Membrane proteins (MPs) play pivotal roles in a variety of cellular functions, many of which are essential to survival . Despite their physiological importance, the study of MPs is lagging due largely to the fact that they are difficult to express in a functional form and at levels needed for biochemical and structural studies. Escherichia coli is a popular host for MP overexpression due to its well understood genetics and rapid growth . However, as with other expression systems, high-level MP production is typically toxic to the cell and the yields of biologically active material are generally poor.
Based on the observation that the overexpression of MPs in E. coli leads to their aggregation and to reduced levels of host membrane and secretory proteins , it has been suggested that MP toxicity is due to the overloading of the Sec-dependent translocation machinery which handles both the post-translational export of secretory proteins and the co-translational insertion of most inner MP into the lipid bilayer . Recently, we have shown that eliminating the signal recognition particle (SRP) -Trigger Factor (TF) competition by making use of TF-deficient (Δtig) expression strains can significantly improve the accumulation of functional MP in the bacterial inner membrane . A more common approach, however, has been to make use of E. coli C41(DE3) and C43(DE3) , two BL21(DE3) derivatives containing a mutation in the lacUV5 promoter that decreases the production of chromosomally-encoded T7 RNA polymerase, and hence the transcription rate of MP genes cloned downstream of the T7 promoter . The same net effect can be achieved by making use of plasmids co-expressing T7Lys (e.g. pLemo, pLysS, pLysE) [7, 8], a T7 RNA polymerase inhibitor that reduces the overall transcription rates of genes placed under T7 promoter control.
In addition to being too strong for MP expression, a drawback of the T7 promoter is its lack of tight repression in the absence of inducer, which may be problematic since even basal levels of MPs can be toxic to the cell [6, 8]. The arabinose-inducible P
BAD promoter of the araBAD operon  is a moderately strong and tightly repressed promoter that has been successfully used for producing MPs in E. coli [5, 10–12]. P
BAD is negatively regulated by AraC when no L-arabinose is present in the medium, and it is positively regulated by both AraC in the presence of arabinose and the cyclic AMP (cAMP) receptor protein (CRP, also known as CAP, catabolite gene activator protein) in the absence of glucose . When loaded with cAMP, the CRP homodimer binds to a consensus sequence located upstream of more than 100 E. coli promoters . This binding favors transcription initiation  both by bending the DNA [16, 17] and by recruiting RNA polymerase to facilitate its interaction with the core promoter .
Here, we describe the isolation of a single nucleotide transversion in one of the CRP interaction domains of the P
BAD promoter that reduces the transcription rates by about 70% and can improve the yields of polytopic MPs, particularly when combined with a chaperone pathway reprogramming strategy relying on the use of Trigger Factor (TF) deficient mutants .