Proteins fulfill essential functions in living cells. To produce sufficient amounts of a protein is essential to study the structure and function of a protein, or to use it for medical purposes. Escherichia coli is a Gram-negative bacterium that is widely used for recombinant protein production. The aim of my PhD studies was to enhance membrane and secretory protein production yields using E. coli. The T7-based protein production system BL21(DE3)/pET was mainly used in my studies. BL21(DE3) contains a strong IPTG-inducible lacUV5 promoter governing the expression of the t7rnap gene encoding the T7RNAP on its chromosome. The target gene is under control of the T7 promoter on the pET plasmid. T7RNAP specifically recognizes the T7 promoter and transcribes the target gene more efficiently than the bacterial RNAP. Unfortunately, the biogenesis machinery for membrane and secretory proteins is usually saturated by the high protein production intensity when the BL21(DE3)/pET system is induced with IPTG, thereby negatively affecting protein production yields. In the first study, we found that when using the BL21(DE3)/pET system omitting the inducer IPTG improved membrane and secretory protein production yields. In previous studies, Lemo21(DE3) was developed to facilitate the production of membrane and secretory proteins. Lemo21(DE3) contains the pLemo plasmid in which the gene encoding the inhibitor of T7RNAP, T7 lysozyme, is under the control of the rhaBAD promoter. The activity of T7RNAP is regulated by synthesizing different levels of T7 lysozyme by adding different amounts of rhamnose. Thus, the production intensity can be modulated such that the biogenesis machinery of membrane and secretory proteins is not saturated upon IPTG induction. In the second study, we combined the key elements from both the pLemo and pET vectors to create the pReX (Regulated eXpression) plasmid to facilitate the use of helper plasmids encoding e.g., chaperones when it is necessary. In the third study, we used the rhaBAD promoter to direct the production of membrane and secretory proteins in a rhamnose metabolism and active uptake deficient strain. The protein production rate can be truly tuned in this setup. Therefore, the production of membrane and secretory proteins can be enhanced by using the right amount of rhamnose in the culture medium. BL21(DE3) contains the λDE3 prophage that carries the t7rnap gene under the control of the lacUV5 promoter. The λDE3 prophage is thought to be stably inserted into the chromosome, but the lytic cycle of the prophage can still be induced by the SOS response inducing antibiotic mitomycin C in the mitomycin C-based bacteriophage test. In the fourth study, we engineered BL21T7 by deleting in BL21(DE3) lysis related genes from the prophage. BL21T7 has similar recombinant protein production characteristics as its ancestor BL21(DE3).

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.