Critique of the use of fluorescence-based reporters in Escherichia coli as a screening tool for the identification of peptide inhibitors of Aβ42 aggregation.

High-throughput screens that dispense with the need for expensive synthetic Aβ peptide would be invaluable for identifying novel anti-aggregants as potential treatments for Alzheimer's disease. A biosynthetic in vivo approach, using a recombinant fluorescent green fluorescent protein (GFP) reporter for the aggregation state of Aβ in Escherichia coli, has been reported by other workers. Here, inducible Aβ-GFP expression in E. coli was coupled to the concurrent constitutive production of a quasi-random peptide library to screen for anti-aggregant activity. To attempt to introduce greater robustness, mCherry was also co-expressed as an internal fluorescence standard to allow ratiometric comparison between samples. However, fluctuations in mCherry expression levels, as well as a low dynamic range of GFP output between positive and negative anti-aggregant peptides, highlighted limitations with the approach. Despite this, two novel peptides were identified that showed an equivalent in vitro anti-aggregant activity to that of epigallocatechin-3-gallate. Thus, although biosynthetic in vivo strategies show promise as screens for novel activities, unforeseen problems can arise because of the variability inherent in any biological system.