Critique of the use of fluorescence-based reporters in Escherichia coli as a screening tool for the identification of peptide inhibitors of Aß42 aggregation.
J Pept Sci
; 19(2): 74-83, 2013 Feb.
Article
en En
| MEDLINE
| ID: mdl-23255255
ABSTRACT
High-throughput screens that dispense with the need for expensive synthetic Aß 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 Aß in Escherichia coli, has been reported by other workers. Here, inducible Aß-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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Fragmentos de Péptidos
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Péptidos
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Péptidos beta-Amiloides
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Proteínas Fluorescentes Verdes
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Escherichia coli
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Ensayos Analíticos de Alto Rendimiento
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Mediciones Luminiscentes
Tipo de estudio:
Diagnostic_studies
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Screening_studies
Idioma:
En
Revista:
J Pept Sci
Asunto de la revista:
BIOQUIMICA
Año:
2013
Tipo del documento:
Article
País de afiliación:
Reino Unido