Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL, USA.
Methods Mol Biol. 2023;2701:209-227. doi: 10.1007/978-1-0716-3373-1_14.
Isolation of a protein/complex is important for its biochemical and structural characterization with mechanistic insights. TAP (tandem affinity purification) strategy allows rapid isolation of cellular proteins/complexes with a high level of purity. This methodology involves an immuno-affinity-based purification followed by a conformation-based isolation to obtain a highly homogeneous protein/complex. Here, we describe the TAP-mediated isolation of endogenous FACT (facilitates chromatin transcription; a heterodimer), an essential histone chaperone associated with BER (base excision repair). However, it is not clearly understood how FACT regulates BER. Such knowledge would advance our understanding of BER with implications in disease pathogenesis, since BER is an evolutionarily conserved process that is linked to various diseases including ageing, neurodegenerative disorders, and cancers. Using isolated FACT by TAP methodology, one can study the mechanisms of action of FACT in BER. Further, isolated FACT can be used for studies in other DNA transactions such as transcription and replication, as FACT is involved in these processes. Furthermore, TAP-mediated isolation strategy can be combined with mass spectrometry to identify the protein interaction partners of FACT.
蛋白质/复合物的分离对于其生化和结构特征的研究具有重要意义，并有助于深入了解其作用机制。TAP（串联亲和纯化）策略可用于快速分离具有高纯度的细胞内蛋白质/复合物。这种方法涉及基于免疫亲和的纯化，然后是基于构象的分离，以获得高度同质的蛋白质/复合物。在这里，我们描述了 TAP 介导的内源性 FACT（促进染色质转录；异二聚体）的分离，FACT 是一种与 BER（碱基切除修复）相关的必需组蛋白伴侣。然而，FACT 如何调节 BER 尚不清楚。这方面的知识将有助于我们理解 BER 在疾病发病机制中的作用，因为 BER 是一种进化上保守的过程，与各种疾病有关，包括衰老、神经退行性疾病和癌症。使用 TAP 方法分离的 FACT，人们可以研究 FACT 在 BER 中的作用机制。此外，分离的 FACT 可用于转录和复制等其他 DNA 转导研究，因为 FACT 参与这些过程。此外，TAP 介导的分离策略可以与质谱结合使用，以鉴定 FACT 的蛋白质相互作用伙伴。
