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.