Final Report Summary - PROARGUS (Protein Aggregation - a quantitative assessment) The PROARGUS project, a Marie Curie Reintegration Grant, enabled Alina Vasilescu, to resume successfully her research activity after working in industry. The project, hosted by the International Centre of Biodynamics, Romania, aimed to the evaluation of oligomer profile in aggregated proteins with identification of marker oligomers and to the development of an integrated sensing platform for soluble aggregates including dedicated sensors for the chosen markers. Protein aggregation is an important issue both in the manufacturing of biopharmaceuticals and when studying aging or amyloid diseases. Early detection of soluble aggregates (i.e. revealing aggregation before mature fibrils are formed) is required to enable prompt intervention in the manufacturing process (where protein aggregation has to be avoided) and because of the higher toxicity of some soluble aggregates compared to mature fibrils. To achieve its aims, the project comprised four connected stages. In Stage 1, a set of analytical methods was defined, allowing to monitor progressive stages in the aggregation process from unfolding and oligomerisation until the formation of mature fibrils. These methods include: Atomic Force Microscopy (AFM), Size Exclusion Chromatography (SEC), Matrix Assisted Laser Desorption Ionisation Time of Flight mass spectrometry (MALDI-TOF-MS), staining with fluorescent dyes such as Thioflavin T, enzymatic activity, gel electrophoresis and electrochemical determinations. While most of the techniques were available within the host laboratories, others, such as SEC, MALDI-TOF-MS, involved collaboration with other research groups widening fellows’ integration in the Romanian scientific community. Important results of this stage were: the forced degradation protocols that have been developed and then tuned to allow aggregation to reach fibril formation in a matter of days; Lysozyme monomer and dimer have been separated and their identity was confirmed by SEC and MALDI-TOF-MS. Stage 2 focused on the development of a sensing platform for quantitative assessment of aggregation. A novel aptamer and surface plasmon resonance (SPR)-based sensor was developed for the label-free detection of lysozyme and was then applied for studying the early stages of protein aggregation. The results emphasize the possibility to detect specifically lysozyme in the range 5-50 μg/mL. We have shown the possibility to detect the onset of aggregation by exploiting the heightened non-specific adsorption of oligomers compared to monomers.Stage 3 was centered on quantitative assessment of monomer and oligomers in buffered protein solutions, further optimisation and validation of the sensing platform. The aptasensor for lysozyme was optimised to achieve maximum sensitivity (detection limit of 2.4 nM) and for detecting lysozyme in real samples (wines were chosen due to the importance of detecting allergenic lysozyme that can be present in them). The aptasensor-based method was validated by comparison with a chromatographic procedure. Next, production of stable solutions of lysozyme oligomers was performed via cross-linking (using dimethyl suberimidate). After chromatographic separation and purification, the oligomers were tested by SDS-PAGE and MALDI-TOF and the identities of a lysozyme dimer and a trimer were confirmed. The binding of lysozyme monomer and dimer to the immobilized aptamer in the previously developed aptasensor was characterized by determining the kinetic constants and performing calibrations. Importantly, further optimisation allowed the possibility to detect, when analyzing with the aptasensor binary mixtures, 0.1% lysozyme dimer in a monomer solution, without separation. Stage 4 focussed on the study of aggregation progress and evaluation of commercial formulations. The studies have been focussed on lysozyme. The dimer was chosen as the main marker of lysozyme aggregation, due to the fragile nature of the aggregates, bound mostly by physical bonds, as evident in SDS-PAGE and Size Exclusion Chromatography studies. The aggregation process of lysozyme was further monitored using both the classic methods developed in the first stage of the project and also using the SPR aptasensor, optimised for the detection of lysozyme dimer. The project demonstrated that the aptasensor can be used as a sensitive tool to determine the formation and dynamics of lysozyme dimer and higher oligomers. The developed method can be applied in the future for therapeutic proteins/peptides, for which specific biorecognition elements exist. The results obtained in PROARGUS project have been published in 6 papers, in one book chapter, and presented in 4 oral communications and 2 posters at international conferences. The multidisciplinary approach in PROARGUS allowed the researcher and the host institution to establish new collaborations with researchers in Romania, France and Canada. The successful re-integration of the Researcher in the European research environment as result of PROARGUS project is further substantiated by active involvement of the fellow in the initiation and fulfillment of research projects. Notably, 1 national project supporting the establishment of fellows’ research team, 1 collaborative project, intended to minimize the gap between lab research and real industrial applications and 1 bilateral (Romania-France) project were initiated and managed by the Researcher in the last 4 years. Moreover, the PROARGUS project provided the perfect framework to initiate and strengthen the collaborative network of the fellow, which materialized in 3 European grant applications in whose preparation she was involved.
