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Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3)

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Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). / Schlegel, Susan; Löfblom, John; Lee, Chiara; Hjelm, Anna; Klepsch, Mirjam; Strous, Marc; Drew, David; Slotboom, Dirk Jan; de Gier, Jan-Willem.

In: J MOL BIOL, Vol. 423, No. 4, 02.11.2012, p. 648-59.

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

Harvard

Schlegel, S, Löfblom, J, Lee, C, Hjelm, A, Klepsch, M, Strous, M, Drew, D, Slotboom, DJ & de Gier, J-W 2012, 'Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3)', J MOL BIOL, vol. 423, no. 4, pp. 648-59. https://doi.org/10.1016/j.jmb.2012.07.019

APA

Schlegel, S., Löfblom, J., Lee, C., Hjelm, A., Klepsch, M., Strous, M., Drew, D., Slotboom, D. J., & de Gier, J-W. (2012). Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J MOL BIOL, 423(4), 648-59. https://doi.org/10.1016/j.jmb.2012.07.019

Vancouver

Schlegel S, Löfblom J, Lee C, Hjelm A, Klepsch M, Strous M et al. Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J MOL BIOL. 2012 Nov 2;423(4):648-59. https://doi.org/10.1016/j.jmb.2012.07.019

Bibtex

@article{0ba866f6a30949f592d6b317ac2df37b,
title = "Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3)",
abstract = "Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.",
keywords = "Bacteriophage T7, DNA-Directed RNA Polymerases, Escherichia coli, Gene Expression, Gene Expression Regulation, Bacterial, Membrane Proteins, N-Acetylmuramoyl-L-alanine Amidase, Promoter Regions, Genetic, Recombinant Fusion Proteins, Viral Proteins, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't",
author = "Susan Schlegel and John L{\"o}fblom and Chiara Lee and Anna Hjelm and Mirjam Klepsch and Marc Strous and David Drew and Slotboom, {Dirk Jan} and {de Gier}, Jan-Willem",
note = "Copyright {\textcopyright} 2012 Elsevier Ltd. All rights reserved.",
year = "2012",
month = nov,
day = "2",
doi = "10.1016/j.jmb.2012.07.019",
language = "English",
volume = "423",
pages = "648--59",
journal = "J MOL BIOL",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "4",

}

RIS

TY - JOUR

T1 - Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3)

AU - Schlegel, Susan

AU - Löfblom, John

AU - Lee, Chiara

AU - Hjelm, Anna

AU - Klepsch, Mirjam

AU - Strous, Marc

AU - Drew, David

AU - Slotboom, Dirk Jan

AU - de Gier, Jan-Willem

N1 - Copyright © 2012 Elsevier Ltd. All rights reserved.

PY - 2012/11/2

Y1 - 2012/11/2

N2 - Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.

AB - Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.

KW - Bacteriophage T7

KW - DNA-Directed RNA Polymerases

KW - Escherichia coli

KW - Gene Expression

KW - Gene Expression Regulation, Bacterial

KW - Membrane Proteins

KW - N-Acetylmuramoyl-L-alanine Amidase

KW - Promoter Regions, Genetic

KW - Recombinant Fusion Proteins

KW - Viral Proteins

KW - Journal Article

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

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

U2 - 10.1016/j.jmb.2012.07.019

DO - 10.1016/j.jmb.2012.07.019

M3 - SCORING: Journal article

C2 - 22858868

VL - 423

SP - 648

EP - 659

JO - J MOL BIOL

JF - J MOL BIOL

SN - 0022-2836

IS - 4

ER -