Characterization of protein-protein interaction of single domain proteins in solution

Protein-based drugs show large potential as therapeutics due to their specificity and low toxicity. Almost all pharmaceutical companies have at least one protein drug in their development. Controlling protein stability and aggregation, however, remains challenging for the development. Formulation plays a major role in protein stability. Finding the right formulation is still mostly a trial and error approach, making the quest for the optimum formulation costly and time consuming.
The consortium ‘Protein-excipient Interaction and Protein-Protein Interaction in formulation’ (PIPPI) (EU-Project Number: 675074) was founded with the goal to develop a public database (https://pippi-data.kemi.dtu.dk/) of protein properties of different kinds of protein drugs in a systematic screen. Protein drugs with different three-dimensional folds were chosen for this systematic investigation. In the future, this knowledge should support the development of biopharmaceutics. At the same time, training of future experts in the field of protein formulation was pursued by the project, where 15 PhD students with different computational and experimental expertise were working on various aspects of protein formulation within the ‘PIPPI’ project.
This PhD project focused on the structural and biophysical characterization of single-domain protein drugs using a combination of high throughput methods and structure determination to acquire a better understanding of the biological system. In single-domain proteins, a high proportion of amino acids are solvent exposed on the protein surface. Aggregation due to protein-protein interaction plays a major role when studying these systems. To control protein aggregation, it is important to structurally characterize the protein aggregates and protein-protein interactions and to understand, which external factors affect aggregation. Within this project, three proteins, the fungal (Pseudoplectania nigrella) anti-microbial peptide plectasin, human interferon alpha-2a and Thermomyces lanuginosus lipase were analyzed in a systematic screen. The three proteins were structurally characterized in selected conditions, primarily by using dynamic light scattering and small angle X-ray scattering, in addition to other techniques. The characterized proteins resemble different types of protein drugs. Plectasin is a small 4.4 kDa peptide with a distinct secondary structure. Within this project the wild type and three variants were characterized. Interferon alpha-2a (19.2 kDa), a purely alpha helical protein, belongs to the group of cytokines. The Thermomyces lanuginosus lipase (31.8 kDa) is a well-characterized enzyme that catalyzes the hydrolysis of triacylglycerols and has various industrial applications.