Recombinant mouse Bcl-2(1-203). Two domains connected by a long protease-sensitive linker.
Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apoptotic activity but whose mechanism of action is poorly understood. The purpose of this paper was to obtain large amounts of soluble Bcl-2 protein for structural and functional studies. Mouse Bcl-2(1-203) (missing the COOH-terminal hydrophobic tail) was produced in bacterial inclusion bodies, solubilized in guanidine, and refolded by dialysis. The resulting protein was monomeric in nondenaturing solution and was active in protecting mouse T hybridoma cells from glucocorticoid-induced apoptosis. Refolded Bcl-2(1-203) showed no tendency to homodimerize by gel filtration or analytical ultracentrifugation. Limited proteolysis experiments identified a region between the BH3 and BH4 homology domains of Bcl-2(1-203) which was extremely susceptible to digestion by several common proteases, but not by a cell extract known to contain CPP-32-like (interleukin-1beta-converting enzyme family) protease activity. The protease-sensitive sites were located within a 50-residue stretch that contained most of the nonconserved and proline residues of Bcl-2(1-203). Trypsin-cleaved Bcl-2(1-203) eluted in the same position as the undigested protein on gel filtration in nondenaturing solution, indicating that the two portions of the molecule connected by the protease-sensitive region associate stably and noncovalently. The solution properties of Bcl-2(1-203) suggest that it consists of two noncovalently associated domains connected by a long protease-sensitive linker and that its structure is similar to that of Bcl-xL, which has been determined by x-ray and NMR analysis.
Vance
B A
BA
Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892-1360, USA.
Zacharchuk
C M
CM
Segal
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017510:Protein Folding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry*; D011994:Recombinant Proteins; D014461:Ultracentrifugation
D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D010450:Endopeptidases