Abstract
Here we describe a protocol for the generation of amyloid aggregates of target amyloidogenic proteins using a bacteria-based system called curli-dependent amyloid generator (C-DAG). C-DAG relies on the natural ability of Escherichia coli cells to elaborate surface-associated amyloid fibers known as curli. An N-terminal signal sequence directs the secretion of the major curli subunit CsgA. The transfer of this signal sequence to the N terminus of heterologous amyloidogenic proteins similarly directs their export to the cell surface, where they assemble as amyloid fibrils. Notably, protein secretion through the curli export pathway facilitates acquisition of the amyloid fold specifically for proteins that have an inherent amyloid-forming propensity. Thus, C-DAG provides a cell-based alternative to widely used in vitro assays for studying amyloid aggregation, and it circumvents the need for protein purification. In particular, C-DAG provides a simple method for identifying amyloidogenic proteins and for distinguishing between amyloidogenic and non-amyloidogenic variants of a particular protein. Once the appropriate vectors have been constructed, results can be obtained within 1 week.