga Tekniska Högskolan, Stockholm, Sweden.
Biotechnol Appl Biochem. 2009 Jan;52(Pt 1):17-27. doi: 10.1042/BA20070178.
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.
c-Jun是一种高度致癌的转录因子，参与不同类型癌症的发展。在本研究中，我们从一个噬菌体展示文库中生成了c-Jun结合亲和蛋白，该文库由基于非免疫球蛋白支架蛋白的组合工程开发的所谓“Affibody配体”组成。同源二聚体c-Jun蛋白在大肠杆菌中重组产生，在筛选之前，通过结合分析研究了靶蛋白的质量，结果表明其与含有c-Jun反应元件的双链DNA发夹构建体特异性结合，但不与对照序列结合。从噬菌体筛选中分离出的Affibody变体在大肠杆菌中表达，通过亲和层析纯化，并分析它们与c-Jun的相互作用。在生物传感器分析中，一种名为Z(cJun518)的Affibody配体与固定化的c-Jun蛋白相互作用，表观解离常数为5微摩尔。通过构建Z(cJun518)的头对头同源二聚体形式，其对c-Jun的表观亲和力可提高三倍，这表明在与固定化c-Jun蛋白的结合中存在协同效应。对Z(cJun518) Affibody分子的进一步表征表明，在亲和捕获和蛋白质印迹实验中，即使c-Jun靶标存在于由细菌细胞裂解物组成的复杂蛋白质背景中，它也能够选择性地与c-Jun相互作用。Z(cJun518)还可用于通过共聚焦荧光显微镜对过表达c-Jun的细胞系C8161进行染色。竞争实验结果表明，Z(cJun518) Affibody分子在c-Jun上的结合表位与针对无结构N端结构域的多克隆抗体以及含有c-Jun反应元件的双链DNA发夹的结合位点不同。本文讨论了针对转录因子和其他致癌因子的Affibody配体在细胞内的潜在应用。
