During this first half of the project, our team has focused on the necessary methodological developments.
We have perfected our expression platform for large-scale mammalian protein production, based on our previously published lentiviral plasmid suite, by introducing numerous improvements to enable production of heteromeric proteins and protein complexes. We are now using this technology for the production of all our soluble and membrane target proteins. A second development is the implementation of a unique workflow for synthetic Nb discovery starting from highly diverse (~1012 unique sequences) in vitro RNA libraries, based on ribosome display, yeast display, and magnetic and fluorescence-enabled cell sorting.
These methods are enabling engineering of transient, low-affinity neuronal ligand-receptor interactions. The ability to increase the affinity between two interaction surfaces is key to future structural studies of transient protein-protein interactions, which are abundant in the synaptic cleft. The strategy is based on introducing sequence variability in interaction interfaces using genetic techniques, followed by screening using our yeast display platform.
We are using the synthetic Nb procedure to identify binders against a variety of postsynaptic and presynaptic signaling proteins, which will enable the rapid testing of new synthetic synaptic organiser protein designs.
Finally, we are working on screening proteins putatively interacting with ionotropic glutamate receptors (iGluRs). We have confirmed a number of candidate molecules, and are in the process of quantifying the interaction strength of these complexes, before turning to their structure determination.
