Purification and biochemical properties of soluble recombinant human Bax

Protein Expression and Purification
S LewisB Antonsson

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.

References

Jun 21, 1985·Science·Y TsujimotoC M Croce
Nov 4, 1966·Science·J W Saunders
Nov 1, 1994·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·C BornerJ C Martinou
Mar 1, 1995·Trends in Genetics : TIG·S J Korsmeyer
May 9, 1995·Proceedings of the National Academy of Sciences of the United States of America·S HaldarC M Croce
Jan 1, 1993·Molecular Carcinogenesis·T J McDonnell
Feb 2, 1996·The Journal of Biological Chemistry·C Y Chen, D V Faller
Apr 12, 1996·The Journal of Biological Chemistry·J J Hunter, T G Parslow
Feb 1, 1996·Current Opinion in Genetics & Development·S N Farrow, R Brown
Nov 29, 1996·The Journal of Biological Chemistry·B A VanceD M Segal
Jan 23, 1997·Nature·A J MinnC B Thompson
Apr 15, 1997·Proceedings of the National Academy of Sciences of the United States of America·Y T HsuR J Youle
May 13, 1997·Proceedings of the National Academy of Sciences of the United States of America·S L SchendelJ C Reed
Jul 18, 1997·Science·B AntonssonJ C Martinou

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Citations

Mar 18, 2005·Molecular Biology of the Cell·Laurent M DejeanKathleen W Kinnally
Mar 7, 2001·The EMBO Journal·K ThressS Kornbluth
Jul 20, 2001·Journal of Virology·R Sundararajan, E White
Mar 23, 2017·Biochimica Et Biophysica Acta. Biomembranes·Lilit SimonyanStéphen Manon
Sep 17, 2005·Journal of Bioenergetics and Biomembranes·Sonia Martinez-CaballeroKathleen W Kinnally
Mar 15, 2006·Critical Reviews in Clinical Laboratory Sciences·Hellinida Thomadaki, Andreas Scorilas
Jan 5, 1999·The Journal of Biological Chemistry·P B PoommipanitZ N Oltvai
Feb 10, 2004·The Journal of Biological Chemistry·Shyra J GardaiPeter M Henson
Mar 2, 1999·Protein Expression and Purification·S MontessuitB Antonsson
Dec 21, 2002·Molecular Immunology·Christoph Borner

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BCL-2 Family Proteins

BLC-2 family proteins are a group that share the same homologous BH domain. They play many different roles including pro-survival signals, mitochondria-mediated apoptosis and removal or damaged cells. They are often regulated by phosphorylation, affecting their catalytic activity. Here is the latest research on BCL-2 family proteins.