Protein fishing using magnetic nanobeads containing calmodulin site-specifically immobilized via an azido group.
J Biochem
; 154(2): 159-65, 2013 Aug.
Article
em En
| MEDLINE
| ID: mdl-23653405
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.
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Base de dados:
MEDLINE
Assunto principal:
Química Encefálica
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Calmodulina
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Proteômica
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Proteínas Imobilizadas
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Nanopartículas de Magnetita
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Proteínas do Tecido Nervoso
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article