A toxin can be described as a foreign substance that inflicts damages to living organisms. Naturally occurring proteinaceous toxins can derive from bacteria, fungi, plants, animal venoms and even viruses. Identifying the toxins’ underlying mechanisms of action has been a major research interest in order to develop inhibitors against their effects. Nonetheless, various findings have sparked the use of toxic moieties for the medical benefit resulting in treatment options as for example for cancers. To gain novel insights into the structure and function of a toxin, the toxin itself has to be synthesized. In vivo production can involve high laboratory safety standards as well as a low total protein amount since the toxin might harm the overexpressing cell. An alternative to circumvent these drawbacks is cell-free protein synthesis (CFPS). Within this doctoral thesis CFPS was established as a platform technology for the production and application of proteinaceous toxins in diagnostic and medical fields. As a first step, various bacterial toxins were analyzed. The mechanisms of action of the tripartite pore-forming toxins (PFT) Hbl and Nhe were studied by hemolytic activity assays, cell-based toxicity assessments and electrophysiological recordings. Next, the PFT CytK was analyzed to identify its potential as a biological nanopore that can be used as a diagnostic tool. This thesis identified the CytK1 variant as a candidate for a nanopore development. Further, two AB5 toxins, namely the cholera toxin and the heat-labile enterotoxin, were modified. These modified toxins could be fluorescently labeled and tested for their functional activity. These data are a proof-of-concept for using CPFS for intracellular trafficking of toxins and coupling of payloads for drug delivery. In a second step, a targeted toxin combining the plant-derived toxin Dianthin and the epidermal growth factor (EGF) was assessed for its potency as a potential cancer therapeutic. The medical benefit of this Dianthin-EGF targeted toxin was demonstrated on human squamous cell carcinoma samples. 0.1 nM Dianthin-EGF in combination with an endosomal escape enhancer suppressed the growth of carcinoma colonies by almost 50%. As a third and last step, CFPS was assessed for its potential as a rapid response system against novel viral pathogens using SARS-CoV2 viral proteins. All SARS-CoV2 proteins could be synthesized and analyzed. The cytotoxic behaviors of the nsp1 and envelope protein were determined. The nucleocapsid protein was quantitatively detected by specific antibodies thereby facilitating cell-free systems for the validation of available antibodies. All in all, this thesis successfully developed a platform technology for the cell-free synthesis, functional characterization and application of toxic proteins in clinical and diagnostic fields.
