High-yield expression and purification of p18 form of Bax as an MBP-fusion protein.
Bax is a pro-apoptotic member of the Bcl-2 family of proteins, which is present in the cytosol of various types of cells in full-length form (p21 Bax). During apoptosis, the N-terminal truncated version of Bax (p18 Bax) is often formed via cleavage of the p21 Bax by the calcium-dependent enzyme, calpain. p18 Bax is a membrane protein found primarily in the mitochondrial fraction of apoptotic cells. Although noticeable amounts of p18 Bax appear relatively late in apoptosis, it may still play a role in the apoptotic cascade. The role of p18 Bax in the apoptotic cascade, particularly, in the release of cytochrome c from mitochondria has not been studied. The goal of this study was to produce reasonable amounts of p18 Bax and study its effect on isolated mitochondria. The expression and purification of membrane proteins such as p18 Bax represents a substantial challenge due to insolubility. We report here that the apoptotic form of Bax, p18 Bax, is highly soluble in the absence of detergents upon fusion with maltose-binding protein (MBP). We describe a scheme for expression and simple metal-affinity based purification of MBP-p18Bax. The MBP-p18Bax triggers the release of cytochrome c from isolated mitochondria in a concentration-dependent, Bcl-2-sensitive manner. The MBP tag of the MBP-p18Bax can be cleaved off with 3C protease to produce pure p18 Bax, although the solubility of p18 Bax becomes very limited. The highly soluble, MBP-fused form of p18 Bax provides a convenient tool to study this apoptotic form of Bax protein.
Eliseev
Roman
R
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. roman_eliseev@urmc.rochester.edu
Alexandrov
Andrey
A
Gunter
Thomas
T
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D058619:Maltose-Binding Proteins; D008930:Mitochondria, Liver / Q000201:enzymology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D002352:Carrier Proteins; D017931:DNA Primers; D058619:Maltose-Binding Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c