Abstract
BackgroundThe overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored.ResultsIn E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF and AcrB), which are also recalcitrant contaminants crystallizing even at low concentration. Engineered expression hosts presented an enhanced fitness and improved folding of target membrane proteins, which correlated with an altered membrane fluidity. We demonstrated the scope of this approach by overproducing several membrane proteins (4 different ABC transporters, YidC and SecYEG).ConclusionsIn summary, E. coli membrane engineering unprecedentedly increases the quality and yield of membrane protein preparations. This strategy opens a new field for membrane protein production, complementary to gene expression tuning.
Highlights
All sequenced genomes contain about 20–30% of genes encoding membrane proteins (MP) [1]
The comparison to the genome of the parental strain C41(DE3) (GenBank ID: Overexpression of ABC transporters in E. coli C41(DE3), C41(DE3)∆(ompF) and C41(DE3)∆(ompF‐acrAB) Our aim was not to perform a precise quantification of protein production but rather to check that recombinant MP production was not impaired in our mutants
For five ABC transporters, i.e. hemolysin B (HlyB)∆CLD, HlyB, ABC2, ABC3, and ABC4, production levels appeared improve by using the OmpF-depleted strain
Summary
All sequenced genomes contain about 20–30% of genes encoding membrane proteins (MP) [1] They are still underrepresented in biochemical and structural studies, despite their undeniable physiological and medical importance—about 70% of all drug targets are membrane proteins. We postulated that the deletion of both, OmpF and AcrAB, should be advantageous for two reasons It should modify the membrane composition and its tolerance to MP overproduction; and second, we expected an improvement in the purity of the recombinant MP. Most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored
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