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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...

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Detalles Bibliográficos
Autores principales: Guo, Zhengguang, Wang, Xiaorong, Li, Huihua, Gao, Youhe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
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
Descripción
Sumario: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.