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Purification of the COP9 Signalosome Complex and Binding Partners from Human T Cells

Details

Publisher
Mary Ann Liebert, Inc.
Copyright
Copyright 2012, Mary Ann Liebert, Inc.
Subject
Original Article
ISSN
1536-2310
eISSN
1557-8100
D.O.I.
10.1089/omi.2011.0158
Publisher site
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Purification of the COP9 Signalosome Complex and Binding Partners from Human T Cells

Abstract

The COP9 Signalosome (CSN) is a highly conserved eight subunit protein complex associated with a wide range of essential biological functions in eukaryotic cells, and directly involved in processes including deneddylation, phosphorylation, and ubiquitination. Despite its significant role, very few studies have been undertaken to reveal the interactions between the CSN and its binding partners, and none in human T cells. Here we present a purification method for the CSN and binding proteins via the Streptavidin-Binding Peptide (SBP) fused to CSN Subunit 1 (CSN1). Using this method, coupled with liquid chromatography-mass spectrometry analysis, we identified all eight subunits of the CSN, as well as expected and putative novel binding partners such as a tumor suppressor under the control of Cullin4a-ligase complex; Neurofibromin 2 (Merlin). This work presents a method for fast, reliable, and specific affinity-based purification of a protein complex from a nonadherent cell line. The purification of the CSN and binding partners from T cells can elucidate the roles of CSN in a cell type where it has never been studied before. This proteomic-based approach can broaden our understanding of the functions of the CSN in contexts such as viral–host interactions or immune activation in their natural milieu.
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