PURIFICATION AND CHARACTERIZATION OF THE BCL-2 PROTEIN

The oncogene product bcl-2 functions as a repressor of programmed cell death and is a 26-kDa protein with a single predicted transmembrane s egment located at the carboxyl terminus. The bcl-2 protein seems to fu nction in different subcellular compartments, as evidenced by several biochemical and ultrastructural studies. The present study was perform ed to purify bcl-2 protein in significant quantities necessary for str uctural and functional studies. For this purpose, the bcl-2 gene was o verexpressed in either baculovirus system or lymphocytes. Initially, a ttempts were undertaken to purify bcl-2 protein using conventional met hods such as ion exchange or gel filtration chromatography. During the se purification attempts we determined that bcl-2 protein is highly hy drophobic and prone to aggregation as might be expected for an integra l membrane protein. By ion exchange and gel filtration chromatography, this protein could be partially purified. In order to purify bcl-2 to apparent homogeneity and avoid the aggregation problem, we prepared i mmunoaffinity columns using a monoclonal antibody developed against a synthetic peptide chosen from residues 61-76 of the amino acid sequenc e of human bcl-2. The antibody was either coupled to CNBr-activated Se pharose 4B or cross-linked into protein A-Sepharose by dimethylpimelim idate dihydrochloride. Cellular extract equivalent to 10(8) bcl-2-over expressing insect cells or lymphocytes was applied to immunoaffinity c olumns. Approximately 500 mu g purified bcl-2 protein could be recover ed as estimated by silver staining and immunoblotting. Furthermore, pu rified bcl-2 protein was electroporated into Pre-B lymphocytes which d o not express this protein in sufficient quantity to delay the onset o f glucocorticoid-induced apoptosis. Following electroporation of homog eneously pure bcl-2 protein, the cells were found to prolong cell surv ival in response to glucocorticoids. (C) 1994 Academic Press, Inc.