Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone.
Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384-11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.
Donnelly
M I
MI
Environmental Research Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Stevens
P W
PW
Stols
L
L
Su
S X
SX
Tollaksen
S
S
Giometti
C
C
Joachimiak
A
A
D000818:Animals; D018835:Chaperonin 10 / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D000431:Ethanol / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D021382:Protein Sorting Signals / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000096:biosynthesis; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D018835:Chaperonin 10; D021382:Protein Sorting Signals; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D000431:Ethanol