The monitoring and affinity purification of proteins using dual tags with tetracysteine motifs.
Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.
Giannone
Richard J
RJ
Biosciences Division, Oak Ridge National Laboratory, 37831, Oak Ridge, TN, USA.
Liu
Yie
Y
Wang
Yisong
Y
D000345:Affinity Labels; D020816:Amino Acid Motifs; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification; D025941:Protein Interaction Mapping; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry; D035341:Telomeric Repeat Binding Protein 2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000345:Affinity Labels; D017931:DNA Primers; D046912:Multiprotein Complexes; D011506:Proteins; D011993:Recombinant Fusion Proteins; C504753:TERF2 protein, human; D035341:Telomeric Repeat Binding Protein 2; D003545:Cysteine