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Optimized mammalian expression system for the ubiquitin E3 ligase E6AP/UBE3A

  1. Author:
    Schafer,Annie
    Muli,Christine
    Heikal,Rehab
    Dyba,Marzena
    Tarasov,Sergey
    Stratton, Margaret M
    Strieter, Eric R
    Walters,Kylie

  2. Author Address

    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, Amherst, MA 01003, USA., Biophysics Resource, Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA., Molecular and Cellular Biology Graduate Program, College of Natural Sciences, University of Massachusetts Amherst, Amherst, MA 01003, USA., Department of Chemistry, University of Massachusetts Amherst, Amherst, MA 01003, USA; Molecular and Cellular Biology Graduate Program, College of Natural Sciences, University of Massachusetts Amherst, Amherst, MA 01003, USA., Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address: kylie.walters@nih.gov.,
    1. Year: 2025
    2. Date: Jan 09
    3. Epub Date: 2025 01 09
  2. Journal: Protein Expression and Purification
    1. Pages: 106661
  4. Type of Article: Article
  5. Article Number: 106661
  1. 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. Copyright © 2025. Published by Elsevier Inc.

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External Sources

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  2. DOI: 10.1016/j.pep.2025.106661
  3. PMID: 39798888
  4. PII : S1046-5928(25)00003-8

Library Notes

  1. Fiscal Year: FY2024-2025
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