Schafer Johanna M, Muli Christine S, Heikal Rehab A, Dyba Marzena A, Tarasov Sergey G, Stratton Margaret M, Strieter Eric R, Walters Kylie J
Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA; Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA.
Protein Expr Purif. 2025 Apr;228:106661. doi: 10.1016/j.pep.2025.106661. Epub 2025 Jan 9.
E6AP/UBE3A is the founding member of the HECT (Homologous to the E6-AP Carboxyl Terminus) ubiquitin E3 ligase family, which add ubiquitin post-translationally to protein substrates. E6AP has been structurally defined in complex with human papillomavirus (HPV) oncoprotein E6 and its gain-of-function substrate tumor suppressor p53; however, there is currently no report of E6AP being expressed and purified from mammalian cells, as studies to date have isolated E6AP from E. coli or insect cells. Here, we report an optimized protocol for purifying E6AP from suspended Human Embryonic Kidney (HEK) cells. Biophysical characterization by Q-TOF confirmed sample purity while mass photometry indicated that purified E6AP forms a monomer-oligomer mixture. E6AP produced by this method is catalytically active and amenable to structural characterization by cryo-electron microscopy (cryo-EM), biochemical assays, and small molecule screening campaigns.
E6AP/UBE3A是HECT（与E6-AP羧基末端同源）泛素E3连接酶家族的创始成员，该家族在翻译后将泛素添加到蛋白质底物上。E6AP已在与人类乳头瘤病毒（HPV）癌蛋白E6及其功能获得性底物肿瘤抑制因子p53的复合物中进行了结构定义；然而，目前尚无从哺乳动物细胞中表达和纯化E6AP的报道，因为迄今为止的研究都是从大肠杆菌或昆虫细胞中分离E6AP。在此，我们报告了一种从悬浮的人胚肾（HEK）细胞中纯化E6AP的优化方案。通过Q-TOF进行的生物物理表征证实了样品的纯度，而质量光度法表明纯化的E6AP形成了单体-寡聚体混合物。通过这种方法产生的E6AP具有催化活性，适用于通过冷冻电子显微镜（cryo-EM）、生化分析和小分子筛选活动进行结构表征。
