Research ExplorerPublicationsTuning Escherichia coli for membrane protein overexpression

Tuning Escherichia coli for membrane protein overexpression

Standard

Tuning Escherichia coli for membrane protein overexpression. / Wagner, Samuel; Klepsch, Mirjam M; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J; Slotboom, Dirk J; Persson, Jan O; de Gier, Jan-Willem.

In: P NATL ACAD SCI USA, Vol. 105, No. 38, 23.09.2008, p. 14371-6.

Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review

Harvard

Wagner, S, Klepsch, MM, Schlegel, S, Appel, A, Draheim, R, Tarry, M, Högbom, M, van Wijk, KJ, Slotboom, DJ, Persson, JO & de Gier, J-W 2008, 'Tuning Escherichia coli for membrane protein overexpression', P NATL ACAD SCI USA, vol. 105, no. 38, pp. 14371-6. https://doi.org/10.1073/pnas.0804090105

APA

Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Högbom, M., van Wijk, K. J., Slotboom, D. J., Persson, J. O., & de Gier, J-W. (2008). Tuning Escherichia coli for membrane protein overexpression. P NATL ACAD SCI USA, 105(38), 14371-6. https://doi.org/10.1073/pnas.0804090105

Vancouver

Wagner S, Klepsch MM, Schlegel S, Appel A, Draheim R, Tarry M et al. Tuning Escherichia coli for membrane protein overexpression. P NATL ACAD SCI USA. 2008 Sep 23;105(38):14371-6. https://doi.org/10.1073/pnas.0804090105

Bibtex

@article{092c893cf2d7403fa9af1adec10a4021,
title = "Tuning Escherichia coli for membrane protein overexpression",
abstract = "A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used {"}Walker strains{"} C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.",
keywords = "DNA-Directed RNA Polymerases, Escherichia coli, Escherichia coli Proteins, Gene Expression, Gene Expression Regulation, Bacterial, Kinetics, Lac Operon, Membrane Fusion Proteins, Membrane Proteins, Membrane Transport Proteins, Promoter Regions, Genetic, Proteome, Viral Proteins, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't",
author = "Samuel Wagner and Klepsch, {Mirjam M} and Susan Schlegel and Ansgar Appel and Roger Draheim and Michael Tarry and Martin H{\"o}gbom and {van Wijk}, {Klaas J} and Slotboom, {Dirk J} and Persson, {Jan O} and {de Gier}, Jan-Willem",
year = "2008",
month = sep,
day = "23",
doi = "10.1073/pnas.0804090105",
language = "English",
volume = "105",
pages = "14371--6",
journal = "P NATL ACAD SCI USA",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "38",

}

RIS

TY - JOUR

T1 - Tuning Escherichia coli for membrane protein overexpression

AU - Wagner, Samuel

AU - Klepsch, Mirjam M

AU - Schlegel, Susan

AU - Appel, Ansgar

AU - Draheim, Roger

AU - Tarry, Michael

AU - Högbom, Martin

AU - van Wijk, Klaas J

AU - Slotboom, Dirk J

AU - Persson, Jan O

AU - de Gier, Jan-Willem

PY - 2008/9/23

Y1 - 2008/9/23

N2 - A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

AB - A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

KW - DNA-Directed RNA Polymerases

KW - Escherichia coli

KW - Escherichia coli Proteins

KW - Gene Expression

KW - Gene Expression Regulation, Bacterial

KW - Kinetics

KW - Lac Operon

KW - Membrane Fusion Proteins

KW - Membrane Proteins

KW - Membrane Transport Proteins

KW - Promoter Regions, Genetic

KW - Proteome

KW - Viral Proteins

KW - Journal Article

KW - Research Support, N.I.H., Extramural

KW - Research Support, Non-U.S. Gov't

U2 - 10.1073/pnas.0804090105

DO - 10.1073/pnas.0804090105

M3 - SCORING: Journal article

C2 - 18796603

VL - 105

SP - 14371

EP - 14376

JO - P NATL ACAD SCI USA

JF - P NATL ACAD SCI USA

SN - 0027-8424

IS - 38

ER -