Optimizing E. coli-based membrane protein production using Lemo21(DE3) or pReX and GFP-Fusions

Grietje Kuipers; Markus Peschke; Nurzian Bernsel Ismail; Anna Hjelm; Susan Schlegel; David Vikström; Joen Luirink; Jan Willem de Gier

doi:10.1007/978-1-4939-6887-9_7

Optimizing E. coli-based membrane protein production using Lemo21(DE3) or pReX and GFP-Fusions

Grietje Kuipers, Markus Peschke, Nurzian Bernsel Ismail, Anna Hjelm, Susan Schlegel, David Vikström, Joen Luirink, Jan Willem de Gier^*

^*Corresponding author for this work

Research output: Chapter in Book / Report / Conference proceeding › Chapter › Academic › peer-review

Abstract

Optimizing the conditions for the production of membrane proteins in E. coli is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain or the pReX T7-based expression vector with membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the optimization of membrane protein production yields. Both Lemo21(DE3) and pReX allow precise regulation of expression intensities of genes encoding membrane proteins, which is critical to identify the optimal production condition for a membrane protein. The use of GFP-fusions allows direct monitoring and visualization of membrane proteins at any stage during the production optimization process.

Original language	English
Title of host publication	Heterologous Gene Expression in E.coli
Subtitle of host publication	Methods and Protocols
Editors	Nicola A. Burgess-Brown
Publisher	Humana Press Inc
Pages	109-126
Number of pages	18
ISBN (Electronic)	9781493968879
ISBN (Print)	9781493968855, 9781493983285
DOIs	https://doi.org/10.1007/978-1-4939-6887-9_7
Publication status	Published - 2017

Publication series

Name	Methods in Molecular Biology
Publisher	Humana Press
Volume	1586
ISSN (Print)	1064-3745

Funding

This work was supported by grants from the Swedish Research Council, the Carl Tryggers Stiftelse, the Marianne and Marcus Wallenberg Foundation, NIH grant 5R01GM081827-03, and the SSF supported Center for Biomembrane Research to JWdG and the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/under REA grant agreement n°607072 (NK and MK) and a SystemsX Transition Postdoc Fellowship (SusS).

Funders	Funder number
SSF supported Center for Biomembrane Research
National Institutes of Health	5R01GM081827-03
Seventh Framework Programme
FP7 People: Marie-Curie Actions
Research Executive Agency	607072
Carl Tryggers Stiftelse för Vetenskaplig Forskning
Vetenskapsrådet
Marcus och Amalia Wallenbergs minnesfond

Keywords

E. coli
Fluorescence
GFP
Lemo21(DE3)
Membrane protein
pReX
Production

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10.1007/978-1-4939-6887-9_7

https://www.researchgate.net/publication/256332716_Optimizing_E_coli-Based_Membrane_Protein_Production_Using_Lemo21DE3_and_GFP-Fusions

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Kuipers, G., Peschke, M., Ismail, N. B., Hjelm, A., Schlegel, S., Vikström, D., Luirink, J., & de Gier, J. W. (2017). Optimizing E. coli-based membrane protein production using Lemo21(DE3) or pReX and GFP-Fusions. In N. A. Burgess-Brown (Ed.), Heterologous Gene Expression in E.coli: Methods and Protocols (pp. 109-126). (Methods in Molecular Biology; Vol. 1586). Humana Press Inc. https://doi.org/10.1007/978-1-4939-6887-9_7

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Kuipers, G, Peschke, M, Ismail, NB, Hjelm, A, Schlegel, S, Vikström, D, Luirink, J & de Gier, JW 2017, Optimizing E. coli-based membrane protein production using Lemo21(DE3) or pReX and GFP-Fusions. in NA Burgess-Brown (ed.), Heterologous Gene Expression in E.coli: Methods and Protocols. Methods in Molecular Biology, vol. 1586, Humana Press Inc, pp. 109-126. https://doi.org/10.1007/978-1-4939-6887-9_7

Optimizing E. coli-based membrane protein production using Lemo21(DE3) or pReX and GFP-Fusions. / Kuipers, Grietje; Peschke, Markus; Ismail, Nurzian Bernsel et al.
Heterologous Gene Expression in E.coli: Methods and Protocols. ed. / Nicola A. Burgess-Brown. Humana Press Inc, 2017. p. 109-126 (Methods in Molecular Biology; Vol. 1586).

Research output: Chapter in Book / Report / Conference proceeding › Chapter › Academic › peer-review

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AU - Kuipers, Grietje

AU - Peschke, Markus

AU - Ismail, Nurzian Bernsel

AU - Hjelm, Anna

AU - Schlegel, Susan

AU - Vikström, David

AU - Luirink, Joen

AU - de Gier, Jan Willem

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AB - Optimizing the conditions for the production of membrane proteins in E. coli is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain or the pReX T7-based expression vector with membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the optimization of membrane protein production yields. Both Lemo21(DE3) and pReX allow precise regulation of expression intensities of genes encoding membrane proteins, which is critical to identify the optimal production condition for a membrane protein. The use of GFP-fusions allows direct monitoring and visualization of membrane proteins at any stage during the production optimization process.

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KW - Fluorescence

KW - GFP

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Kuipers G, Peschke M, Ismail NB, Hjelm A, Schlegel S, Vikström D et al. Optimizing E. coli-based membrane protein production using Lemo21(DE3) or pReX and GFP-Fusions. In Burgess-Brown NA, editor, Heterologous Gene Expression in E.coli: Methods and Protocols. Humana Press Inc. 2017. p. 109-126. (Methods in Molecular Biology). Epub 2017 May 4. doi: 10.1007/978-1-4939-6887-9_7

Optimizing E. coli-based membrane protein production using Lemo21(DE3) or pReX and GFP-Fusions

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