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Abstract：
Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca(2+) release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.
摘要：
低聚通常是蛋白质实现其特定细胞功能的结构要求。例如,ryanodine受体（RyR）的四聚化对于形成功能性Ca（2）释放通道孔是必需的。这里,我们描述了评估蛋白质自缔合的详细方案，包括酵母双杂交（Y2H），共免疫沉淀(co-IP)和化学交联测定。在Y2H系统中，在共表达GAL4诱饵和测试蛋白的靶融合体中,通过β-半乳糖苷酶测定检测蛋白自身相互作用。使用在哺乳动物HEK293细胞中共表达的测试蛋白的HA-和cMyc-标记的融合物,通过co-IP进一步评估蛋白自身相互作用。在HEK293细胞中表达后,通过交联和SDS-PAGE分析检查蛋白质同源寡聚体的精确化学计量。使用这些不同但互补的技术，我们一直观察到RyRN末端结构域的自缔合，并证明了其形成四聚体的内在能力。这些方法可应用于其他哺乳动物内在膜蛋白的蛋白质-蛋白质相互作用和同源寡聚化研究。
