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Title: Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone.

Abstract

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.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
National Institute of Standards and Technology (NIST); USDOE Office of Science (SC); OUS
OSTI Identifier:
943061
Report Number(s):
ANL/ER/JA-37188
TRN: US201002%%567
DOE Contract Number:  
DE-AC02-06CH11357
Resource Type:
Journal Article
Journal Name:
Protein Expr. Purif.
Additional Journal Information:
Journal Volume: 22; Journal Issue: 3 ; Aug. 2001
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; AMINO ACID SEQUENCE; APOPTOSIS; ELECTROPHORESIS; ESCHERICHIA COLI; ETHANOL; GELS; GROWTH; HUMAN POPULATIONS; LYSIS; PEPTIDES; PROTEINS; SIGNALS; TOXICITY; VECTORS

Citation Formats

Donnelly, M I, Stols, L, Stevens, P W, Su, S X, Tollaksen, S, Giometti, C S, and Joachimiak, A. Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone.. United States: N. p., 2001. Web. doi:10.1006/prep.2001.1442.
Donnelly, M I, Stols, L, Stevens, P W, Su, S X, Tollaksen, S, Giometti, C S, & Joachimiak, A. Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone.. United States. https://doi.org/10.1006/prep.2001.1442
Donnelly, M I, Stols, L, Stevens, P W, Su, S X, Tollaksen, S, Giometti, C S, and Joachimiak, A. 2001. "Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone.". United States. https://doi.org/10.1006/prep.2001.1442.
@article{osti_943061,
title = {Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone.},
author = {Donnelly, M I and Stols, L and Stevens, P W and Su, S X and Tollaksen, S and Giometti, C S and Joachimiak, A},
abstractNote = {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.},
doi = {10.1006/prep.2001.1442},
url = {https://www.osti.gov/biblio/943061}, journal = {Protein Expr. Purif.},
number = 3 ; Aug. 2001,
volume = 22,
place = {United States},
year = {Wed Aug 01 00:00:00 EDT 2001},
month = {Wed Aug 01 00:00:00 EDT 2001}
}