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 t he E. coli cochaperone GroES reduced Bax's toxicity significantly and allow ed good expression. The peptide, which was attached to the N-terminus, incl uded the amino acid sequence of the mobile loop of GroES that has been demo nstrated to interact with the chaperonin, GroEL. Under normal growth condit ions, expression of this construct was still toxic, but generated a small a mount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular ch aperones GroEL and DnaK, toxicity was reduced and good overexpression occur red. 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. (C) 2001 Academic Press.