Change search
ReferencesLink to record
Permanent link

Direct link
Expression and purification of full-length anti-apoptotic Bcl-2 using cell-free protein synthesis
Swedish NMR Centre, University of Gothenburg, Gothenburg, Sweden.
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Gerhard Gröbner)
Swedish NMR Centre, University of Gothenburg, Gothenburg, Sweden.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2011 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 77, no 2, 220-223 p.Article in journal (Refereed) Published
Abstract [en]

The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the α-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family.

Place, publisher, year, edition, pages
Elsevier Inc , 2011. Vol. 77, no 2, 220-223 p.
Keyword [en]
Apoptosis, Bcl-2, Cell-free protein synthesis, Full-length protein, Optimized gene
National Category
Organic Chemistry
URN: urn:nbn:se:umu:diva-40403DOI: 10.1016/j.pep.2011.02.003PubMedID: 21315822OAI: diva2:402949
Available from: 2011-03-10 Created: 2011-02-22 Last updated: 2012-11-08Bibliographically approved
In thesis
1. Insight into the mitochondrial apoptotic pathway: The interplay of the pro-apoptotic Bax protein with oxidized phospholipids and its counterplayer, the pro-survival Bcl-2 protein
Open this publication in new window or tab >>Insight into the mitochondrial apoptotic pathway: The interplay of the pro-apoptotic Bax protein with oxidized phospholipids and its counterplayer, the pro-survival Bcl-2 protein
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis plays a crucial role in multicellular organisms by preserving tissue homeostasis and removing harmful cells. The anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and the pro-apoptotic Bcl-2-associated X protein (Bax) act as major regulators of the mitochondrial apoptotic pathway. Activation of Bax via stress signals causes its translocation to the mitochondrial outer membrane (MOM). There, Bax forms homo-oligomeric pores, leading to the release of apoptogenic factors, caspase activation and ultimately cell death. However, the underlying mechanism for the recruitment and pore forming activity of Bax is still not elucidated. Nevertheless, the mitochondrial membrane system seems to play an active and crucial role, presumably being directly involved in the onset of the mitochondrial apoptosis. Since the formation of reactive oxygen species (ROS) is a common stress signal and one of the hallmarks of the mitochondrial apoptosis, direct damage can occur to these membranes by the generation of oxidized phospholipids (OxPls), whose presence can crucially influence the pro-apoptotic action of Bax there. To better understand the impact of OxPls on membranes as well as their potential role in the mitochondrial apoptotic process, defined OxPl species were incorporated into phospholipid vesicles and studied with various biophysical techniques. Differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy were used to gain insight into changes in membrane properties in the presence of OxPls. In addition to circular dichroism (CD) spectroscopy, DSC and solid state NMR were furthermore performed to elucidate the impact of OxPls on Bax-membrane interactions. The occurrence of OxPls gave rise to dramatic changes in membrane organization and dynamics, manifested as lateral phase separation into OxPl-rich and -poor domains and modified hydration at the membrane interface. The presence of OxPls also had a great impact on the interaction between Bax and mitochondria-mimicking vesicles, strongly promoting the association of the protein with the membrane.

At the MOM, Bax is believed to be inhibited by Bcl-2. How this inhibition occurs is still a mystery due to the lack of biophysical information on Bcl-2, in particular on the full-length protein variant. Since Bcl-2 is also one of the main culprits in the progression of various forms of cancer, knowledge of the structural and mechanistic properties of the full-length protein is essential for a fundamental understanding of its function at a molecular level. To this end, a method for the production of full-length Bcl-2 was developed. By performing cell-free protein synthesis, preparative amounts of the protein were obtained, which enabled a biophysical characterization of the putative interaction between Bax and Bcl-2 using CD and fluorescence spectroscopy. A protocol for the reconstitution of Bcl-2 into proteoliposomes was also developed, promising for future studies of the full-length protein in its native membrane environment; a prerequisite to fully understand its pro-survival functions as well as providing crucial information for the design of novel anti-cancer drugs.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2012. 62 p.
apoptosis, Bax, Bcl-2, CD, cell-free protein synthesis, DSC, membrane, mitochondria, oxidized phospholipids, reconstitution, solid state NMR
National Category
Research subject
biological chemistry
urn:nbn:se:umu:diva-61290 (URN)978-91-7459-490-4 (ISBN)
Public defence
2012-11-30, KBC-huset, KB3B1, Umeå universitet, Umeå, 10:00 (English)
Available from: 2012-11-09 Created: 2012-11-07 Last updated: 2012-11-08Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Wallgren, MarcusGröbner, Gerhard
By organisation
Department of Chemistry
In the same journal
Protein Expression and Purification
Organic Chemistry

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 962 hits
ReferencesLink to record
Permanent link

Direct link