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J Biochem. 2013 Aug;154(2):159-65. doi: 10.1093/jb/mvt038. Epub 2013 May 7.

Protein fishing using magnetic nanobeads containing calmodulin site-specifically immobilized via an azido group.

Author information

1
Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.

Abstract

We developed a novel method for capturing proteins that interact with a target protein. This method utilizes a protein containing a site-specifically incorporated 3-azidotyrosine (N3-Y) and FG beads for immobilization of the protein via an azido group. Using calmodulin (CaM) as the target protein, we introduced N3-Y at position 72 and conjugated it to FG beads by copper-free click chemistry. From the Ca(2+)/CaM-binding proteins captured from mouse brain cell lysate and analysis by mass spectrometry, we identified six proteins: alpha-enolase (ENOA), glucose-6-phosphate isomerase (GPI), annexin A5 (ANXA5), malate dehydrogenase 1 (MDH1), glyceraldehyde-3-phosphate dehydrogenase and the well-known CaM-binding protein phosphoglycerate kinase 1 (PGK1). The presence of photo-crosslinking products via N3-Y for all the captured proteins except GPI indicated that they bound directly to CaM. In this study, ENOA, ANXA5 and MDH1 were identified as novel CaM-binding proteins, and PGK1 was bound to Ca(2+)/CaM and also Ca(2+)-free CaM. This method should prove useful for capturing novel interacting proteins and serve as a useful tool for proteomic analyses.

KEYWORDS:

3-azidotyrosine; FG beads; calmodulin; photo-crosslinking; protein–protein interaction

PMID:
23653405
DOI:
10.1093/jb/mvt038
[Indexed for MEDLINE]

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