Document Detail


Identification of calpain substrates by ORF phage display.
MedLine Citation:
PMID:  21339709     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Nora B Caberoy; Gabriela Alvarado; Wei Li
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2011-02-21
Journal Detail:
Title:  Molecules (Basel, Switzerland)     Volume:  16     ISSN:  1420-3049     ISO Abbreviation:  Molecules     Publication Date:  2011  
Date Detail:
Created Date:  2011-02-22     Completed Date:  2011-04-27     Revised Date:  2013-07-15    
Medline Journal Info:
Nlm Unique ID:  100964009     Medline TA:  Molecules     Country:  Switzerland    
Other Details:
Languages:  eng     Pagination:  1739-48     Citation Subset:  IM    
Affiliation:
Department of Ophthalmology, Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Calcium-Binding Proteins / metabolism
Calpain / antagonists & inhibitors,  genetics,  metabolism*
Isoenzymes / antagonists & inhibitors,  genetics,  metabolism
Open Reading Frames*
Peptide Library*
Peptides / genetics,  metabolism*
Rats
Grant Support
ID/Acronym/Agency:
P30-EY014801/EY/NEI NIH HHS; R01 EY016211/EY/NEI NIH HHS; R01 EY016211-05/EY/NEI NIH HHS; R01EY016211/EY/NEI NIH HHS; R01EY016211-05S1/EY/NEI NIH HHS
Chemical
Reg. No./Substance:
0/Calcium-Binding Proteins; 0/Isoenzymes; 0/Peptide Library; 0/Peptides; 79079-11-1/calpastatin; EC 3.4.22.-/Calpain
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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