A bacterial export system for generating extracellular amyloid aggregates

Here, we describe a detailed protocol for generating amyloid aggregates of target amyloidogenic proteins using a bacteria-based system. This system, which we call Curli-dependent amyloid generator (C-DAG), relies on the natural ability of E. coli cells to elaborate surface associated amyloid fibers known as curli that are composed of the amyloidogenic proteins CsgA and CsgB. A bipartite N-terminal signal sequence directs CsgA and CsgB across the inner and outer membranes to the outside of the cell. 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. Importantly, 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 that circumvents the need to purify individual proteins of interest. 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.