Membrane and secretory protein production in Escherichia coli

Abstract: Escherichia coli (E. coli) is the most widely used bacterium for the production of recombinant proteins. However, the production of proteins in its cytoplasmic membrane or periplasm is challenging. Therefore, the aim of this doctoral thesis was to develop setups for enhancing the production of membrane and secretory proteins in E. coli. Production of membrane and secretory proteins in E. coli requires precise tuning of protein production rates in order to avoid the deleterious saturation of the secretion apparatus. Here, we engineered two tunable protein production setups. We engineered the pReX expression vector, which is a simplified version of the setup used for creating the T7 RNA polymerase-based Lemo21(DE3) strain, and a setup based on the use of the rhamnose promoter in a Δrha strain background. Both setups can be used to enhance membrane and secretory protein production yields. Based on our current knowledge, it is challenging to predict which signal peptide should be utilized to produce a recombinant protein in the periplasm. Using the tunable rhamnose promoter-based setup, we developed a combined screen involving different signal peptides and varying production rates. This enables the identification of an optimal signal peptide and production rate combination for the periplasmic production of a recombinant protein. Next, proteome analysis was used to examine cells producing a recombinant protein in the periplasm, when using, an optimal signal peptide ­and protein production rate combination. Interestingly, the proteome analysis showed that cells had increased their protein translocation capacity, i.e., they had adapted. Finally, we investigated the co-translational folding of the periplasmic, disulfide bond-containing, E. coli protein alkaline phosphatase. Using Force Profile Analysis, it was shown that co-translationally translocated PhoA folds via at least two disulfide-stabilized folding intermediates. This thesis highlights the importance of fine-tuning membrane and secretory protein production rates to enhance their production yields, selecting the most optimal signal peptide for the periplasmic production of a protein, using combinatorial protein production screening approaches, studying the effects of recombinant protein production on the cell and developing experimental systems to monitor periplasmic protein folding.