Expression and purification of epitope-tagged multisubunit protein complexes from mammalian cells.
Biochemical characterization and functional studies of mammalian proteins are often hampered by the availability of the purified protein, in particular, when the functional entity is present as a multisubunit protein complex in the cell. To overcome the difficulties in the purification of multisubunit protein complexes from mammalian cells, one may create stable cell lines containing epitope-tagged protein. The first protocol in this unit describes the procedures involved in the establishment of a stable cell line constitutively expressing the FLAG-tagged protein by retrovirus-mediated gene transfer and immunoaffinity purification of the epitope-tagged multisubunit protein complex. The next protocol outlines the steps involved in the establishment of an inducible cell line conditionally expressing the FLAG-tagged protein by a tetracycline-regulated system, and the one-step immunoaffinity purification of the multisubunit protein complex. An alternate protocol provides an excellent example for the purification of different forms of human RNA polymerase II complexes, achieved simply by choosing the appropriate starting material and by varying wash conditions. The isolation of various human TATA-binding protein (TBP)-containing complexes is described in a support protocol, and is a good example of combining the P11 column and immunoaffinity purification. These protocols, collectively, illustrate a powerful methodology in applying epitope tagging and stable cell line approaches for the purification of multisubunit protein complexes from mammalian cells.
Wu
Shwu-Yuan
SY
Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Chiang
Cheng-Ming
CM
D000818:Animals; D002460:Cell Line / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007202:Indicators and Reagents; D007475:Ion Exchange Resins; D008322:Mammals; D046912:Multiprotein Complexes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014161:Transduction, Genetic
D000939:Epitopes; D007202:Indicators and Reagents; D007475:Ion Exchange Resins; D046912:Multiprotein Complexes; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide