Cellular and Molecular Biotechnology, Faculty of Technology , Bielefeld University , Bielefeld 33613 , Germany.
Biofidus AG , Hainteichstrasse 78 , Bielefeld 33613 , Germany.
Biochemistry. 2019 Feb 26;58(8):1043-1047. doi: 10.1021/acs.biochem.9b00021. Epub 2019 Feb 15.
The epidermal growth factor receptor (EGFR) is a transmembrane protein involved in cell signaling processes, and dysregulation of its activity often drives tumor growth. EGFR is a clinically validated tumor marker and target for antibodies and tyrosine kinase inhibitors. We demonstrate that a fusion protein of the natural ligand epidermal growth factor (EGF) with the fluorescent reporter mCherry can be expressed in the cytosol of E. coli in high yields and with a high biological activity. Biophysical characterization by mass spectrometry analysis confirmed three disulfide bonds that are crucial for protein structure. Biolayer interferometry data of the protein-protein interaction of EGF-mCherry with the soluble EGFR are comparable to that of unmodified EGF. Cell culture experiments demonstrated that this fusion replicates all important features of the natural ligand. Finally, fluorescent assays based on EGF-mCherry provided a simple and convenient method to compare EGFR levels on cells and to determine competition of EGFR-binding molecules. These assays will help to rank competitive properties of EGFR inhibitors.
表皮生长因子受体（EGFR）是一种参与细胞信号转导过程的跨膜蛋白，其活性的失调常常驱动肿瘤的生长。EGFR 是一种临床验证的肿瘤标志物，也是抗体和酪氨酸激酶抑制剂的作用靶点。我们证明，表皮生长因子（EGF）与荧光报告蛋白 mCherry 的融合蛋白可以在大肠杆菌的细胞质中以高产率和高生物活性表达。通过质谱分析进行的生物物理特性分析证实了三个对蛋白质结构至关重要的二硫键。EGF-mCherry 与可溶性 EGFR 的蛋白质-蛋白质相互作用的生物层干涉数据与未修饰的 EGF 相当。细胞培养实验表明，这种融合复制了天然配体的所有重要特征。最后，基于 EGF-mCherry 的荧光测定法提供了一种简单方便的方法来比较细胞上 EGFR 的水平，并确定 EGFR 结合分子的竞争。这些测定法将有助于对 EGFR 抑制剂的竞争特性进行排序。
