| Autor: |
Lewis, Shareta, Bethell, Susanne S., Patel, Sahil, Martinou, Jean-Claude, Antonsson, Bruno |
| Zdroj: |
Protein Expression and Purification; June 1998, Vol. 13 Issue: 1 p120-126, 7p |
| Abstrakt: |
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 glutathioneS-transferase (GST) fusion protein inEscherichia 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 anin vitrosubstrate for specific serine/threonine protein kinases. |
| Databáze: |
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