UNIT 16.13 Expression and Purification of Epitope-Tagged Multisubunit Protein Complexes from Mammalian Cells
Published Online: 1 NOV 2002
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Wu, S.-Y. and Chiang, C.-M. 2002. Expression and Purification of Epitope-Tagged Multisubunit Protein Complexes from Mammalian Cells. Current Protocols in Molecular Biology. 60:III:16.13:16.13.1–16.13.17.
- Published Online: 1 NOV 2002
- Published Print: OCT 2002
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.