Identification of calpain substrates by ORF phage display

Nora B. Caberoy, Gabriela Alvarado, Wei Li

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1739-1748
Number of pages10
JournalMolecules
Volume16
Issue number2
DOIs
StatePublished - Feb 1 2011

Fingerprint

Bacteriophages
Calpain
Open Reading Frames
Display devices
protease
Reading Frames
Substrates
Peptide Hydrolases
peptides
Gene Library
proteins
Peptide Library
Peptides
Proteins
Streptavidin
biotin
Biotin
Substrate Specificity
inhibitors
Clone Cells

Keywords

  • Calpain
  • Calpastatin
  • ORF phage display
  • Protease
  • Substrate

ASJC Scopus subject areas

  • Organic Chemistry

Cite this

Identification of calpain substrates by ORF phage display. / Caberoy, Nora B.; Alvarado, Gabriela; Li, Wei.

In: Molecules, Vol. 16, No. 2, 01.02.2011, p. 1739-1748.

Research output: Contribution to journalArticle

Caberoy, Nora B. ; Alvarado, Gabriela ; Li, Wei. / Identification of calpain substrates by ORF phage display. In: Molecules. 2011 ; Vol. 16, No. 2. pp. 1739-1748.
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