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Identification of Calpain Substrates by ORF Phage Display
Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motif...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3175615/ https://www.ncbi.nlm.nih.gov/pubmed/21339709 http://dx.doi.org/10.3390/molecules16021739 |
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author | Caberoy, Nora B. Alvarado, Gabriela Li, Wei |
author_facet | Caberoy, Nora B. Alvarado, Gabriela Li, Wei |
author_sort | Caberoy, Nora B. |
collection | PubMed |
description | Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases. |
format | Online Article Text |
id | pubmed-3175615 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-31756152011-09-19 Identification of Calpain Substrates by ORF Phage Display Caberoy, Nora B. Alvarado, Gabriela Li, Wei Molecules Article Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases. MDPI 2011-02-21 /pmc/articles/PMC3175615/ /pubmed/21339709 http://dx.doi.org/10.3390/molecules16021739 Text en © 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Caberoy, Nora B. Alvarado, Gabriela Li, Wei Identification of Calpain Substrates by ORF Phage Display |
title | Identification of Calpain Substrates by ORF Phage Display |
title_full | Identification of Calpain Substrates by ORF Phage Display |
title_fullStr | Identification of Calpain Substrates by ORF Phage Display |
title_full_unstemmed | Identification of Calpain Substrates by ORF Phage Display |
title_short | Identification of Calpain Substrates by ORF Phage Display |
title_sort | identification of calpain substrates by orf phage display |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3175615/ https://www.ncbi.nlm.nih.gov/pubmed/21339709 http://dx.doi.org/10.3390/molecules16021739 |
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