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

Johanna M Schafer¹, Christine S Muli¹, Rehab A Heikal², Marzena A Dyba³, Sergey G Tarasov³, Margaret M Stratton⁴, Eric R Strieter⁵, Kylie J Walters⁶

Affiliations

¹ Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
² 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.
³ Biophysics Resource, Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
⁴ Molecular and Cellular Biology Graduate Program, College of Natural Sciences, University of Massachusetts, Amherst, MA, 01003, USA; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, 01003, USA.
⁵ Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA; Molecular and Cellular Biology Graduate Program, College of Natural Sciences, University of Massachusetts, Amherst, MA, 01003, USA.
⁶ 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.

PMID: 39798888
PMCID: PMC11779574 (available on 2026-04-01)
DOI: 10.1016/j.pep.2025.106661

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

Johanna M Schafer et al. Protein Expr Purif. 2025 Apr.

. 2025 Apr:228:106661.

doi: 10.1016/j.pep.2025.106661. Epub 2025 Jan 9.

Authors

Johanna M Schafer¹, Christine S Muli¹, Rehab A Heikal², Marzena A Dyba³, Sergey G Tarasov³, Margaret M Stratton⁴, Eric R Strieter⁵, Kylie J Walters⁶

Affiliations

¹ Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
² 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.
³ Biophysics Resource, Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
⁴ Molecular and Cellular Biology Graduate Program, College of Natural Sciences, University of Massachusetts, Amherst, MA, 01003, USA; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, 01003, USA.
⁵ Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA; Molecular and Cellular Biology Graduate Program, College of Natural Sciences, University of Massachusetts, Amherst, MA, 01003, USA.
⁶ 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.

PMID: 39798888
PMCID: PMC11779574 (available on 2026-04-01)
DOI: 10.1016/j.pep.2025.106661

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.

Keywords: E6AP; Mammalian cell culture; Proteasome; Recombinant expression; UBE3A; Ubiquitin.

Published by Elsevier Inc.

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References

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

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

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