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 segment located at the carboxyl terminus. The bcl-2 protein seems to function in different subcellular compartments, as evidenced by several biochemical and ultrastructural studies. The present study was performed to purify bcl-2 protein in significant quantities necessary for structural and functional studies. For this purpose, the bcl-2 gene was over-expressed in either baculovirus system or lymphocytes. Initially, attempts were undertaken to purify bcl-2 protein using conventional methods such as ion exchange or gel filtration chromatography. During these purification attempts we determined that bcl-2 protein is highly hydrophobic and prone to aggregation as might be expected for an integral 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 immunoaffinity columns using a monoclonal antibody developed against a synthetic peptide chosen from residues 61-76 of the amino acid sequence of human bcl-2. The antibody was either coupled to CNBr-activated Sepharose 4B or cross-linked into protein A-Sepharose by dimethylpimelimidate dihydrochloride. Cellular extract equivalent to 10(8) bcl-2-overexpressing insect cells or lymphocytes was applied to immunoaffinity columns. Approximately 500 micrograms purified bcl-2 protein could be recovered as estimated by silver staining and immunoblotting. Furthermore, purified bcl-2 protein was electroporated into Pre-B lymphocytes which do not express this protein in sufficient quantity to delay the onset of glucocorticoid-induced apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Haldar
S
S
Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Jena
N
N
DuBois
G C
GC
Takayama
S
S
Reed
J C
JC
Fu
S S
SS
Croce
C M
CM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000166:cytology; D016367:Baculoviridae; D018274:Electroporation; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D018411:Spodoptera
D005938:Glucocorticoids; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins