Purification and biochemical properties of soluble recombinant human Bax.
Protein Expr Purif
; 13(1): 120-6, 1998 Jun.
Article
em En
| MEDLINE
| ID: mdl-9631524
ABSTRACT
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.
Buscar no Google
Base de dados:
MEDLINE
Assunto principal:
Proteínas Proto-Oncogênicas
Limite:
Humans
Idioma:
En
Ano de publicação:
1998
Tipo de documento:
Article