dc.contributor.author
Ramm, Franziska
dc.date.accessioned
2022-11-21T14:53:02Z
dc.date.available
2022-11-21T14:53:02Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/36850
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-36563
dc.description.abstract
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.
en
dc.format.extent
XV, 173, xxiii Seiten
dc.rights.uri
https://creativecommons.org/licenses/by/4.0/
dc.subject
Cell-free Synthesis
en
dc.subject
Proteinaceous Toxins
en
dc.subject
Bacterial Enterotoxins
en
dc.subject
Targeted Toxins
en
dc.subject
Viral Cytotoxins
en
dc.subject
Protein Analytics
en
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::572 Biochemie
dc.title
Development of a platform technology for the cell-free synthesis and functional characterization of toxic proteins with clinical and diagnostic relevance
dc.contributor.gender
female
dc.contributor.firstReferee
Kubick, Stefan
dc.contributor.furtherReferee
Wahl, Markus
dc.date.accepted
2022-10-20
dc.identifier.urn
urn:nbn:de:kobv:188-refubium-36850-5
dc.title.subtitle
Applications of bacterial enterotoxins, ribosome-inactivating proteins and viral cytotoxins
refubium.affiliation
Biologie, Chemie, Pharmazie
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access