Recombinant Mouse Bcl-2(1-203)

作者: Barbara A. Vance , Charles M. Zacharchuk , David M. Segal

DOI: 10.1074/JBC.271.48.30811

关键词: ProteasesProteasePeptide sequenceProtein foldingIntegral membrane proteinBiochemistryInclusion bodiesLinkerGuanidineBiologyCell biologyMolecular biology

摘要: Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apoptotic activity but whose mechanism of action poorly understood. The purpose this paper was to obtain large amounts soluble for structural and functional studies. Mouse Bcl-2(1-203) (missing the COOH-terminal hydrophobic tail) produced in bacterial inclusion bodies, solubilized guanidine, refolded by dialysis. resulting monomeric nondenaturing solution active protecting mouse T hybridoma cells from glucocorticoid-induced apoptosis. Refolded showed no tendency homodimerize gel filtration or analytical ultracentrifugation. Limited proteolysis experiments identified region between BH3 BH4 homology domains which extremely susceptible digestion several common proteases, not cell extract known contain CPP-32-like (interleukin-1beta-converting enzyme family) protease activity. protease-sensitive sites were located within 50-residue stretch that contained most nonconserved proline residues Bcl-2(1-203). Trypsin-cleaved eluted same position as undigested on solution, indicating two portions molecule connected associate stably noncovalently. properties suggest it consists noncovalently associated long linker its structure similar Bcl-xL, has been determined x-ray NMR analysis.

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