Optimized isolation of enzymatically active ubiquitin E3 ligase E6AP/UBE3A from mammalian cells

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-01-09 DOI:10.1016/j.pep.2025.106661

Johanna M. Schafer , Christine S. Muli , Rehab A. Heikal , Marzena A. Dyba , Sergey G. Tarasov , Margaret M. Stratton , Eric R. Strieter , Kylie J. Walters

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引用次数: 0

Abstract

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.

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优化了泛素E3连接酶E6AP/UBE3A的哺乳动物表达体系。

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与人乳头瘤病毒（HPV）癌蛋白E6及其功能获得性底物肿瘤抑制因子p53在结构上已被确定；然而，目前还没有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是从大肠杆菌或昆虫细胞中分离出E6AP。在这里，我们报告了从悬浮的人胚胎肾（HEK）细胞中纯化E6AP的优化方案。Q-TOF生物物理表征证实了样品纯度，质谱分析表明纯化后的E6AP形成了单体-低聚物混合物。通过这种方法生产的E6AP具有催化活性，并且可以通过低温电子显微镜（cryo-EM），生化分析和小分子筛选活动进行结构表征。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.