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An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor
The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure...
Autores principales: | , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2008
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330230/ https://www.ncbi.nlm.nih.gov/pubmed/18319286 http://dx.doi.org/10.1093/nar/gkm1163 |
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author | Dirnberger, Dietmar Messerschmid, Monika Baumeister, Ralf |
author_facet | Dirnberger, Dietmar Messerschmid, Monika Baumeister, Ralf |
author_sort | Dirnberger, Dietmar |
collection | PubMed |
description | The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol. |
format | Text |
id | pubmed-2330230 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-23302302008-05-05 An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor Dirnberger, Dietmar Messerschmid, Monika Baumeister, Ralf Nucleic Acids Res Methods Online The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol. Oxford University Press 2008-04 2008-03-04 /pmc/articles/PMC2330230/ /pubmed/18319286 http://dx.doi.org/10.1093/nar/gkm1163 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Dirnberger, Dietmar Messerschmid, Monika Baumeister, Ralf An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor |
title | An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor |
title_full | An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor |
title_fullStr | An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor |
title_full_unstemmed | An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor |
title_short | An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor |
title_sort | optimized split-ubiquitin cdna-library screening system to identify novel interactors of the human frizzled 1 receptor |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330230/ https://www.ncbi.nlm.nih.gov/pubmed/18319286 http://dx.doi.org/10.1093/nar/gkm1163 |
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