High-yield expression and purification of soluble forms of the anti- apoptotic Bcl-x sub(L) and Bcl-2 as TolAIII-fusion proteins

A method is presented to produce large amounts of Bcl-2 and Bcl-x sub(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 sub(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 sub(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 >12 mg of Bcl-x sub(L) Delta C or >6 mg of Bcl-2(2) Delta C per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x sub(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-x sub(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x sub(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 TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.