Evidence map›Paper›PMID 39798888›Full record

ArticleProtein expression and purification2025

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

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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Article in Protein expression and purification, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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1 · What the graph read from it

What it found

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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

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3 · Its place in the literature

Who cites it

0 citing papers in PubMed.

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4 · The record

Corrections and comments

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5 · Who and what money

Authors and funding

8 authors.

Johanna M SchaferProtein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
Christine S MuliProtein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
Rehab A HeikalProtein 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.
Marzena A DybaBiophysics Resource, Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
Sergey G TarasovBiophysics Resource, Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
Margaret M StrattonMolecular 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.
Eric R StrieterDepartment 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.
Kylie J WaltersProtein 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.

Funding

Myosin VI: a new ubiquitin receptorZIABC011627 · NCI · DIVISION OF BASIC SCIENCES - NCI · PI WALTERS, KYLIE · 2015 to 2025
$4.6M
Unraveling the molecular events driven by CaMKII in Ca2+-coupled cellsR35GM145376 · NIGMS · UNIVERSITY OF MASSACHUSETTS AMHERST · PI Margaret M Stratton · 2022 to 2026
$2.1M
Defining the Function of Proteasomal DeubiquitinasesR35GM149532 · NIGMS · UNIVERSITY OF MASSACHUSETTS AMHERST · PI ERIC Robert STRIETER · 2023 to 2026
$1.5M
Intramural NIH HHS ZIA BC011627NIGMS NIH HHS R35 GM145376NIGMS NIH HHS R35 GM149532
6 · The paper itself

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.

Indexed as

Ubiquitin-Protein LigasesBiophysical PhenomenaCell LineEnzyme ActivationGene ExpressionHumansPlasmidsUBE3A protein, humanUbiquitin-Protein LigasesE6APMammalian cell cultureProteasomeRecombinant expressionUBE3AUbiquitin

Identifiers

PMID39798888
PMCPMC11779574

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.