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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wagner S, Bader ML, Drew D et al (2006) Rationalizing membrane protein overexpression. Trends Biotechnol 24:364–371
Hjelm A, Schlegel S, Baumgarten T et al (2013) Optimizing E. coli-based membrane protein production using Lemo21(DE3) and GFP-fusions. Methods Mol Biol 1033:381–400
Schlegel S, Hjelm A, Baumgarten T et al (2014) Bacterial-based membrane protein production. Biochim Biophys Acta 1843:1739–1749
Samuelson JC (2011) Recent developments in difficult protein expression: a guide to E. coli strains, promoters, and relevant host mutations. Methods Mol Biol 705:195–209
Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130
Wagner S, Klepsch MM, Schlegel S et al (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105:14371–14376
Giacalone MJ, Gentile AM, Lovitt BT et al (2006) Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. Biotechniques 40:355–364
Wagner S, Baars L, Ytterberg AJ et al (2007) Consequences of membrane protein overexpression in Escherichia coli. Mol Cell Proteomics 6:1527–1550
Schlegel S, Löfblom J, Lee C et al (2012) Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J Mol Biol 423:648–659
Drew DE, von Heijne G, Nordlund P et al (2001) Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett 507:220–224
Drew D, Slotboom DJ, Friso G et al (2005) A scalable, GFP-based pipeline for membrane protein overexpression screening and purification. Protein Sci 14:2011–2017
Drew D, Lerch M, Kunji E et al (2006) Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods 3:303–313
Geertsma ER, Groeneveld M, Slotboom DJ et al (2008) Quality control of overexpressed membrane proteins. Proc Natl Acad Sci U S A 105:5722–5727
Feilmeier BJ, Iseminger G, Schroeder D et al (2000) Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J Bacteriol 182:4068–4076
Drew D, Sjöstrand D, Nilsson J et al (2002) Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis. Proc Natl Acad Sci U S A 99:2690–2695
Nugent T, Jones DT (2009) Transmembrane protein topology prediction using support vector machines. BMC Bioinformatics 10:159
Peters C, Tsirigos KD, Shu N et al (2016) Improved topology prediction using the terminal hydrophobic helices rule. Bioinformatics 32:1158–1162
Tsirigos KD, Peters C, Shu N et al (2015) The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Res 43:W401–W407
Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14:673–681
Luirink J, Yu Z, Wagner S et al (2012) Biogenesis of inner membrane proteins in Escherichia coli. Biochim Biophys Acta 1817:965–976
Hsieh JM, Besserer GM, Madej MG et al (2010) Bridging the gap: a GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini. Protein Sci 19:868–880
Löw C, Jegerschöld C, Kovermann M et al (2012) Optimisation of over-expression in E. coli and biophysical characterisation of human membrane protein synaptogyrin 1. PLoS One 7:e38244
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234
Shaner NC, Lambert GG, Chammas A et al (2013) A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10:407–409
Acknowledgments
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Kuipers, G. et al. (2017). Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) or pReX and GFP-Fusions. In: Burgess-Brown, N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6887-9_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6887-9_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6885-5
Online ISBN: 978-1-4939-6887-9
eBook Packages: Springer Protocols