Lookup NU author(s): Isa Gokce,
Dr Helen Waller,
Professor Jeremy Lakey
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A method is presented to produce large amounts of Bcl-2 and Bcl-xL, two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-xL proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10 mg of more than 90% pure TolAIII-Bcl-xLΔC and TolAIII-Bcl-2(2)ΔC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing >12 mg of Bcl-xLΔC or >6 mg of Bcl-2(2)ΔC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-xLΔC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)ΔC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an α-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-xL proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-xLΔC and Bcl-2(2)ΔC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins. © 2008 Elsevier Inc. All rights reserved.
Author(s): Nedelkina S, Gokce I, Ridley H, Weckerle C, Magnin T, Vallette F, Pattus F, Lakey JH, Bechinger B
Publication type: Article
Publication status: Published
Journal: Protein Expression and Purification
ISSN (print): 1046-5928
ISSN (electronic): 1096-0279
Publisher: Academic Press
PubMed id: 18522870
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