Purification and biochemical properties of soluble recombinant human Bax.
Bax is a member of the Bcl-2 protein family with proapoptotic properties. The proteins of this family contain three highly conserved regions termed BH1, BH2, and BH3 as well as a hydrophobic COOH-terminal domain, which is responsible for the membrane attachment of the proteins. We have expressed human Bax truncated of the 20 amino acid COOH-terminal hydrophobic domain to obtain large amounts of soluble protein suitable for biochemical and structural studies. The truncated protein was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The GST-Bax fusion protein was bound to glutathione-Sepharose, and Bax was released by thrombin cleavage and further purified by sequential chromatography on heparin-Sepharose and DEAE-Sepharose. The purified protein was present in solution as a heptamer and multimers of the heptamer complex. Limited tryptic digestion cleaved the protein in the region preceding the BH3 domain and produced a specific stable protein fragment of 15 kDa. Phosphorylation has been proposed as a possible regulatory mechanism of the bcl-2 proteins. The Bax protein was an in vitro substrate for specific serine/threonine protein kinases.
Lewis
S
S
Geneva Biomedical Research Institute, Glaxo Wellcome R & D S.A., Plan-les-Ouates, Switzerland.
Bethell
S S
SS
Patel
S
S
Martinou
J C
JC
Antonsson
B
B
D000595:Amino Acid Sequence; D017209:Apoptosis; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein