Next Article in Journal
Structure of Dihydrochalcones and Related Derivatives and Their Scavenging and Antioxidant Activity against Oxygen and Nitrogen Radical Species
Next Article in Special Issue
Diversity of Phage-Displayed Libraries of Peptides during Panning and Amplification
Previous Article in Journal
Carotenoids and Their Isomers: Color Pigments in Fruits and Vegetables
Previous Article in Special Issue
Design and Screening of M13 Phage Display cDNA Libraries
Molecules 2011, 16(2), 1739-1748; doi:10.3390/molecules16021739
Article

Identification of Calpain Substrates by ORF Phage Display

,  and *
Received: 3 January 2011; Accepted: 18 February 2011 / Published: 21 February 2011
(This article belongs to the Special Issue Phage Display of Combinatorial Libraries)
Download PDF [223 KB, uploaded 18 June 2014]
Abstract: 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.
Keywords: calpain; protease; substrate; ORF phage display; calpastatin calpain; protease; substrate; ORF phage display; calpastatin
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Export to BibTeX |
EndNote


MDPI and ACS Style

Caberoy, N.B.; Alvarado, G.; Li, W. Identification of Calpain Substrates by ORF Phage Display. Molecules 2011, 16, 1739-1748.

AMA Style

Caberoy NB, Alvarado G, Li W. Identification of Calpain Substrates by ORF Phage Display. Molecules. 2011; 16(2):1739-1748.

Chicago/Turabian Style

Caberoy, Nora B.; Alvarado, Gabriela; Li, Wei. 2011. "Identification of Calpain Substrates by ORF Phage Display." Molecules 16, no. 2: 1739-1748.


Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert