Low-Level Expression of a Folding-Incompetent Protein in Escherichia coli: Search for the Molecular Determinants of Protein Aggregation In Vivo

Aggregation of peptides and proteins into insoluble amyloid fibrils or related intracellular inclusions is the hallmark of many degenerative diseases, including Alzheimer's disease, Parkinson's disease, and various forms of amyloidosis. In spite of the considerable progress carried out in vitro in elucidating the molecular determinants of the conversion of purified and isolated proteins into amyloid fibrils, very little is known on factors governing this process in the complex environment of living organisms. Taking advantage of increasing evidence that bacterial inclusion bodies consist of amyloid-like aggregates, we have expressed in Escherichia coli both wild type and 21 single-point mutants of the N-terminal domain of the E. coli protein HypF. All variants were expressed as folding-incompetent units in a controlled manner, at low and comparable levels. Their solubilities were measured by quantifying the protein amount contained in the soluble and insoluble fractions by Western blot analysis. A significant negative correlation was found between the solubility of the variants in E. coli and their intrinsic propensity to form amyloid fibrils, predicted using an algorithm previously validated experimentally in vitro on a number of unfolded peptides and proteins, and considering hydrophobicity, β-sheet propensity, and charge as major sequence determinants of the aggregation process. These findings show that the physicochemical parameters previously recognized to govern amyloid formation by fully or partially unfolded proteins are largely applicable in vivo and pave the way for the molecular exploration of a process as complex as protein aggregation in living organisms.