Expression and purification of full-length human Bax alpha.
Bax is a proapoptotic ion channel forming protein of the Bcl-2 family. In cells the protein is found in the cytosol and in the mitochondria membrane where it presumably is involved during apoptosis in disruption of the mitochondrial membrane potential and release of cytochrome c. The protein has a hydrophobic domain at the C-terminus, which renders it a limited solubility. Thus, all studies on recombinant Bax has so far been performed on C-terminal truncated protein. We have expressed and purified the full-length human Bax alpha. The protein was expressed with a His tag at the N-terminus and purified by affinity chromatography on Ni-NTA-agarose followed by ion-exchange chromatography on Q-Sepharose. The protein was more than 98% pure on SDS-PAGE and in the presence of 1% (w/v) octyl glucoside it could be concentrated up to 0.5 mg/ml. Full-length Bax was 25-fold more efficient, compared to C-terminal truncated Bax, in forming ion channels and trigger carboxyfluorescein release from liposomes.
Montessuit
S
S
Serono Pharmaceutical Research Institute, Ares-Serono International S.A., 14 ch. des Aulx, Plan-les-Ouates, CH-1228, Switzerland.
Mazzei
G
G
Magnenat
E
E
Antonsson
B
B
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D008081:Liposomes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007473:Ion Channels; D008081:Liposomes; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; C024098:6-carboxyfluorescein