Proteins fulfil a wide variety of essential functions in the cell. Recombinant protein production in the Gram-negative bacterium Escherichia coli (E. coli) facilitates structural and functional studies of proteins and it has been instrumental in biotechnology for the manufacturing of e.g. many protein-based drugs. However, to obtain sufficient amounts of active recombinant protein is not always a trivial task. The production of proteins that reside in membranes is limited by their complex biogenesis and their hydrophobic nature. Consequently, despite the importance of membrane proteins in health and disease (approx. 70% of today’s drugs act on membrane proteins), structures of only a very small fraction of the existing membrane proteins have yet been solved. Many soluble proteins are also difficult to produce, e.g. those containing disulfide bonds (e.g. antibody fragments and most hormones). Disulfide bond-containing proteins have to be produced in the periplasm of E. coli to be able to fold properly. To reach the periplasm, these proteins have to be ‘secreted’ across the inner membrane, which makes that also their biogenesis is complex. The aim of my PhD studies has been to improve E. coli-based production of recombinant membrane and secretory proteins. I have found that (i) the previously developed Lemo21(DE3) protein production strain can be used to set the expression intensity of a gene encoding a membrane protein such that the protein is optimally produced in the cytoplasmic membrane without causing any notable stress. Also, (ii) membrane protein production using the Lemo21(DE3) strain can be improved and simplified using carefully optimized culturing and induction conditions, demonstrated by the development of the ‘MemStar recipe’. Furthermore, I found that (iii) when using the standard BL21(DE3)/pT7 expression system for the production of membrane and secretory proteins, omitting the inducer IPTG leads to drastically improved yields as compared to when IPTG is added, owing to a lower initial target protein production rate. In the fourth study, I found that (iv) when using the PrhaBAD promoter for expression of the target gene, protein accumulation rates appear to be mostly unaffected by the inducer concentration. Using a strain-engineering approach, PrhaBAD-based protein production rates could be made constant and rhamnose concentration dependent. This dramatically improved production yields of both membrane and secretory proteins, using only very low amounts of inducer. Taken together, in accordance with previous studies, lowering production rates is an efficient strategy to increase production yields of both membrane- and secretory proteins. This is mostly due to alleviating saturation of the machinery involved in the biogenesis of these proteins. Finally, I also conducted a study (v) where, in both E. coli and Salmonella, I orchestrated the production of two membrane proteins (one that mediates the production of antigens on the surface of Gram-negative bacteria and another that makes defined pore-structures in the Gram-negative bacterial cell envelope) for the development of a safe (non-living) vaccine platform.

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).