The Gram-negative bacterium E. coli is the most widely used host for the production of recombinant proteins. Disulfide bond containing recombinant proteins are usually produced in the periplasm of E. coli since in this compartment of the cell - in contrast to the cytoplasm - disulfide bond formation is promoted. To reach the periplasm recombinant proteins have to be translocated across the cytoplasmic membrane by the protein translocation machinery. To obtain sufficient yields of active recombinant protein in the periplasm is always challenging. The Ph.D. studies have aimed at developing strategies to enhance recombinant protein production yields in the periplasm, to better understand what happens when a protein is produced in the periplasm, and to shed light on the protein folding process in the periplasm. It has been shown that evolving translation initiation regions (TIRs) can enhance periplasmic protein production yields of a variety of proteins. Furthermore, it has been shown that the protein translocation machinery can adapt for enhanced periplasmic recombinant protein production. Force profile analysis was used to study co-translational folding of the periplasmic disulfide-bond containing protein alkaline phosphatase (PhoA) in the periplasm. It was shown that folding-induced forces can be transmitted via the nascent chain from the periplasm to the peptidyl transferase center in the ribosome and that PhoA appears to fold co- translationally via disulfide-stabilized folding intermediates. Finally, the S. pneumoniae neuraminidases NanA, NanB, and NanC were produced in E. coli and subsequently isolated. The activity of these neuraminidases was monitored at different pH as well as their oligomeric state was studied.

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.