We will describe the development of first- and second-generation E. coli SuptoxD and SuptoxR, two specialized strains for high-level recombinant membrane protein (MP) production. These engineered strains can: (1) suppress the toxicity that frequently accompanies MP overexpression, thus enabling enhanced levels of final bacterial biomass; and (2) markedly increase the cellular accumulation of membrane-embedded protein. Combined, these two positive effects result in dramatically enhanced volumetric yields for various prokaryotic and eukaryotic recombinant MPs.

Membrane proteins play important roles in cell signaling, the influx and efflux of nutrients and metabolites, and many are potential drug targets. For their structural and functional analysis, high yields of correctly folded and modified protein are needed. Multidrug resistance protein 4 (MRP4/ABCC4) is a multi-substrate primary transporter that has been expressed using both recombinant baculovirus infection of Sf9 insect cells, and baculovirus-mediated transduction of Freestyle HEK cells.

Membrane proteins (MPs), important drug targets, display crucial functions in organisms but their study remains difficult (hydrophobicity and low abundance). Their overexpression is mandatory for structural and functional characterizations but this strategy could encounter obstacles. Using the NICE system and the L. lactis strains, in the last twenty years, more than 100 MPs were expressed allowing either their functional and/or structural characterization. Recently, one eukaryotic membrane protein was expressed at a high-expression yield and allowed the formation of intracellular vesicles. In conclusion, L. lactis represents an interesting system for expression of MPs.

The baculovirus expression vector system leads to high yields of Virus-like-Particles (VLPs). Yet, it´s time-intensive, inflexible in regard of protein ratios, its quality can be hampered by low cell viability, and last but not least, purification is challenging due to simultaneously produced baculoviral particles. The here-presented alternative of baculovirus-free VLP production in insect cells avoids the pitfalls of BEVS and produces VLPs in high quality and quantity for antibody development.

Recombinant proteins can be artificially self-assembled in the form of functional structures with promising applications in the field of drug delivery via nanostructured protein-only drugs. The antigenic RBD domain of the SARS-CoV-2 spike was produced in several expression systems such as bacteria, insect, and mammalian cells, to be used as a model to investigate the influence of the protein source in the production of nanoparticles and secretory microparticles. Both structures were generated in all cases but with different biophysical properties indicating thus that the selected expression system is a relevant factor for protein assembly into supramolecular structured and functional materials.

Animal venoms contain a plethora of biologically active toxins that have the potential to revolutionize antivenom development. However, producing functional toxins with the correct disulfide pattern is challenging. Our approach to engineering co-chaperon systems in Escherichia coli and leveraging Pichia pastoris' secretory capacity to produce natural and designed toxins. Our optimized strategies allow efficient toxin folding and purification, enabling downstream biotechnological applications, such as directed biotinylation for phage display.

There is a need for prophylactic SARS-CoV-2 blocking agents that are invulnerable to mutational viral variation and economical to produce. TriSb92 is a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein. TriSb92 potently neutralizes SARS-CoV-2, including the latest Omicron subvariants. In mice, intranasal administration of TriSb92 as early as 8 hours before but also 4 hours after SARS-CoV-2 challenge can protect from infection. Triggering of a conformational shift in the spike trimer was revealed as the inhibitory mechanism. TriSb92 could be useful as a nasal spray for protecting from SARS-CoV-2 infection.

Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. We investigated the recombinant production and purification of PpL and characterized the product in detail. In my talk I present a comprehensive roadmap for the production of the versatile protein PpL in E. coli.