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Bax to the future – A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2019 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 158, p. 20-26Article in journal (Refereed) Published
Abstract [en]

Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.

Place, publisher, year, edition, pages
Elsevier, 2019. Vol. 158, p. 20-26
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-156983DOI: 10.1016/j.pep.2019.02.004ISI: 000462805100004PubMedID: 30738180Scopus ID: 2-s2.0-85061543221OAI: oai:DiVA.org:umu-156983DiVA, id: diva2:1293193
Funder
Swedish Research CouncilSwedish Cancer SocietyThe Kempe FoundationsKnut and Alice Wallenberg FoundationAvailable from: 2019-03-04 Created: 2019-03-04 Last updated: 2019-04-15Bibliographically approved

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Dingeldein, Artur P. G.Lindberg, Mikael J.Ådén, JörgenGröbner, Gerhard

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Dingeldein, Artur P. G.Lindberg, Mikael J.Ådén, JörgenGröbner, Gerhard
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Protein Expression and Purification
Biochemistry and Molecular Biology

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