ETD

• bacterial two-hybrid
• intrabodies
• intrabody
• PISA
• post translational modifications
• ptms
• two-hybrid

Post-translational modifications (PTMs) of proteins are crucial for modulating the functional states of proteins in health and disease. Molecular tools able to differentiate distinct pools of functional states of the same protein, would be extremely useful to understand the nuances of molecular pathways and to formulate new drugs that act on the disease-specific states of the involved protein.
Intracellular antibodies (intrabodies) represent a class of molecules that could be exploited to selectively target PTMs. The P.I.S.A (Post-translational Intracellular Silencing Antibodies) technology developed in our group, is a tool that allows the selection of PTM-directed antibodies intracellularly.
We now plan on developing a 2.0 version of the P.I.S.A platform, by using the expanded genetic code to insert specific PTMs, decided a priori, at any given position in our bait co-translationally. This project will be developed in a bacterial-two hybrid (B2H) system, in lieu of the previously employed yeast-two hybrid system, as most of the work on the expansion of genetic code to date has been done in E. coli. The aims of this thesis are the creation of a naive camelid nanobody library to be used in the screenings, that will be tested in a well-stated B2H strain available in our laboratory; the integration of two orthogonal amber-suppression tRNA/synthetase pairs in the aforementioned B2H system, and their functional validation by readthrough sfGFP fluorescent protein expression.