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Identification of Calpain Substrates by ORF Phage Display
Department of Ophthalmology, Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, USA
* Author to whom correspondence should be addressed.
Received: 3 January 2011; Accepted: 18 February 2011 / Published: 21 February 2011
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
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MDPI and ACS Style
Caberoy, N.B.; Alvarado, G.; Li, W. Identification of Calpain Substrates by ORF Phage Display. Molecules 2011, 16, 1739-1748.
Caberoy NB, Alvarado G, Li W. Identification of Calpain Substrates by ORF Phage Display. Molecules. 2011; 16(2):1739-1748.
Caberoy, Nora B.; Alvarado, Gabriela; Li, Wei. 2011. "Identification of Calpain Substrates by ORF Phage Display." Molecules 16, no. 2: 1739-1748.