Recombinant mouse Bcl-2(1-203). Two domains connected by a long protease-sensitive linker.

The Journal of Biological Chemistry
B A VanceD M Segal

Abstract

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 ...Continue Reading

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Citations

Jun 4, 2014·Chemistry and Physics of Lipids·Juan C Gómez-Fernández
Nov 5, 1999·International Immunology·M M TomaykoM P Cancro
Dec 31, 2003·The Journal of Biological Chemistry·Christine M Sorenson
Feb 20, 1999·The Journal of Biological Chemistry·A ConstantinouS G Clarkson
Jun 19, 1998·Protein Expression and Purification·S LewisB Antonsson

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