title: Critique of the use of fluorescence-based reporters in Escherichia coli as a screening tool for the identification of peptide inhibitors of Aβ42 aggregation.
abstract: 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.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, New Museums Site, Pembroke Street, Cambridge, CB2 3RA, UK.
authors.forename: Oliver
authors.initials: O
authors.lastname: Wright
authors.forename: Liao
authors.initials: L
authors.lastname: Zhang
authors.forename: Yun
authors.initials: Y
authors.lastname: Liu
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yoshimi
authors.forename: Yizhi
authors.initials: Y
authors.lastname: Zheng
authors.forename: Alan
authors.initials: A
authors.lastname: Tunnacliffe
mesh_terms: D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000032:analysis* / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects
chemical_list: D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; C075222:amyloid beta-protein (1-42); D049452:Green Fluorescent Proteins