Detection of a Peptide Biomarker by Engineered Yeast Receptors.
ACS Synth Biol
; 7(2): 696-705, 2018 02 16.
Article
em En
| MEDLINE
| ID: mdl-29366326
ABSTRACT
Directed evolution of membrane receptors is challenging as the evolved receptor must not only accommodate a non-native ligand, but also maintain the ability to transduce the detection of the new ligand to any associated intracellular components. The G-protein coupled receptor (GPCR) superfamily is the largest group of membrane receptors. As members of the GPCR family detect a wide range of ligands, GPCRs are an incredibly useful starting point for directed evolution of user-defined analytical tools and diagnostics. The aim of this study was to determine if directed evolution of the yeast Ste2p GPCR, which natively detects the α-factor peptide, could yield a GPCR that detects Cystatin C, a human peptide biomarker. We demonstrate a generalizable approach for evolving Ste2p to detect peptide sequences. Because the target peptide differs significantly from α-factor, a single evolutionary step was infeasible. We turned to a substrate walking approach and evolved receptors for a series of chimeric intermediates with increasing similarity to the biomarker. We validate our previous model as a tool for designing optimal chimeric peptide steps. Finally, we demonstrate the clinical utility of yeast-based biosensors by showing specific activation by a C-terminally amidated Cystatin C peptide in commercially sourced human urine. To our knowledge, this is the first directed evolution of a peptide GPCR.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
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Saccharomyces cerevisiae
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Engenharia de Proteínas
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Evolução Molecular Direcionada
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Proteínas de Saccharomyces cerevisiae
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Receptores de Fator de Acasalamento
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Cistatina C
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
ACS Synth Biol
Ano de publicação:
2018
Tipo de documento:
Article