Department of BioEngineering and BioMedical Sciences, Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Biomolecular Science & Engineering (BMSE) program, University of California in Santa Barbara, Santa Barbara, CA, USA.
Methods Mol Biol. 2023;2701:199-207. doi: 10.1007/978-1-0716-3373-1_13.
Affinity pulldown is a powerful technique to discover novel interaction partners and verify a predicted physical association between two or more proteins. Pulldown assays capture a target protein fused with an affinity tag and analyze the complexed proteins. Here, we detail methods of pulldown assays for two high-affinity peptide fusion tags, Flag tag (DYKDDDDK) and hexahistidine tag (6xHis), to study protein-protein interactions of human NEIL1 glycosylase and the checkpoint protein complex RAD9-RAD1-HUS1 (9-1-1). We uncover unique interactions between 9-1-1 and NEIL1, which suggest a possible inhibitory role of the disordered, phosphorylated C-terminal region of RAD9 in regulating NEIL1 activity in base excision repair through lack of physical association of 9-1-1 and NEIL1.
亲和下拉是一种强大的技术，可用于发现新的相互作用伙伴，并验证两个或更多蛋白质之间预测的物理关联。下拉测定法捕获与亲和标签融合的靶蛋白，并分析复合蛋白。在这里，我们详细介绍了两种高亲和力肽融合标签（Flag 标签[DYKDDDDK]和六组氨酸标签[6xHis]）的下拉测定法，以研究人 NEIL1 糖基化酶与检查点蛋白复合物 RAD9-RAD1-HUS1（9-1-1）的蛋白-蛋白相互作用。我们揭示了 9-1-1 和 NEIL1 之间独特的相互作用，这表明 RAD9 无规则、磷酸化的 C 末端区域可能通过缺乏 9-1-1 和 NEIL1 的物理关联来抑制碱基切除修复中 NEIL1 的活性。
