Fiche publication
Date publication
août 2008
Auteurs
Membres identifiés du Cancéropôle Est :
Pr BECHINGER Burkhard
Tous les auteurs :
Nedelkina S, Gokce I, Ridley H, Weckerle C, Magnin T, Vallette F, Pattus F, Lakey JH, Bechinger B
Lien Pubmed
Résumé
A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), 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-x(L) 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 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC 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.
Référence
Protein Expr Purif. 2008 Aug;60(2):214-20
