Advertisement

LuMPIS: Luciferase-Based MBP-Pull-Down Protein Interaction Screening System

  • María G. Vizoso PintoEmail author
  • Armin Baiker
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 815)

Abstract

Analyzing the putative interaction partners of an individual protein is one approach to elucidate its function. In the LuMPIS protocol, bait and prey proteins are expressed with N-terminal maltose binding protein (MBP)- and eGFP-luciferase (eGFP-luc) tags, respectively. Positive protein–protein interactions (PPIs) can be detected after pull-down of the MBP-tagged prey protein using amylose beads followed by the bioluminescence detection of the bound eGFP-luc-tagged bait protein. The LuMPIS technology offers the following advantages: the PPIs are detected in the mammalian cell context, the use of two long protein tags (i.e., MBP and eGFP-luc) increases the expression levels of genes whose gene expression levels are known to be frequently impaired, the use of amylose beads for pull-down is much more economic as compared to sepharose beads in combination with monoclonal antibodies and finally, the use of an eGFP-luc-tag enables the qualitative control of transfection efficiencies by fluorescence microscopy prior to starting the assay.

Key words

Protein–protein interactions High-throughput Luciferase detection Maltose binding protein 

References

  1. 1.
    Stellberger, T., Hauser, R., Baiker, A., Pothineni, V. R., Haas, J., and Uetz, P. (2010) Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome, Proteome Sci 8, 8.PubMedCrossRefGoogle Scholar
  2. 2.
    Barrios-Rodiles, M., Brown, K. R., Ozdamar, B., Bose, R., Liu, Z., Donovan, R. S., Shinjo, F., Liu, Y., Dembowy, J., Taylor, I. W., Luga, V., Przulj, N., Robinson, M., Suzuki, H., Hayashizaki, Y., Jurisica, I., and Wrana, J. L. (2005) High-throughput mapping of a dynamic signaling network in mammalian cells, Science 307, 1621–1625.PubMedCrossRefGoogle Scholar
  3. 3.
    Vizoso Pinto, M. G., Villegas, J. M., Peter, J., Haase, R., Haas, J., Lotz, A. S., Muntau, A. C., and Baiker, A. (2009) LuMPIS – a modified luminescence-based mammalian interactome mapping pull-down assay for the investigation of protein–protein interactions encoded by GC-low ORFs, Proteomics 9, 5303–5308.PubMedCrossRefGoogle Scholar
  4. 4.
    Seldeen, K. L., McDonald, C. B., Deegan, B. J., and Farooq, A. (2008) Evidence that the bZIP domains of the Jun transcription factor bind to DNA as monomers prior to folding and homodimerization, Arch Biochem Biophys 480, 75–84.PubMedCrossRefGoogle Scholar
  5. 5.
    Hu, C. D., Chinenov, Y., and Kerppola, T. K. (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation, Mol Cell 9, 789–798.PubMedCrossRefGoogle Scholar
  6. 6.
    Dyer, B. W., Ferrer, F. A., Klinedinst, D. K., and Rodriguez, R. (2000) A noncommercial dual luciferase enzyme assay system for reporter gene analysis, Anal Biochem 282, 158–161.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of VirologyMax von Pettenkofer-InstituteMünichGermany
  2. 2.Bavarian Health and Food Safety AuthorityOberschleissheimGermany

Personalised recommendations