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 10 mg of more than 90% pure TolAIII-Bcl-x(L)Delta C and TolAIII-Bcl-2(2)Delta C proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12mg of Bcl-X-L Delta C or > 6mg of Bcl-2(2)Delta C per liter of E coli cell culture with a purity of more than 95%. Whereas Bcl-X-L Delta C is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII-Bcl-2(2)Delta C 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-XL proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified BCl-X-L Delta C and Bcl-2(2)Delta C 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 TbIAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins. (c) 2008 Elsevier Inc. All rights reserved.