| First Author | Vance BA | Year | 1996 |
| Journal | J Biol Chem | Volume | 271 |
| Issue | 48 | Pages | 30811-5 |
| PubMed ID | 8940062 | Mgi Jnum | J:36809 |
| Mgi Id | MGI:84222 | Doi | 10.1074/jbc.271.48.30811 |
| Citation | Vance BA, et al. (1996) Recombinant mouse Bcl-2(1-203). Two domains connected by a long protease-sensitive linker. J Biol Chem 271(48):30811-5 |
| abstractText | 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. |
