Eukaryotic Aggresomes: Protocols and Tips for Their Production, Purification , and Handling.

Abstract

Aggresomes are insoluble protein aggregates found in eukaryotic cells when the intracellular machinery is overtitered by, for example, the overexpression of a recombinant protein. These protein nanoparticles have become excellent models in studies devoted to elucidate protein aggregation processes in eukaryotic cells, like those involved in "conformational disorders" linked to neurodegenerative diseases. Since the presence of such protein aggregates is a hallmark of these conditions, they constitute an excellent target for new therapeutic approaches for such devastating pathologies. Moreover, and following the pathway opened a few years ago by bacterial inclusion bodies, eukaryotic aggresomes have been proposed as a new type of carrier-free, self-immobilized biocatalysts for use in biotechnology and biomedicine. Altogether, unraveling the characteristics and putative applications of naturally occurring protein aggregates has received an increasing interest during the last years. For that, availability of protocols allowing the production and purification of aggresomes constitute a valuable tool to boost research in the abovementioned fields. In this chapter, we describe both upstream and downstream protocols to obtain aggresomes produced in human cells, using as a model the recombinant human enzyme alpha-galactosidase A (GLA), together with technical tips and advices when working and analyzing eukaryotic aggresomes.