PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons

Related Articles
Retrospective: Producing Proteins the PSI:Biology Way
July 2015
Immunity: Conformational Capture
June 2014
Coaxing Rare Codons
February 2014
Caught in the Act
December 2013
Membrane Proteome: Tuning Membrane Protein Expression
December 2012
Membrane Proteome: Every Protein Has Its Tag
November 2012
Insert Here
October 2012
S is for solubility
June 2012
Overexpressed problems
February 2012
Gentle membrane protein extraction
January 2012
No more labeled lipids
May 2011
Five good reasons to use single protein production for membrane proteins
January 2010
One system to express them all
November 2009
Crystallizing glycoproteins
September 2009
GFP to the rescue
July 2009
Solve your solubility problems
June 2009
Tips for crystallizing membrane proteins
June 2009
Speedy cloning and mutagenesis for structural genomics
January 2009
Tuning membrane protein overexpression
October 2008

Technology Topics Protein Expression

Overexpressed problems

SBKB [doi:10.1038/sbkb.2011.64]
Technical Highlight - February 2012
Short description: An analysis of the effects of protein overexpression gives insight into the difficulties of preparing intramembrane proteins for crystallization.

Electron micrographs of E. coli wild-type cells (top) and cells overexpressing an IMP (middle) or soluble protein (bottom). Figure courtesy of Francesca Gubellini.

Despite the recent publication of several breakthrough, high-resolution crystal structures of intramembrane proteins (IMPs), the production of IMPs for structural determination remains challenging. Protein toxicity is a frequent roadblock, widely believed to be related to the limited capacity of the host cell to properly fold and accommodate overexpressed IMPs. However, some IMPs can be highly expressed while others are toxic even at low levels; thus, these limitations cannot be generalized.

To understand the basis of overexpressed IMP toxicity, Hunt and colleagues (PSI NYCOMPS) have now extensively analyzed the response of Escherichia coli to the overexpression of six proteins (including four IMPs). Two of the most toxic IMPs, E. coli GlpT and Helicobacter pylori HP1206, caused similar global gene expression changes, and overexpression caused a more elongated morphology compared to control cells. However, cells overexpressing soluble proteins shared most of these changes, although to a lesser extent than those overexpressing IMPs. Surprisingly, cells overexpressing either soluble proteins or IMPs did not present the standard stationary-phase transcriptional response, nor were the heat-shock response and/or envelope-stress systems upregulated, and no major changes were seen in the expression of genes encoding metabolic enzymes. The absence of these expected responses indicates that there is no inherent deterrent to IMP production in E. coli.

Instead of a general barrier, the results suggest that the toxicities due to IMP overexpression may be related to the behavior of the target proteins themselves. This was supported by the finding that the toxicity of E. coli GlpT overexpression was related to its glycerol transport activity and by the discovery of a spontaneous mutation in E. coli MsbA that reduced its toxicity and its transport activity, as well as spontaneous missense mutations in HP1206. Given that the toxicity of a target IMP likely depends on its activity, the authors suggest that using alternative strains that are less susceptible to the effects of a specific protein may result in improved expression of IMPs. They also suggest that selection of activity-reducing mutations during overexpression may be an unappreciated source of heterogeneity impeding IMP crystallization.

Steve Mason


  1. F. Gubellini et al. Physiological response to membrane protein overexpression in E. coli.
    Mol. Cell. Proteomics. 10, M111.007930 (2011). doi:10.1074/mcp.M111.007930

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health