Controlled Design and Production of Tunable Modular Protein Nanoparticles

<p>Bacterial inclusion bodies (IBs) are insoluble protein aggregates formed during the overexpression of recombinant genes in bacteria. Although usually considered as unfolded waste material, recent studies have shown that IBs can contain biologically functional proteins. Moreover, the fusion of aggregation-inducing or ‘Pull-down’ tags has been used as a strategy to produce catalytically active IBs. This project aims to optimise the production and purification of p40 inclusion bodies (p40-IBs) for the controlled design of modular protein nanoparticles for industrial applications. Especially, it focuses on the study of several parameters such as concentration of inducer, temperature and time of induction to achieve control over the yield, size, and morphology of p40-IBs. Another aspect of the project studied the intracellular formation of fluorescent mCherry-p40 IBs in the host cell <em>Escherichia coli. </em>Expression kinetics of mCherry-p40 IBs in <em>E. coli </em>cells showed that production parameters such as temperature, time of induction, and the concentration of inducer have a significant role in tuning the IBs properties. Further studies were conducted to analyse the biophysical characteristics of the mCherry-p40 IBs obtained under different production conditions. Finally, the reversible reaggregation of p40-IBs after complete solubilisation with a chaotropic agent was studied as a potential material biofunctionalisation strategy.</p>