Development of strategies for the characterisations of biologics

The aim of this PhD project was the development of strategies for the optimization of the expression and biophysical characterization of human proteins of pharmaceutical interest. On one hand, the aim was to design, implement and optimize protocols for the production and purification of recombinant human proteins in Escherichia coli. Particular effort was put on the improvement of yield, purity and stability of the protein samples. Moreover, special attention was paid to the expression of isotopically enriched proteins in minimal medium, which is known to be less straightforward and more expensive, given also the usually low yields. On the other hand, the other complementary target was the development of strategies based on the use NMR methodologies, both in solution and solid-state. This was relevant for many reasons: i) characterizing proteins; ii) studying interactions; iii) designing new therapeutics; iv) and investigating protein involvement not only in the insurgence and the progression of pathologies, but also in the prevention, diagnosis, and treatment. In this work, my main focus was the expression in E. coli of human proteins of pharmaceutical interest such as transthyretin (TTR), L-asparaginase II (ANSII), the programmed cell death protein 1 (PD1) and its ligand programmed death ligand 1 (PD-L1).