High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-x(L) and Bcl-2 as TolAIII-fusion proteins.
A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.
Nedelkina
Svetlana
S
Université Louis Pasteur/CNRS LC3-UMR 7177, Institut de Chimie Strasbourg, Faculté de Chimie, Institut le Bel, 4 rue Blaise Pascal, F-67070 Strasbourg, France.
Gokce
Isa
I
Ridley
Helen
H
Weckerle
Céline
C
Magnin
Thierry
T
Vallette
François
F
Pattus
Franc
F
Lakey
Jeremy H
JH
Bechinger
Burkhard
B
D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification; D008930:Mitochondria, Liver / Q000201:enzymology; D010957:Plasmids; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D051020:bcl-X Protein / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D029968:Escherichia coli Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein