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

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.