Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Caberoy, Nora B., Alvarado, Gabriela, Li, Wei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2011
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
_version_ 1782212166190891008
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
work_keys_str_mv AT caberoynorab identificationofcalpainsubstratesbyorfphagedisplay
AT alvaradogabriela identificationofcalpainsubstratesbyorfphagedisplay
AT liwei identificationofcalpainsubstratesbyorfphagedisplay