Photorhabdus Toxin Complex (PTC) as variable protein injection apparatus

• Applicant: Professorin Dr. Gudula Schmidt
• Subject Area: Pharmacology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 441232035

In recent publications, we showed that the Photorhabdus toxin complex (PTC) can be used for injection of proteins into mammalian cells. The complex is composed of recombinant proteins an assembles in the culture medium. Now, we intend to use PTC for the injection of affibodies to block signaling pathways in mammalian cells. Affibodies are single chained small proteins showing high affinity to target proteins. Unpublished work of my group shows that a PTCinjected affibody binding to Raf 1 significantly reduces the EGFstimulated proliferation of cancer cells. An affibody against Ras showed weaker activity. The direct proof of affibodies within the cytosol is still missing. Because of the impressive cell biological effects of the affibodies we are interested to answer the questions how long the affibodies would be functional and how exactly the block the EGFR-Ras-MAPK pathway. Moreover, we would like to improve the injection conditions to enhance the amount of injected affibodies. We identified Plexin as possible cellular protein receptor for PTC by use of a knockout library and want to study, whether Plexin indeed is involved in PTC binding and uptake. A long lasting aim of this proposal is to change the cell specificity of PTC. This proposal additionally aims to build up an affibody library. With this we intend to efficiently use PTC and affibodies to block further signaling pathways. To isolate target specific binding affibodies, we express them on the surface of bacteria. We established a first system, however we intend to compare others. The isolated affibodies should be characterized biochemically and functionally.

• DFG Programme: Research Grants