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Screening E3 Substrates Using a Live Phage Display Library
Ubiquitin ligases (E3s) determine specificity of ubiquitination by recognizing target substrates. However, most of their substrates are unknown. Most known substrates have been identified using distinct approaches in different laboratories. We developed a high-throughput strategy using a live phage...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790729/ https://www.ncbi.nlm.nih.gov/pubmed/24124579 http://dx.doi.org/10.1371/journal.pone.0076622 |
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author | Guo, Zhengguang Wang, Xiaorong Li, Huihua Gao, Youhe |
author_facet | Guo, Zhengguang Wang, Xiaorong Li, Huihua Gao, Youhe |
author_sort | Guo, Zhengguang |
collection | PubMed |
description | Ubiquitin ligases (E3s) determine specificity of ubiquitination by recognizing target substrates. However, most of their substrates are unknown. Most known substrates have been identified using distinct approaches in different laboratories. We developed a high-throughput strategy using a live phage display library as E3 substrates in in vitro screening. His-ubiquitinated phage, enriched with Ni-beads, could effectively infect E. coli for amplification. Sixteen natural potential substrates and many unnatural potential substrates of E3 MDM2 were identified through 4 independent screenings. Some substrates were identified in different independent experiments. Additionally, 10 of 12 selected candidates were ubiquitinated by MDM2 in vitro, and 3 novel substrates, DDX42, TP53RK and RPL36a were confirmed ex vivo. The whole strategy is rather simple and efficient. Non-degradation substrates can be discovered. This strategy can be extended to any E3s as long as the E3 does not ubiquitinate the empty phage. |
format | Online Article Text |
id | pubmed-3790729 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-37907292013-10-11 Screening E3 Substrates Using a Live Phage Display Library Guo, Zhengguang Wang, Xiaorong Li, Huihua Gao, Youhe PLoS One Research Article Ubiquitin ligases (E3s) determine specificity of ubiquitination by recognizing target substrates. However, most of their substrates are unknown. Most known substrates have been identified using distinct approaches in different laboratories. We developed a high-throughput strategy using a live phage display library as E3 substrates in in vitro screening. His-ubiquitinated phage, enriched with Ni-beads, could effectively infect E. coli for amplification. Sixteen natural potential substrates and many unnatural potential substrates of E3 MDM2 were identified through 4 independent screenings. Some substrates were identified in different independent experiments. Additionally, 10 of 12 selected candidates were ubiquitinated by MDM2 in vitro, and 3 novel substrates, DDX42, TP53RK and RPL36a were confirmed ex vivo. The whole strategy is rather simple and efficient. Non-degradation substrates can be discovered. This strategy can be extended to any E3s as long as the E3 does not ubiquitinate the empty phage. Public Library of Science 2013-10-04 /pmc/articles/PMC3790729/ /pubmed/24124579 http://dx.doi.org/10.1371/journal.pone.0076622 Text en © 2013 Guo et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Guo, Zhengguang Wang, Xiaorong Li, Huihua Gao, Youhe Screening E3 Substrates Using a Live Phage Display Library |
title | Screening E3 Substrates Using a Live Phage Display Library |
title_full | Screening E3 Substrates Using a Live Phage Display Library |
title_fullStr | Screening E3 Substrates Using a Live Phage Display Library |
title_full_unstemmed | Screening E3 Substrates Using a Live Phage Display Library |
title_short | Screening E3 Substrates Using a Live Phage Display Library |
title_sort | screening e3 substrates using a live phage display library |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790729/ https://www.ncbi.nlm.nih.gov/pubmed/24124579 http://dx.doi.org/10.1371/journal.pone.0076622 |
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