RECOMBINANT MOUSE BCL-2((1-203)) - 2 DOMAINS CONNECTED BY A LONG PROTEASE-SENSITIVE LINKER

Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apop totic activity but whose mechanism of action is poorly understood. The purpose of this paper was to obtain large amounts of soluble Bcl-2 pr otein for structural and functional studies. Mouse Bcl-2((1-203)) (mis sing the COOH-terminal hydrophobic tail) was produced in bacterial inc lusion bodies, solubilized in guanidine, and refolded by dialysis. The resulting protein was monomeric in nondenaturing solution and was act ive 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 proteolys is experiments identified a region between the BH3 and BH4 homology do mains of Bcl-2((1-203)) which was extremely susceptible to digestion b y several common proteases, but not by a cell extract known to contain CPP-32-like (interleukin-1 beta-converting enzyme family) protease ac tivity. The protease-sensitive sites were located within a 50-residue stretch that contained most of the nonconserved and proline residues o f Bcl-2((1-203)). Trypsin-cleaved Bcl-2((1-203)) eluted in the same po sition as the undigested protein on gel filtration in nondenaturing so lution, 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-sensitiv e linker and that its structure is similar to that of Bcl-x(L), which has been determined by x-ray and NMR analysis.