Team:Tuebingen
Tübingen 2018
Development of a shuttle mechanism using detoxified botulium toxin
Inspiration
It has been demonstrated, that some botulinum neurotoxins (BoNTs) can be used for delivery of enzymatically active proteins.1 However, the remarkable toxicity of BoNTs has stopped this substance class from being employed for this feature. In 2017 Vazquez-Cintron et al. has shown a series of point mutations in botulinum toxin C that reduce its toxicity by a factor of approximately 5 x 106, while still being able to transport the light chain into the target cell cytosol.2 Combining these approaches should enable for constructs trafficking a wide range of molecules into neurons with a BoNTs intrinsic, excellent specificity and activity.
Project idea
Our project tries to explore the range of capabilities for internalization of the nontoxic botulinus toxin C, by fusing various proteins and small molecular compounds to it. By trying to use cleavable linkers, we will attempt to release our fused molecules from the botulinum toxin light chain once it has invaded the target cell cytoplasm. This should increase the activity of especially the small molecular compounds. Finally we wish to make our delivery system as modular as possible.
Coupling and assays
We will develop a library of different detoxified botulinum toxin derivatives which can carry other proteins, small molecules, and fluorochromes by specific linkers.
| Coupling | Assay |
|---|---|
| Omomyc-GSPGS_linker | Caspase-Glo® 3/7 Assay (Promega) |
| Cre-GSPGS_linker | loxP-GFP-loxP-RFP |
| SOD1 | ROS-Glo-Assay (Promega) |
| Eslicarbazepine | Patch clamp |
| Fluor488 | Microscopy |
| Phlourin2 (Sortase) | Microscopy |
| HiBit-LBit (Nano-Glo® Promega) | Luminescence (Plate reader) with positive control (Fuse-It Kit, IBIDI), Luciferase Assay |
- SNAP-25 cleavage assay for proof of the detoxification
Modelling and VR
We try to predict changes of the tertiary structure of botulinum toxin C1 caused by the detoxifying point mutations introduced in the toxin. Furthermore we are developing a virtual reality (VR) protein viewer for smartphones, that is easily and cheaply accessible for anyone, giving scientists an immersive experience while working with proteins.
We want you - for cooperations
| What we want | What we offer |
|---|---|
| Read out systems for living cells | pH-dependent GFP |
| Proteins or other molecules for coupling | Human cell line SH-SY5Y |
- Vazquez-Cintron, E. J., Beske, P. H., Tenezaca, L., Tran, B. Q., Oyler, J. M., Glotfelty, E. J., … Ichtchenko, K. (2017). Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery. Scientific Reports, 7, 42923. http://doi.org/10.1038/srep42923