Understanding the mechanism of protein aggregation in thermal and refolding studies

Biopharmaceutical production of protein for therapeutic use is an expanding practice for treatment of numerous diseases in medicine. However the full benefits of this technique have not yet been fully realised due to a number of barriers. The largest of these barriers is that of protein aggregation, where mis-folded protein monomers self associate to form non functional macromolecules; aggregates. Further understanding of protein aggregation may lead to an improvement in the effectiveness and availability of these therapeutic treatments. Here is presented work which utilises novel or under-used techniques to elucidate information on the structure of protein aggregates, their formation mechanisms and the kinetics and thermodynamics of their growth.

Results presented in the chapter on aggregate nucleation indicate that the nucleation stage of aggregation in bovine serum albumin has a temperature dependant mechanism, which in the middle of the temperature range follow mechanisms for stable nuclei population postulated in the literature. However at the extremes of this range, it appears that the nucleation mechanisms deviate from this and that there may be clustering of highly reactive nuclei at high temperatures, and continual formation of aggregate nuclei at low temperatures. Possible explanations for this behaviour are discussed.

Analysis presented on the growth of particulate aggregates show that the model of monomer addition to the aggregate nuclei appears to be a fitting description of the growth process, which is generic across proteins. Furthermore the detailed analysis from an ultra violet light scattering spectroscopy technique provides a numerical method for examining the efficiency of aggregate preventing additives, and also illustrates the mechanism by which the additives prevent aggregation through stabilising the native state.

Finally; results presented in the chapter on aggregation during refolding indicate the use of fluorescence anisotropy to monitor the molten globule state during refolding of proteins. Most strikingly, it is shown there is an obvious relationship between the mobility period of the protein and its propensity to aggregate. It is also shown that the presence of salt and urea can be utilised to moderate the presence of the molten globule state, and therefore the resultant aggregation.