Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA.
Department of Biology, Appalachian State University, Boone, NC, USA.
Methods Mol Biol. 2025;2848:269-297. doi: 10.1007/978-1-0716-4087-6_17.
Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.
转录因子之间的动态相互作用控制着基因表达的变化，这些变化介导了伴随损伤反应和再生的细胞状态的变化。转录因子通常作为必需的二聚体发挥作用，其活性通常受到翻译后修饰的调节。这些关键且通常是短暂的相互作用不易被传统的蛋白质-蛋白质相互作用研究方法检测到。本章讨论了一种融合蛋白的设计和验证，该融合蛋白涉及一种转录因子与邻近标记连接酶 APEX2 的连接。在这种技术中，无论相互作用的时间如何，在感兴趣的转录因子的小半径内，蛋白质都被生物素化。这里我们讨论了确保转录因子邻近标记工具正常工作所需的验证，以及用于生物素化蛋白质的样品制备，以便对假定的蛋白质相互作用物进行质谱分析。
