An efficient protein complex purification method for functional proteomics in higher eukaryotes

Abstract

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes1,2. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast1,3,4. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi)5,6 to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Selection of the Dm exosome.
Figure 2: Selection of a tetrameric complex involved in mRNA nuclear export.
Figure 3: Selection of proteins associating with MGN-Y14 dimers.

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. 1

    Gavin, A.C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002).

    CAS  Article  Google Scholar 

  2. 2

    Ho et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    CAS  Article  Google Scholar 

  3. 3

    Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Puig, O. et al. The tandem affinity purification (tap) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Hannon, G.J. RNA interference. Nature 418, 244–251 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Clemens, J.C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97, 6499–6503 (2000).

    CAS  Article  Google Scholar 

  7. 7

    Mitchell, P. & Tollervey, D. Musing on the structural organization of the exosome complex. Nat. Struct. Biol. 10, 843–846 (2000).

    Article  Google Scholar 

  8. 8

    van Hoof, A & Parker, R. The exosome: a proteasome for RNA? Cell 99, 347–350 (1999).

    CAS  Article  Google Scholar 

  9. 9

    Conti, E. & Izaurralde, E. Nucleocytoplasmic transport enters the atomic age. Curr. Opin. Cell Biol. 13, 310–319 (2001).

    CAS  Article  Google Scholar 

  10. 10

    Katahira, J. et al. The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human. EMBO J. 18, 2593–2609 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Herold, A., Klimenko, T. & Izaurralde, E. NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila. RNA 7, 1768–1780 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Fribourg, S., Braun, I.C., Izaurralde, E. & Conti, E. Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor. Mol. Cell. 3, 645–656 (2001).

    Article  Google Scholar 

  13. 13

    Bachi, A. et al. The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. RNA 6, 136–158 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Palacios, I.M. RNA processing: splicing and the cytoplasmic localization of mRNA. Curr. Biol. 12, R50–R52 (2002).

    CAS  Article  Google Scholar 

  15. 15

    Le Hir, H., Gatfield, D., Braun, I.C., Forler, D. & Izaurralde, E. The protein Mago provides a link between splicing and mRNA localization. EMBO Rep. 2, 1119–1124 (2001).

    CAS  Article  Google Scholar 

  16. 16

    Tuschl, T. Expanding small RNA interference. Nat. Biotechnol. 20, 446–448 (2002).

    CAS  Article  Google Scholar 

  17. 17

    Benting, J., Lecat, S., Zacchetti, D. & Simons, K. Protein expression in Drosophila Schneider cells. Anal. Biochem. 278, 59–68 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Dignam, D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids. Res. 11, 1475–1489 (1983).

    CAS  Article  Google Scholar 

  19. 19

    Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    CAS  Article  Google Scholar 

  20. 20

    Wilm, M., Neubauer, G. & Mann, M. Parent ion scans of unseparated peptide mixtures. Anal. Chem. 68, 527–533 (1996).

    CAS  Article  Google Scholar 

  21. 21

    Mann, M. & Wilm, M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390–4399 (1994).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This study was supported by the European Molecular Biology Organization (EMBO) and by the Bundesministerium für Bildung und Forschung (BMBF), BioFuture grant no 0311862.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Matthias Wilm.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Forler, D., Köcher, T., Rode, M. et al. An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat Biotechnol 21, 89–92 (2003). https://doi.org/10.1038/nbt773

Download citation

Further reading